WO2025171397A1 - Methods to stabilize chemokine proteins in solution and for point of care detection of stabilized chemokine proteins - Google Patents

Abstract

A method of stabilizing a chemokine protein with a C-X-C or a C-C structural motif such as CXCL13 and CCL21 is provided. The method comprises the step of introducing the chemokine proteins into a solution comprising a buffer a non- ionic or amphoteric surfactant; and an aqueous chaotropic agent. A method of detecting a chemokine protein with a C-X-C or a C-C structural motif such as CXCL13 and CCL21 conjugated to antibodies thereto labelled with a fluorescent label in a solution is also provided. The method comprises the following steps: providing a test substrate comprising an elongate nitro cellulose membrane.

Description

Title

METHODS TO STABILIZE CHEMOKINE PROTEINS IN SOLUTION AND FOR POINT OF CARE

DETECTION OF STABILIZED CHEMOKINE PROTEINS

Field

This invention relates to methods to stabilize lymphoid proteins in order to determine real-time specimen adequacy in biopsy procedures and for detecting the stabilized lymphoid proteins on a point of care cartridge.

Background

Lung cancer is a prevalent malignancy globally and is a leading cause of cancer-related deaths in both males and females It is estimated that 2.2 million patients are diagnosed annually.¹

Patients suspected of having lung cancer are typically first assessed with CT and/or x-ray imaging, followed by a biopsy of lymph nodes in the identified regions of concern. Lymph node tissue taken from the biopsy is subsequently diagnosed by pathology as malignant or benign If malignant, the type of cancer is identified along with its staging. With this information, an appropriate course of patient treatment is planned.

Essential to this process is the ability to obtain sufficient lymph node tissue for diagnosis by pathology analysis. Trans Bronchial Needle Aspiration, whether by conventional methods (c-TBNA),² or Endo Bronchial UltraSound-guided (EBUS-TBNA), is a minimally invasive procedure used to perform biopsies.³⁴ During EBUS-TBNA, the patient is sedated, and an ultrasound-guided bronchoscope is used to position an aspiration needle at each targeted lymph node. The needle punctures the bronchial wall (from the inside) to sample the lymph node situated on the exterior of the bronchial passage. Multiple aspiration attempts, or “passes”, are made for each lymph node to obtain specimen volume sufficient for pathology diagnosis. Generally, EBUS-TBNA is used for real-time imaging and aspiration biopsy of mediastinal and hilar tissues in the upper respiratory tract. Related procedures Endoscopic UltraSound-guided Fine Needle Aspiration (EUS-FNA) and Endoscopic UltraSound with Bronchoscope-guided Fine Needle Aspiration (EUS-B-FNA) are used to assess the posteroinferior mediastinum.⁵'⁶ In all cases, the obtained specimens are sent for analysis at a pathology laboratory, typically a central laboratory remote to the biopsy suite. Specimens that are sufficient for pathological analysis are deemed to be “diagnostic”, and those insufficient are “nondiagnostic”.

While ultrasound-guided biopsy procedures offer significant benefits - minimally invasive, provide realtime imaging, enable comprehensive staging - achieving consistent and adequate tissue specimens during sampling procedures has proven to be a challenge. Rates of nondiagnostic specimens can be as high as 40%.⁷ Medical team experience, diverse specimen acquisition techniques and a lack of procedural standardization contribute to elevated rates of nondiagnostic specimens, and a need to repeat the bronchoscopy procedure.

Rapid On-Site Evaluation (ROSE) is a procedure in which a portion of the aspirated specimen is rapidly assessed by a cytopathologist (or a cytotechnician under their authority) during the biopsy to provide real-time feedback to the medical team, helps optimize specimen diagnostic yield and reduces the rate of repeat bronchoscopy procedures.⁸ Additionally, ROSE helps to reduce the need for excessive number of aspiration passes and lowers the risk to the patient by contributing to a shorter procedure and reduced risk of bleeding.

Despite its benefits, the availability of ROSE is limited primarily by the availability cytopathologists and cytotechnicians⁹’¹⁰ and secondarily the lack of standardized and quantitative criteria for cytological interpretation. The current low value of reimbursement OPT codes creates additional barriers to the implementation of ROSE.¹¹ A 2018 survey of members of the American Society of Cytopathology reports that 59% of respondents use a type of ROSE procedure.¹²

Olympus, Cook and Boston Scientific are market leaders in the provision of EBUS-TBNA equipment and needle kits. EBUS-TBNA instrumentation and each needle kit (one per patient per procedure) cost approximately $100,000 and $3,500 respectively. The market leader, Olympus, is estimated to have worldwide annual sales of 400,000 kits.

Several distinct technological approaches have been explored to improve the cytotechnician- or cytopathologist-run ROSE procedure, or to develop alternate methods to ROSE that increase specimen diagnostic yield.

Telecytology provides an ability to extend the ROSE procedure to diverse and remote sites by reducing the need for a cytopathologist to be physically present in the biopsy suite.¹⁰ In this approach, cytolology slides are prepared in the biopsy suite, digitized, and enabled to be reviewed remotely over the internet by a cytopathologist. This technique enables a single cytopathologist to support multiple biopsy suites in diverse physical locations, and may become economical by eliminating cytopathologist travel and other down time.¹⁰'¹²

Milestone Medical (Via Fatebenefratelli 1-5, 24010 - Sorisole (BG) ITALY, P.IVA: IT01879330163) has attempted to improve efficiency of histology within the biopsy suite by creating a standardized mobile workstation for ROSE.¹³ The RoseSTATION standardizes and collects all required equipment and consumables on a single mobile cart.

With a similar focus on workflow efficiency, ASP Health has developed an automated ROSE sample preparation platform.¹⁴ The platform incorporates all the histology consumable supplies and an automated slide preparation and slide reader instrument which has the potential to reduce the specimen diagnostic adequacy assessment in the biopsy suite. This device has not been cleared by the FDA.¹⁵

Ishiwata has described a biomarker approach to ROSE.¹⁶ Instead of a histological slide review, cytokine protein biomarkers CXCL13 (C-X-C motif chemokine ligand 13) and CCL21 (C-C motif chemokine ligand 21), previously known to be specific to the lymph node,^{17 18} were assessed using commercially available ELISA sandwich immunoassay kits. A correlation was established between elevated concentrations of biomarkers and specimen diagnostic adequacy determined by pathology. While this approach showed promise, the delivery of the immunoassay results is limited by the length of time required to run these tests (3.5-4.75 hours); this turnaround time exceeds significantly the requirements of 10 minutes or less to fit within the 30-60 minute duration of the EBUS-TBNA procedure.

The structures of CXCL13¹⁹ and CCL21²⁰'²¹ have been characterized by protein x-ray diffraction crystallography. Both structures are characterized by a core domain consisting of an N-loop region follow by a three-stranded [3-sheet and a C-terminal a-helix. The proteins are stabilized by a pair of disulfide bonds. Chemokines have propensity to form oligomers, with each other and with other chemokines and chemokine receptors The function effects of oligomerization are not fully understood.²²

The stability of proteins in solutions is essential to analytical performance in the immunoassay format, especially in a point of care format where the time to result delivery is typically under 15 minutes. The solution in which Ishiwata collected the lung biopsy specimens is a diluted, unbuffered saline solution. It is unclear whether this matrix is ideal for protein stability and control of oligomerization, and whether it can preserve chemokine structure and stability throughout freeze-thaw cycle(s) during sample processing prior to analysis.²³

None of the above provides a product with: (1) timely result of sample adequacy within the timeframe of the biopsy (2) a method to determine sample adequacy independent of highly trained cytopathologist or cytotechnician, (3) methodology to provide stability of the biomarker proteins, (4) a methodology for detecting the sample quantitatively and qualitatively on a point of care cartridge. There is a need for a method that overcomes the above-mentioned limitations and that includes the features enumerated above.

While the proteins contain structural motifs, specifically disulfide bonds which are expected to stabilize proteins, the proteins are unstable in conventional matrices and are unable to be detected at clinically relevant concentrations using standard point of care technologies in less than 15 minutes. There is a need for a method for detecting the proteins in less than 15 minutes.

There is a need for the determination of biomarker proteins at clinically relevant concentrations within the time frame of biopsy procedures, such as EBUS-TBNA, to enable the determination that the biopsy specimen contains lymphoid tissue sufficient for subsequent diagnosis of malignancy and additionally support the staging of cancer.

Summary

The invention provides in an aspect, methods for stabilizing natural or recombinant proteins in various liquid matrices. Also provided in other aspects are methods and systems to determine the concentrations of natural or recombinant proteins in liquid matrices using a point of care testing format. Also provided in other aspects, are methods and systems to determine whether obtained samples originate from lymph nodes in the context of cancer screening.

The method can for example be used to verify the adequacy of samples obtained during a biopsy procedure, in which the determination of concentration of biomarker proteins is required to be executed during the time frame of the biopsy procedure, typically 30-60 minutes.

The features preferably include a formulation comprising a pH-buffer solution component, a surfactant component, and a chaotropic component. More preferably, the formulation also comprises an osmolyte.

The buffer may be any of a number of buffers known to persons skilled in the art that maintain the pH of a solution in a pH range of from about pH 6 to about pH 9 with sufficient buffer capacity to handle the addition of acidic or alkaline components where the buffer is compatible with maintaining proteins in the buffer solution.

The buffer components are preferably selected from saline, 1 X Phosphate buffer, phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane buffer (Tris) and 2-[4-(2-hydroxyethyl)piperazin-1- yl]ethanesulfonic acid (HEPES). pH contributes to the stability of proteins.

The surfactant component preferably includes a surfactant selected from: polyvinylpyrrolidone (PVP), TWEEN® 20, TWEEN® 80, polyethylene glycol) 8000 (PEG8000), IGEPAL® CA-630, Tergitol™ NP- 40S, 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) and N,N- Dimethyl-N-dodecylglycine betaine, N-(Alkyl Cw-Ci6)-N,N-dimethylglycine betaine (EMPIGEN® BB). Surfactant components contain both hydrophilic and hydrophobic regions and serve to solubilize proteins without denaturation.

The chaotropic component preferably includes a chaotropic agent selected from EDTA, glycine, ethanol, urea. Chaotropic components may disrupt hydrogen bonding networks in aqueous solutions and create disorder in the water network surrounding proteins. For the purposes of the present disclosure, the terms chaotropic components and chaotropes have the same meaning and can be used interchangeably.

The solution may include osmolytes which are compounds that stabilize cellular components, such as red blood cells, of the patient sample via modulation of the osmotic pressure. Acceptable osmolytes include trehalose, mannose, raffinose, sorbitol, mannitol, xylitol, glycerol, TMAO, TMAO dihydrate, proline, betaine and DMSO. The chirality of sugars that function as osmolytes does not affect the functionality of the osmolyte for stabilizing cellular components, such as red blood cells. As such the function of D-mannitol and D-sorbitol as osmolytes is equivalent to the function of mannitol, and sorbitol for example.

According to an aspect, there is provided a method of stabilizing chemokine proteins with C-X-C and C-C structural motifs is provided. The method comprises the step of introducing the chemokine proteins into a solution comprising a buffer; a surfactant; and a chaotropic agent.

According to an aspect, there is provided a method of stabilizing a chemokine protein with a C-X-C or a C-C structural motif. The method comprises the step of introducing the chemokine protein into a solution comprising a buffer; a surfactant preferably selected from 1 % polyvinylpyrrolidone (PVP), 0.25% Tween 20, 0.25% Tween 80, 0.5% Tween 80, 1% poly(ethylene glycol) 8000 PEG 8000, 0.5% IGEPAL CA-630, 1.0% (wt/vol) EMPIGEN BB, 0.5% to 0.75% Tergitol NP-40S and 0.5% CHAPSO; and a chaotropic agent preferably selected from 30 mM ethylenediaminetetraacetic acid (EDTA), 1 % glycine, 1 % ethanol and 0.5M urea.

According to another aspect, there is provided a solution for stabilizing chemokine proteins with C-X-C and C-C structural motifs comprising a buffer; a non-ionic or amphoteric surfactant; and an aqueous chaotropic agent.

According to another aspect, there is provided a solution for stabilizing a chemokine protein with a C- X-C or a C-C structural motif comprising a buffer; a surfactant preferably selected from 1% polyvinylpyrrolidone (PVP), 0.25% Tween 20, 0.25% Tween 80, 0.5% Tween 80, 1 % poly(ethylene glycol) 8000 PEG 8000, 0.5% IGEPAL CA-630, 1.0% (wt/vol) EMPIGEN BB, 0.5% to 0.75% Tergitol NP-40S and 0.5% CHAPSO; and a preferably chaotropic agent selected from 30 mM ethylenediaminetetraacetic acid (EDTA), 1 % glycine, 1% ethanol and 0.5M urea.

According to an aspect, the buffer is preferably selected from 1 X Phosphate buffer, 1 X phosphate buffer saline (1X PBS), 0.05M tris(hydroxymethyl)aminomethane (Tris) and 0.01 M 2-[4-(2- hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

According to an aspect, the buffer is preferably 1 X Phosphate, pH 7.4.

According to another aspect, there is provided a method of detecting at least one chemokine protein with a C-X-C or a C-C structural motif conjugated to antibodies thereto labelled with a fluorescent label in a solution comprising the following steps: providing a test substrate comprising an elongate nitrocellulose membrane, the nitrocellulose membrane having a first end for receiving the solution and a second end whereby the solution flows from the first end to the second end by means of wicking, the substrate including a sample pad located at the first end of the nitrocellulose membrane and an absorbent pad located on the second end of the nitrocellulose membrane, at least one of a test line for the at least one chemokine protein with a C-X-C or a C-C structural motif being printed on the nitrocellulose membrane between the sample pad and the absorbent pad, the nitrocellulose membrane further including a control line printed thereon between the sample pad and the absorbent pad; treating the sample pad with Stabilization Buffer (SB); treating the conjugate release pad with Conjugate Dilution Buffer (CDB); applying the solution to the first end of the nitrocellulose membrane; reading the level of fluorescent intensity of at least one of a test line and the control line with a reader; determining the level of the at least one chemokine protein with a C-X-C or a C-C structural motif present in the solution based on the reading.

According to another aspect, there is provided a test substrate comprising an elongate nitrocellulose membrane, the nitrocellulose membrane having a first end for receiving the solution and a second end whereby the solution flows from the first end to the second end by means of wicking, the substrate including a sample pad located at the first end of the nitrocellulose membrane and an absorbent pad located on the second end of the nitrocellulose membrane, at least one of a test line for a chemokine protein with a C-X-C or a C-C structural motif being printed on the nitrocellulose membrane between the sample pad and the absorbent pad, the nitrocellulose membrane further including a control line printed thereon between the sample pad and the absorbent pad; wherein the sample pad is treated with Stabilization Buffer (SB), wherein the conjugate release pad is treated with Conjugate Dilution Buffer (CDB).

According to another aspect of the present invention, Stabilization Buffer (SB) and the Conjugate Dilution Buffer (CDB) are preferably tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5.

According to another aspect, the chemokine protein with a C-X-C or a C-C structural motifs to be detected is selected from the group consisting of CCL21 , CXCL 13 and CCL19.

According to another aspect, there is provided a method for detecting the sample quantitatively and qualitatively on a point of care cartridge wherein the chemical and biological composition of the stabilizing components in solution are extended to cartridge elements of the point of care cartridge. The cartridge elements are treated with the Stabilization Buffer or Conjugate Dilution Buffer solution, preferably by soaking, followed by a drying (curing) step prior to being assembled in the cartridge.

Brief Description of the Drawings

FIG. 1 is a plot of the CCL21 signal intensity of different CCL21 concentrations in multiple buffer solution formulations, as measured in a point of care device.

FIG. 2 is a plot of the CCL21 signal intensity of different CCL21 concentrations in 0.05M Tris buffer solutions containing surfactant additives, as measured in a point of care device.

FIG. 3 is a plot of the CCL21 signal intensity of different CCL21 concentrations in 0.05M Tris buffer, pH 7.4 with 0.50% Tergitol NP-40S solutions containing chaotropic additives, as measured in a point of care device.

FIG. 4 is a plot of the CCL21 signal intensity of different CCL21 concentrations in solutions of 0.05M Tris buffer, 0.50% Tergitol NP-40S and 0.5M urea, at different pH levels, as measured in a point of care device.

FIG. 5 is a plot of the CCL21 signal intensity of different CCL21 concentrations in solutions of 0.05M Tris buffer, pH 8.0, 0.50% Tergitol NP-40S solutions containing chaotropic additives, as measured in a point of care device.

FIG. 6 is a plot of CXCL13 and CCL21 biomarker concentrations in clinical specimens obtained from five lymph node sites from two patients during lung biopsy procedure and solubilized in stabilizing solution, as measured in a point of care device.

FIG. 7A is a plot of CCL21 and CXCL13 signal intensity of different CCL21 and CXCL13 concentrations in Tris and HEPES buffered solutions.

FIG. 7B is a plot of CCL21 and CXCL13 signal intensity of different CCL21 and CXCL13 concentrations in Tris, HEPES and Phosphate buffered solutions containing 0.5M Urea, 0.625% Tergitol NP-40S, and a preservative (either 0.03% ProClin300 or 0.09% sodium azide). FIG. 8 is a plot of CCL21 signal intensity at 2E4 pg/mL CCL21 concentration in presence of whole blood, with and without D-Mannitol in buffer.

FIG. 9 is a plot of buffer condition versus signal intensity relating to the addition of ProClin300 to buffer.

FIG. 10 is a photograph of a point of care test cartridge showing visual readings of response to whole blood (1 , 2, 5, 10% in buffer).

FIG. 11 is a plot of CCL21 signal intensity at 30 ng/mL CCL21 concentration for Tergitol in buffer.

FIG. 12 is a plot of CCL21 signal intensity for osmolytes Betaine and TMAO in buffer.

FIG. 13 is a plot of CCL21 signal intensity for buffer with 0.3M D-Sorbitol plus urea and urea replacements.

FIG. 14 is a plot of CCL21 signal intensity at 30 ng/mL CCL21 concentration for buffer containing 0.3M D-mannitol and D-Trehalose, D-mannose, xylitol, sorbitol.

Fig 15 is a plot of CCL21 signal intensity for sugar osmolytes.

FIG 16 a plot of CCL21 signal intensity at 30 ng/mL CCL21 concentration for osmolytes DMSO and proline in buffer.

FIG. 17 is a front perspective view of internal components of a lateral flow test cartridge

FIG 18 is a plot of CCL21 signal intensity at different concentrations for different sample pad conditions.

FIG 19 is a plot of CCL21 signal intensity at different concentrations for different sample pad conditions. FIG 20 is a plot of CCL21 signal intensity at different concentrations for different sample pad conditions.

FIG 21 is a plot of CCL21 signal intensity at different concentrations for different sample pad conditions.

FIG 22 is a plot of CCL21 signal intensity at different concentrations for different sample pad conditions. FIG 23 is a plot of CCL21 signal intensity at different concentrations for different conjugate release pad conditions.

FIG 24 is a plot of CCL21 signal intensity at different concentrations for different conjugate release pad conditions.

FIG 25 is a plot of CXCL13 and CCL21 signal intensity as function of conjugation ratios (1 :10, 1 :20).

Detailed Description

The present disclosure relates to methods for stabilization of chemokine proteins in aqueous solution for the purpose of assessing presence of chemokine proteins in aqueous solution both quantitatively and qualitatively by diagnostic devices.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Terms, definitions and terminology used in the description in this disclosure are for describing particular embodiments only and is not intended to limit the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated in their entirety by reference.

Terminology o standard deviation

ABS Acrylonitrile Butadiene Styrene

CCL19 C-C motif chemokine ligand 19

CCL21 C-C motif chemokine ligand 21

CDB Conjugate Dilution Buffer

CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate

CPT Current Procedural Terminology

CT Computed Tomography CXCL13 C-X-C motif chemokine ligand 13

%CV Percentage coefficient of variation, calculated as 100% x (o I mean)

DMSO Dimethyl sulfoxide

EBUS-TBNA Endo Bronchial UltraSound-guided Trans Bronchial Needle Aspiration

EDTA Ethylenediaminetetraacetic acid, C10H16N2O8

ELISA Enzyme-linked immunosorbant assay

EMPIGEN BB N,N-Dimethyl-N-dodecylglycine betaine, N-(Alkyl C10-C16)-N,N-dimethylglycine betaine

Eu Europium

EUS-B-FNA Endoscopic UltraSound with Bronchoscope-guided Fine Needle Aspiration

EUS-FNA Endoscopic UltraSound-guided Fine Needle Aspiration

FDA Food and Drug Administration

HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-

Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)

HRP Horse Radish Peroxidase

LoD Limit of Detection. Calculated as the averaged signal of a blank solution + 3x standard deviation of the measured blank solution.

NaN₃ Sodium Azide PEG8000 Poly(ethylene glycol), average molecular weight 8,000 g/mol, CAS 25322-68-3

PBS Phosphate Buffered Saline

PVP PolyVinylPyrrolidone

RFU Relative Fluorescent Units

ROSE Rapid OnSite Evaluation

TMAO Trimethylamine N-oxide

Tris tris(hydroxymethyl)aminomethane, (HOCHshCNHz

In one aspect, the present disclosure provides methods for stabilizing one or more chemokine proteins in solution. In some embodiments, this stabilizing solution is a standard that is used to calibrate a diagnostic device measuring quantitatively [semi-quantitatively; qualitatively] one or more chemokine protein biomarkers. In other embodiments, this solution is a quality control material that is used in conjunction with a diagnostic device to ensure the device meets quality objectives for quantitative and/or semi-quantitative; and/or qualitative measurement of one or more chemokine protein biomarkers.

In some embodiments, the stabilizing solution is a transfer medium into which a tissue [biopsy] specimen obtained from a lymph node by needle biopsy [needle aspiration, fine needle aspiration, EBUS-TBNA, EUS-FNA, EUS-B] is placed in order to measure quantitatively and/or semi- quantitatively; and/or qualitatively, one or more chemokine protein biomarkers in the specimen.

In other embodiments, the stabilizing solution is a transfer medium into which a tissue [biopsy] specimen obtained from a lymph node by needle biopsy [needle aspiration, fine needle aspiration, EBUS-TBNA, EUS-FNA, EUS-B] is placed in order to measure quantitatively and/or semi- quantitatively; and/or qualitatively one or more chemokine protein biomarkers in the specimen by in vitro diagnostics [immunohistochemistry, cytopathology] methods.

Stabilizing components may be produced by any suitable methods known in the art or purchased from commercial sources.

In some embodiments, one or more chemokine protein biomarkers in the stabilizing solution are measured with a point of care device with Lateral Flow fluorescent detection system based on a sandwich assay principle with a measurement time of under 15 minutes from sample loading. Preferred implementation is for 10 minutes, or less than 10 minutes.

The solution for stabilizing a chemokine protein with a C-X-C or a C-C structural motif, such as CXCL13 and CCL21 , includes a buffer, a non-ionic or amphoteric surfactant surfactant and an aqueous chaotropic agent.

Acceptable buffers for the purposes of the present disclosure are buffers known to a person skilled in the art that maintain the pH of solutions in a range of from about pH 6 to about pH 9 with sufficient buffer capacity to handle the addition of acidic or alkaline components where the buffer is compatible with maintaining proteins in the buffer solution.

Acceptable surfactants for the purposes of the present disclosure have the property of being soluble in aqueous solution. The molecular structure is generally linear, containing a polar end and non-polar end. The polar end provides solubility in aqueous solution. These surfactants are non-ionic or amphoteric (zwitterionic). In addition, the acceptable surfactants do not denature proteins. A detergent is a subgroup of surfactants. Certain detergents are acceptable surfactants for the purposes of the present disclosure.

Acceptable chaotropic agents or chaotropes have the common characteristics I properties of being soluble in aqueous solution and of disrupting non-covalent interactions between molecules (water, proteins) such as hydrogen bonding, van der Waals interactions and the hydrophobic effect.

The solution for stabilizing chemokine proteins CXCL13 and CCL21 preferably also includes an osmolyte. Acceptable osmolytes for the purposes of the present disclosure are soluble in aqueous solution, are small organic molecules that maintain the integrity of cells in solution by regulating osmotic pressure through mechanisms of viscosity, melting point and ionic strength of the solution. Acceptable osmolytes are not ions such as Na+, Cl- etc.

Preparation of 1 L of 1X Phosphate Buffer Saline (1X PBS) solution.

A stock solution of 1 L 25X Phosphate Buffer solution was prepared as follows. Add a magnetic stirbar and 700 ml_ deionized water to container. Weigh out 2.72 g of potassium phosphate monobasic and add to 700 mL deionized water. Weigh out 27.9 g of potassium phosphate dibasic and add to 700 mL deionized water. Stir for 15 min on magnetic stir plate. Measure pH of solution with calibrated pH meter and adjust with either 5N HCI or 5N NaOH until pH is 7.8 ±0.1. Bring up solution to 1 L with deionized water.

To a second container with a magnetic stirbar, add 700 mL deionized water. Weigh 8.7 g of sodium chloride and add to 700 mL deionized water and mix. Add 40 mL of 25X Phosphate Buffer stock solution to saline solution and mix. Stir for 15 min on magnetic stirplate. Measure pH of solution with calibrated pH meter and adjust with either 5N HCI or 5N NaOH until pH is 7.2 ±0.1 . Bring up solution to 1 L with deionized water. Filter solution through 0.22 urn filter.

Preparation of 40 mL of 0.05M Tris Buffer pH 7.4, 0.5M Urea, 0.3M D-Mannitol, 0.03% ProCiin 300 solution

The solution was prepared as follows. Add a magnetic stirbar and 32 mL deionized water to a mixing vessel. Weigh out 0.267 g of Tris HCI (C4H11NO3.HCI, mw 157.60 g/mol; CAS 1185-53-1) and add to mixing vessel while solution is stirring. Weigh out 0.037 g of Tris Base ((HOCH₂)₃CNH₂, mw 121.14 g/mol; CAS 77-86-1) and add to mixing vessel while solution is stirring. Add 1.201 g of Urea (NH2CONH2, mw 60.06 g/ml; CAS 57-13-6) to the mixing vessel while solution is stirring. Add 2.186 g of D-Mannitol (CeH^Oe, mw 182.172 g/mol; CAS 66-65-8) to the mixing vessel while solution is stirring. Add 0.250 g of Tergitol NP-40S (mw 1980 g/mol, 70% in H20). Add 0.012 mL of ProCiin 300 to the mixing vessel while solution is stirring. Measure and adjust the pH of the solution to 7.4 ± 0.10 with 5N NaOH or 5N HCI in small increments. Adjust volume to 40 mL by adding deionized water.

Preparation of 40 mL 0.01 M HEPES Buffer pH 7.4, 0.5M Urea, 0.3M D-Mannitol, 0.03% ProCiin 300 solution

The solution was prepared as follows. Add a magnetic stirbar and 32 mL deionized water to a mixing vessel. Weigh out 0.095 g of HEPES (C8H18N2O4S, mw 238.30 g/mol; CAS 7365-45-9) and add to mixing vessel while solution is stirring. Weigh out 1.201 g of Urea (NH2CONH2, mw 60.06 g/ml; CAS 57-13-6) and add to the mixing vessel while solution is stirring. Add 2.186 g of D-Mannitol (C₆HI₄O6, mw 182.172 g/mol; CAS 66-65-8) to the mixing vessel while solution is stirring. Add 0.250 g of Tergitol NP-40S (mw 1980 g/mol, 70% in H20). Add 0.012 mL of ProCiin 300 to the mixing vessel while solution is stirring. Measure and adjust the pH of the solution to 7.4 ± 0.10 with 5N NaOH or 5N HCI in small increments. Adjust volume to 40 mL by adding deionized water.

Preparation of 40 mL of 1X Phosphate Buffer pH 7.4, 0.5M Urea, 0.3M D-Mannitol, 0.03% ProCiin 300 solution

The solution was prepared as follows. Add a magnetic stirbar and 32 mL deionized water to a mixing vessel. Add 1 .6 mL of 25X Phosphate Buffer stock solution to the solution and mix. Weigh out 1.201 g of Urea (NH2CONH2, mw 60.06 g/ml; CAS 57-13-6) and add to the mixing vessel while solution is stirring. Add 2.186 g of D-Mannitol (CeHuOs, mw 182.172 g/mol; CAS 66-65-8) to the mixing vessel while solution is stirring. Add 0.250 g of Tergitol NP-40S (mw 1980 g/mol, 70% in H20). Add 0.012 mL of ProCiin 300 to the mixing vessel while solution is stirring. Measure and adjust the pH of the solution to 7.4 ± 0.10 with 5N NaOH or 5N HCI in small increments. Adjust volume to 40 mL by adding deionized water.

Preparation of solutions containing stabilizing components

Concentration of the stabilizing additive ethyl alcohol is expressed as % volume/volume. For example, an additive of 1 % ethyl alcohol may be achieved by adding 10 mL of ethyl alcohol to 990 mL of 0.05M Tris Buffer pH 7.4 solution. Similarly, the concentration of glycerol is expressed as % volume/volume.

All other stabilizing additives expressed as a percentage refer to % weight/volume. For example, an additive of 1% glycine may be achieved by adding 10 g of glycine into 1 ,000 mL of 0.05M Tris Buffer pH 7.4 solution.

Hemolysis of red blood cells (RBC) presents a challenge for the performance of buffered solutions such as Tris containing detergents (Tergitol, Tween), polymers (polyvinylpyrrolidone) and chaotropic agents (urea, ethanol) in stabilizing added recombinant antigens CCL21 and/or CXCL13 in varying concentrations. The underlying mechanism of hemolysis is related to osmotic pressure. The buffer solutions described herein may not have a sufficiently high ionic strength to match the interior of the red blood cells. To address challenges presented where a sample being tested may contain blood, the antigen-containing buffer has been modified in alternate embodiments from Tris to HEPES, and concentrations of sugars (mannitol, proline, sorbitol, etc.), osmolytes (Betaine, TMAO (trimethylamine- N-Oxide)), preservative Proclin 300 and anticoagulants such as heparin have been added to the buffer solutions.

In general, the preferred embodiments are the ones that maximize the test line signal (highest intensity at a low concentration).

For reference, the Limit of Detection is presented. The LoD is calculated as the average signal of the blank + 3 x standard deviation of the blank determination.

Where the stabilizing components are buffers such as 1X PBS at pH 7.2, or 0.05M Tris at pH 7.4, signal intensities of a solution containing a CCL21 concentration of 1x10⁵ pg/mL were detected above the Limit of Detection (LoD), whereas saline solution containing a CCL21 concentration of 1x10⁵ pg/mL is not detected above the LoD, as shown in figure 1 .

Figure 1 is a graph illustrating the signal intensity of CCL21 in units of Relative Fluorescent Units (RFU) in a point of care detection device as a function of CCL21 protein prepared at different concentration levels in different solutions. Solution 1 is a normal saline solution. Solution 2 is 1X PBS buffer solution, pH 7.2. Solution 3 is a 0.05M Tris buffer solution, pH 7.4. The results as illustrated in figure 1 obtained are shown in table 1.

Table 1

The results shown in figure 1 indicate that 0.05M Tris buffer solution, pH 7.4 is the preferred buffer solution for stabilizing CCL21 of the three solutions tested shown in figure 1.

With addition of surfactants (such as: PVP, PEG8000, IGEPAL® CA-630, EMPIGEN® BB, TWEEN® 20, TWEEN® 80 or Tergitol™ NP-40S) to 0.05M Tris buffer solutions at pH 7.4, signal intensities of a solution containing a CCL21 concentration of 1x10⁴ pg/mL were detected above the LoD, with the strongest signal at that concentration observed for a surfactant additive of 0.5% Tergitol™ NP-40S, as shown in figure 2. It is noted that the percentage for Tergitol™ NP-40S is expressed as weight per volume (wt/vol, in units of g/mL). For example, for 0.5% Tergitol™ NP-40S in preparing 100 ml_ of extraction buffer, 0.5g of Tergitol NP-40S is weighed out.

Figure 2 is a graph illustrating the relative fluorescent intensity of CCL21 in a point of care detection device as a function of CCL21 protein prepared at different concentration levels in a solution of 0.05M Tris buffer, pH 7.4 containing surfactant additives. Solution 4: with 0.25% (wt/vol) TWEEN® 20.

Solution 5: with 0.25% (wt/vol) TWEEN® 80. Solution 6: with 0.50% (wt/vol) TWEEN® 80. Solution 7: with 0.50% (wt/vol) Tergitol NP-40S. The results as illustrated in figure 2 obtained are shown in table 2. Additional data is also presented in table 2, which is not illustrated in figure 2.

Table 2

The results shown in figure 2 indicate that a solution of 0.05M Tris buffer, pH 7.4 containing 0.50% Tergitol NP-40S as a surfactant additive is the preferred surfactant additive solution for stabilizing CCL21 of the four solutions tested shown in figure 2.

Where the stabilizing components are chaotropic agents (such as: 30 mM EDTA, 1% (wt/vol) glycine, 0.5M urea, 1% (vol/vol) ethyl alcohol, or a combination thereof) added to 0.05M Tris buffer solutions at pH 7.4 containing 0.5% Tergitol™ NP-40S, signal intensities of a solution containing a CCL21 concentration of 1x10⁴ pg/mL were detected above the LoD, with the strongest signal at that concentration observed for a chaotropic additive of 1 % (vol/vol) ethyl alcohol as shown in figure 3.

Figure 3 is a graph illustrating the relative fluorescent intensity of CCL21 point of care device as a function of CCL21 protein prepared at different concentration levels in a solution of 0.05M Tris buffer, pH 7.4 with 0.50% (wt/vol) Tergitol NP-40S containing chaotropic additives. Solution 8: no additives. Solution 9: with 1 % (wt/vol) glycine. Solution 10: with 0.5M urea. Solution 11: with 1% (vol/vol) ethyl alcohol. Solution 12: with 1% (wt/vol) glycine, 0.5M urea and 1 % (vol/vol) ethyl alcohol.

The results as illustrated in figure 3 obtained are shown in table 3. Additional data is also presented in table 3, which is not illustrated in figure 3.

Table 3

The results shown in figure 3 indicate that a solution of 0.05M Tris buffer, pH 7.4 with 0.50% Tergitol NP-40S containing 1 % ethyl alcohol as a chaotropic additive is the preferred chaotropic additive solution for stabilizing CCL21 of the five solutions tested shown in figure 3.

As shown in figure 4, upon variation of the pH of 0.05M Tris buffer solutions containing 0.5% Tergitol™ NP-40S and 1 % ethyl alcohol, signal intensities of a solution containing a CCL21 concentration of 1x10⁴ pg/mL were detected above the LoD, with the strongest signal at that concentration observed for a pH of 8.0. This indicates that a pH of 8.0 is a preferred pH level for stabilizing CCL21 in a solution of 0.05M Tris buffer with 0.50% Tergitol NP-40S and 1% ethyl alcohol.

Figure 4 is a graph illustrating the relative fluorescent intensity of CCL21 point of care device as a function of CCL21 protein prepared at different concentration levels in a solution of 0.05M Tris buffer with 0.50% Tergitol NP-40S and 1% ethyl alcohol at different pH levels. Solution 13: pH 7.4. Solution 14: pH 8.0. Solution 15: pH 8.5. Solution 16: pH 9.0.

The results as illustrated in figure 4 obtained are shown in table 4.

Table 4

Figure 5 is a graph illustrating the relative fluorescent intensity of CCL21 point of care device as a function of CCL21 protein prepared at different concentration levels in a solution of 0.05M Tris buffer, pH 8.0 with 0.50% (wt/vol) Tergitol NP-40S containing chaotropic additives. Solution 17: with 1% (vol/vol) ethyl alcohol. Solution 18: with 0.5M urea.

The results as illustrated in figure 5 obtained are shown in table 5.

Table 5

As shown in figure 5, where the stabilizing components are chaotropic agents (such as: 1 % (wt/vol) glycine, 0.5M urea, 1% (vol/vol) ethyl alcohol, or a combination thereof) added to 0.05M Tris buffer solutions at pH 8.0 containing 0.5% (wt/vol) Tergitol™ NP-40S, signal intensities of a solution containing a CCL21 concentration of 5x10³ pg/mL were detected above the LoD, with the strongest signal at that concentration observed for a chaotropic additive of 0.5M urea. Additionally, signal intensities of a solution containing a CCL21 concentration of 1x10³ pg/mL were detected above the LoD. This indicates that a solution of 0.05M Tris buffer, pH 8.0 with 0.50% (wt/vol) Tergitol NP-40S containing 0.5M urea as a chaotropic additive is a preferred chaotropic additive solution at pH 8.0 for stabilizing CCL21.

Figure 6 is a graph illustrating CXCL13 and CCL21 biomarker estimated concentrations in specimens obtained from five lymph node sites from two patients during lung biopsy procedure and solubilized in stabilizing solution 18 as described above with respect to figure 5. A point of care device containing sensors for CXCL13 and CCL21 was used. Concentrations were determined from a calibration curve from linearity studies using solution 18. The Rapid On-Site Evaluation provided by a cytotechnician was recorded as “sufficient” or “insufficient” for further send out of the specimens to the pathology laboratory.

The results as illustrated in figure 6 obtained are shown in table 6.

Table 6

As shown in figure 6, a volume of approximately 100 pL of a lymphoid protein-stabilizing solution of 0.05M Tris buffer solution at pH 8.0 with 0.5% (wt/vol) Tergitol™ NP-40S and 0.5M urea was used to dilute approximately 20 pL of specimen obtained from EBUS-TBNA and EUS-FNA procedures from two patients at 2-3 distinct lymph node sites. The diluted samples were then assessed on a point of care cartridge and read with the fluorescent reader 5 minutes after addition of sample to the cartridge. For those samples identified as “sufficient” by ROSE, a range of CXCL13 concentrations of 0 - 12,870 pg/mL, and a range of CCL21 concentrations of 1 ,870 - 3,842 pg/mL, were observed. For the sample identified as “insufficient” by ROSE, a CXCL13 concentration of 0 pg/mL, and a CCL21 concentration of 1,496 pg/mL, was observed. Elevated concentrations of the lymphoid biomarkers CXCL13 and CCL21 were observed with clinical samples identified as “sufficient” by ROSE.

“Saline” refers to Sodium Chloride, 0.9% (weight/volume), Isotonic Saline Suitable for Use as Blood Bank Saline, available from Ricca Chemical Company.

“Tergitol NP-40S” refers to Tergitol™ solution - Type NP-40S, 70% in H₂O available from Sigma- Aldrich.

Based on the results obtained, a solution of 0.05M Tris buffer solution at pH 8.0 with 0.5% Tergitol™ NP-40S and 0.5M urea as a chaotropic additive and 0.50% TWEEN® 80 as a surfactant additive is a preferred solution for stabilizing CXCL13 and CCL21 .

Addition of CHAPSO

CHAPSO is a zwitterionic surfactant. Addition to the solution may provide stabilizing environment via both surfactant characteristics and charge balancing. Table 7 presents data obtained with 0.05M Tris buffer system (pH 8.0) containing varying concentrations of the chaotropic agent urea, the surfactant Tergitol NP-40S and the zwitterionic surfactant CHAPSO.

Table 7

The data shown in table 7 demonstrates that when replacing the urea component in the buffer with CHAPSO, the cartridge performance is equivalent in CCL21 signal response. The data shown in table 7 further demonstrates that when replacing both urea and Tergitol NP-40S with CHAPSO, the cartridge performance is only slightly lowered in CCL21 signal response. Thus, the zwitterionic surfactant 0.5% CHAPSO is a viable substitute for 0.5M urea, and the zwitterionic surfactant 0.5% CHAPSO is a viable substitute for 0.5M and 0.5%Tergitol NP-40S in buffer formulations.

HEPES, Tris and 1X Phosphate Buffers pH control via HEPES buffer (pH 7.4) is equivalent to TRIS buffer (pH 8.0). The data in table 8A below demonstrates that HEPES buffer performs better than TRIS buffer with approximately 2x signal improvement for both CXCL13 and CCL21.

Figure 7A is a graph illustrating the relative fluorescent intensity of CCL21 and CXCL13 point of care device as a function of CCL21 and CXCL13 protein prepared at different concentration levels in a solution of 0.5% Tergitol NP-40S, 0.5M urea buffered by either 0.05M Tris buffer (pH 8.0) or 0.01 M HEPES buffer (pH 7.4). For both CXCL13 and CCL21 proteins, the 0.01M HEPES buffer solution shows higher signal intensity. The preferred buffer solution in this comparison is 0.01M HEPES pH 7.4.

The results are illustrated in figure 7A and are also shown in table 8A.

Table 8A

Control of pH can also be provided by a phosphate buffer, separate and distinct from the phosphate buffer saline solution. Figure 7B is a graph illustrating the relative fluorescent intensity of CCL21 and CXCL13 point of care device as a function of CCL21 and CXCL13 protein prepared at different concentration levels in a solution of 0.625% Tergitol NP-40S; 0.5M urea; 0.03% (vol/vol) ProCiin 300 or 0.09% (vol/vol) sodium azide (NaN₃) buffered by: 0.05M Tris buffer (pH 8.0), 0.01 M HEPES buffer (pH 7.4) or 1X Phosphate buffer (pH 7.4). For both CXCL13 and CCL21 proteins, the 1 X Phosphate buffer solution shows the highest signal intensities. There is no effect of the biocide (0.03% ProClin300, 0.09% sodium azide) on CXCL13 signal intensity. The use of 0.03% ProClin300 gives a higher CCL21 signal. The preferred buffer solution is 1 X Phosphate pH 7.4, 0.625% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol, 0.03% Proclin300.

The results are illustrated in figure 7B and are also shown in table 8B. Table 8B

HEPES Buffer + Osmolyte (D-Mannitol) with Whole Blood The following solutions were prepared:

1) Diluent Buffer, 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, pH 7.4

2) Diluent Buffer, 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, pH 7.4 + 1% Whole Blood

3) Diluent Buffer, 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, pH 7.4 + 2% Whole Blood

4) Diluent Buffer, 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, pH 7.4 + 5% Whole Blood

5) Diluent Buffer, 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol, pH 7.4

6) Diluent Buffer, 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol, pH 7.4 + 1 %

Whole blood

7) Diluent Buffer, 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol, pH 7.4 + 2% Whole blood

8) Diluent Buffer, 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol, pH 7.4 + 5% Whole blood

Figure 8 is a graph showing a plot of % whole blood in a solution of 0.01M HEPES (pH 7.4) with 0.5% Tergitol and 0.5M urea with and without 0.3M D-mannitol versus relative fluorescent intensity of CCL21. The results show that the presence of an osmolyte (D-mannitol) in HEPES buffer reduces the interference of the CCL21 test line signal in the presence of whole blood.

Addition of Biocide ProClin300 to Buffer

Table 9

Figure 9 is a plot of buffer condition versus signal intensity. Buffer condition 1 is 0.01M HEPES (pH

7.4) containing 0.5% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol with 0.00% ProClin300. Buffer condition 2 is 0.01M HEPES (pH 7.4) containing 0.5% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol with 0.03% ProClin300. The results depicted in figure 9 are listed in table 9. These results show that the addition of a preservative / biocide to the buffer solution does not destabilize the chemokine CCL21 as indicated in the similar signal intensities (less than 1 % difference) at 30 ng/mL.

Addition of Osmolytes to HEPES buffer solution

Osmolytes contribute to the osmotic pressure of the solution and may provide stability to cellular components such as red blood cells. The rupture of red blood cells may release interfering substances into the test solution and give rise to inaccurate test results. Osmolytes are small organic compounds that can be classified into five categories as set out in table 10.

Table 10 - Osmolyte Categories

In addition to D-mannitol, the following osmolytes were assessed analytically only in buffer:

• Betaine

• TMAO

• TMAO Dihydrate

• Trehalose

• Mannose

• Xylitol

• Sorbitol

• Raffinose • DMSO

• Proline

Figure 10 is a photograph of point of care cartridges loaded with a chemokine-stabilizing buffer solution (0.01 M HEPES, 0.5% (g/mL), Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol, 0.03% (vol/vol) Proclin 300, pH 7.4) containing whole blood at concentrations (vol/vol) of 1 % (cartridges 1 , 2), 2% (cartridges 3,4), 5% (cartridges 5,6) and 10% (cartridges 7,8,9). The buffer test solution for cartridge 9 does not contain 0.3M D-Mannitol, 0.03% (vol/vol) Proclin 300. Each of test cartridges 1-8 contains a sample pad treated with conjugate dilution buffer containing 0.75% g/mL Tergitol NP-40S, 0.5 mg/mL anti-red blood cell rabbit antibody. Cartridge 9 does not contain 0.5 mg/mL anti-red blood cell rabbit antibody. The photograph shows that all the red blood cell components are captured and localized on the sample entry pad (“S” in the photograph). The intensity of the color correlates to the concentration of the whole blood in the buffer solution. None of the cartridges 1-8 show presence of red blood cells in the cartridge read window at the Test Line or the Control Line (“T” and “C” respectively in the photograph). Cartridge 9 shows the presence of red blood cells in the read window.

It was assessed that 0.75% Tergitol NP-40S (a surfactant) supports a higher test line signal than 0.50% Tergitol NP-40S. Most of the data presented are from a buffer formulation at 0.5% Tergitol NP- 40S. The optimal concentration is 0.75% Tergitol which is the concentration used in the cartridge. The results are summarized in table 11 below.

Table 11 The results summarized in table 11 are shown graphically in figure 11.

Table 12 lists results for osmolytes in buffer (betaine, trimethylamine-N-oxide, trimethylamine-N-oxide di hydrate).

Condition 1 being the control condition was buffer formulated with 0.01M HEPES, 0.5% Tergitol NP- 40S, 0.5M Urea, 0.3M D-Mannitol, pH 7.4. As set out in Table 12, conditions 2-8 vary the urea concentration and add Betaine and Trimethylamine N-oxide (TMAO). TMAO and TMAO dihydrate were shown to be equivalent.

Conditions 2-6 are equivalent to the control buffer formulation, whereas conditions 7 and 8 show a lower signal for the 30 ng/mL CCL21 signal.

Table 12

The results set out in table 12 are shown graphically in figure 12. Additional data is also presented in table 12, which is not illustrated in figure 12.

Additionally, other combinations of osmolyte sugars and osmolyte/chaotropic agents have been found to provide optimized signal intensity. In a 0.01 M HEPES buffer (pH 7.4) solution containing 0.625% Tergitol NP-40S, 0.03M D-Sorbitol and 0.03% ProClin300, the additives in table 13 below were used to create conditions 1-4. In all cases, replacing the chaotropic agent urea with 0.075M TMAO Dihydrate, as well as TMAO Dihydrate with 0.1M and 0.2M Betaine generated higher signals.

The results set out in table 13 are shown graphically in figure 13.

Table 13

Sugar osmolyte additives are D-trehalose, D-mannose, xylitol, sorbital) in buffer containing 0.3M mannitol.

Figure 14 is a graph showing a plot of signal intensity for CCL21 versus buffer condition for different buffer conditions. The control condition is buffer formulated with 0.01 M HEPES, 0.5% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol, with 0.03% (vol/vol) ProCiin 300, pH 7.4. Condition 6 is with no sugar added. Condition 7: 0.3M D-Trehalose added to buffer. Condition 8: 0.3M D-Mannose added to buffer. Condition 9: 0.3M xylitol added to buffer. Condition 10: 0.3M sorbitol added to buffer.

The results are summarized in table 14. Additional data is also presented in table 14, which is not illustrated in figure 14.

Table 14 The results show that D-Mannose, Xylitol and Sorbital at 0.3M each are equivalent to the control buffer formulation.

As summarized in table 15 and shown graphically in figure 15, the following sugar osmolyte additives at 0.3M concentration were added to a buffer solution of 0.01 M HEPES (pH 7.4) containing 0.5% Tergitol NP-40S surfactant, 0.5M Urea and 0.03% ProClin300: D-Mannitol (control condition), D- Trehalose, D-Mannose, Xylitol and D-Sorbitol. The results indicate that all sugar osmolyte additives serve to stabilize the chemokine protein. D-Sorbitol provides a 13% higher CCL21 signal intensity at 30 ng/mL.

Table 15

Osmolyte additive: glycerol in buffer containing 0.3M D-Sorbitol

Glycerol is a polyl osmolyte and serves as a cryoprotectant in biological solutions. To a buffer solution containing 0.01 M HEPES (pH 7.4) containing 0.625% Tergitol NP-40S, 0.5M Urea, 0.3M D-Sorbitol, 0.03% ProClin300), was added 0 to 5% (vol/vol) of glycerol. The results are summarized in table 16. Compared to the control condition, conditions with % glycerol from 0.5% to 5.0% (vol/vol) showed equivalent performance.

Table 16 Osmolyte additives (DMSO, Proline) in buffer containing 0.3M mannitol

Figure 16 is a graph showing a plot of signal intensity for CCL21 versus buffer condition for different buffer conditions. The control condition is solution formulated with: 0.01 M HEPES buffer, 0.5% Tergitol NP-40S, 0.5M Urea, 0.3M D-Mannitol, pH 7.4. Condition 15 is control condition. Condition 16 is control buffer with 1% DMSO. Condition 17 is control buffer with 0.2M Proline. Also tested (but not plotted): 0.1% DMSO, 0.5% DMSO, 0.05M Proline, 0.1 M Proline.

The results are summarized in table 17. Additional results are included in table 17 that are not shown in Figure 16. Table 17 Conclusion: Additives 1% DMSO and 0.2M Proline are equivalent to the control buffer formulation.

The preferred buffers are 1 X Phosphate, pH 7.4, 1X PBS at pH 7.2, 0.05M Tris at pH 7.4, and 0.01 M HEPES, pH 7.4. Concentrations of Tris buffer from about 0.02M to about 0.2M are acceptable for the purposes of the present disclosure. Concentrations of HEPES of from about 0.002M to about 0.1M are acceptable for the purposes of the present disclosure.

A preferred embodiment of the stabilizing protein solution is shown in Table 18 below. Table 18

Point of Care Testing System

A point of care test carried out on a point of care cartridge is provided that operates on the principle of a sandwich immunofluorescent assay in a lateral flow format to detect lymphoid chemokine proteins CCL21 and CXCL13 in test samples.

The three main cartridge elements of the point of care cartridge are:

1. Sample Pad;

2. Conjugate Release Pad; and

3. Nitrocellulose Strip.

As illustrated in figure 17, a plastic casing of a single-use test cartridge houses a sample pad 2, a conjugate release pad 12 (containing the detector antibody reagents), a nitrocellulose membrane 6 (containing the capture antibody reagents), and an absorbent pad 8. The test sample is delivered to a sample port (not shown) and absorbed into the sample pad 2, wicks through the nitrocellulose membrane 6 to the absorbent pad 8.

The recombinant detector antibodies, preferably mouse monoclonal anti-human CCL21 and CXCL13, are conjugated with a fluorescent dye. These bind with the CCL21 and CXCL13 antigens in the test sample as it wicks through the conjugate release pad and form detector antibody-antigen complexes that then wick through the membrane.

Two different recombinant capture antibodies are coated in separate test lines on the membrane: mouse monoclonal anti-human CCL21 and CXCL13 antibodies. The detector antibody-antigen complexes form sandwich complexes at the test lines, resulting in the development of distinct lines of fluorescent signal when the antigens are present in the test sample. The control line is preferably printed with goat anti-mouse antibodies. Excess conjugated antibodies bind with these goat antibodies, resulting in fluorescent signal on the control line that indicates that the lateral flow test has performed properly.

Additional description of the point of care cartridges used in this study is detailed in Table 19.

Table 19. Details of preferred point of care cartridge.

The test cartridges are read on a commercially available fluorescent reader such as Axxin AX-2X-S Lateral Flow Reader (Axxin Pty Ltd, Adelaide, Australia). The fluorescent intensities are determined by a peak height fit. Typically, the solutions are measured in replicates of 2-10, and the detected signals are averaged.

The chemical and biological composition of the stabilizing components in solution are extended to cartridge elements. The cartridge elements are treated with a buffer solution, preferably 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5, typically by soaking, followed by a drying (curing) step prior to being assembled in the cartridge.

As illustrated in figure 17, a point of care cartridge comprises a sample pad 2, a conjugate release pad 12, a nitrocellulose membrane 6 and an absorbent pad 8.

The nitrocellulose membrane 6 is preferably 25 mm long by 4 mm wide and is placed on a preferably 60 mm x 4 mm plastic backing layer. At a sample entry portion 10, a conjugate release pad 12 is placed on top of the backing layer with overlap to the nitrocellulose membrane 6. The conjugate release pad 12 contains one or more antibodies covalently bonded to a fluorescent label molecule (referred to as the conjugated antibody). The sample pad 2 is placed on the conjugate release pad 12 and the backing layer. At the other end of the nitrocellulose membrane 6 and backing layer is placed the absorbent pad 8.

On the nitrocellulose membrane 6 is a control line 14, which contains antibodies to the conjugated antibodies. In the embodiment shown in figure 13, a first test line 16 for CCL21 is on the nitrocellulose membrane 6. A second test line 18 for CXCL13 is on the nitrocellulose membrane 6 which contains immobilized capture antibodies, which in the presence of a protein-conjugated antibody complex, form a classic immunoassay “sandwich” complex. In an alternate embodiment there may be a single test line for both CCL21 and CXCL13 and possibly CCL19 on the nitrocellulose membrane 6. Alternate embodiments may include only a single test line for either CCL21 or CXCL13 or CCL19.

The membrane assembly is preferably pressure-fitted into a cartridge housing comprised of an ABS base and an ABS top plate.

A test sample, after loading on the sample pad, flows along the nitrocellulose towards the absorbent pad via capillary action. Flow rates and protein stability are determined by the selection of these elements.

A fluorescent reader images the control line and the test lines. The fluorescent reader reports a peak height measurement in units of relative fluorescent units (RFU) preferably after 5 or 10 minutes of addition of the sample to the cartridge. In the data presented herein, the fluorescent reader is an Axxin AX-2X-S fluorescent reader.

As explained above, the test solutions formulations for the detection CCL21 and/or CXCL13 are generally buffered solutions (Tris) containing surfactants (Tergitol, Tween, polyvinylpyrrolidone) and chaotropic agents (urea, ethanol). To these solutions are added recombinant antigens CCL21 and/or CXCL13 in varying concentrations.

Preferably antibodies to red blood cells are added to the sample pad to immobilize any red blood cells and keep them from moving into the nitrocellulose. This is for the purpose of reducing background fluorescent signals due to red blood cells which may be present in the test sample. The antigencontaining test solution also preferably includes HEPES, and added concentrations of sugars (mannitol, proline, sorbitol, etc.), osmolytes (Betaine, TMAO (trimethylamine-N-Oxide)), preservative Proclin and anticoagulants such as heparin.

Sample Pad Treatment

For the results referred to below, the Stabilization Buffer (SB) is 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5.

Figure 18 illustrates results of relative fluorescent intensity of CCL21 in saline solutions containing concentration of 1 x 10⁵ pg/mL of CCL21 and 1 x 10⁶ pg/mL of CCL21 as measured by point of care devices made with different sample pad conditions. Condition 1 is 17mm Ahlstrom 8964 glass fiber. Condition 2: is 17mm Ahlstrom 8964 glass fiber treated with Stabilization Buffer (SB) being 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5. Condition 3 is 17mm Ahlstrom 8964 glass fiber treated with SB containing 1 % polyvinylpyrrolidone (PVP) and 0.25% Tween20. The Limit of Detection (LoD) is calculated from the measurement of a blank (saline solution containing no CCL21).

Table 20 sets out sample pad details for results summarized in figure 18.

Table 20

Table 21 summarizes the measurements from Figure 18. Table 21

The results show similar fluorescent intensity of detection CCL21 with Ahlstrom 8964 glass fiber treated with Stabilization Buffer (SB) as with Ahlstrom 8964 glass fiber treated with SB containing 1% polyvinylpyrrolidone (PVP) and 0.25% Tween 20. Both the treated sample pad conditions show higher signal response than the untreated sample pad. Figure 19 illustrates results of relative fluorescent intensity of CCL21 in buffer solutions containing concentration of 1 x 10³ pg/mL of CCL21 and 5 x 10³ pg/mL of CCL21 as measured by point of care devices made with different sample pad conditions. Condition 4 is 17mm Ahlstrom 8964 glass fiber treated with Stabilization Buffer (SB) containing 1% polyvinylpyrrolidone (PVP) and 0.25% Tween 20. Condition 5 is 17mm Ahlstrom 8964 glass fiber treated with SB containing 0.25% Tergitol NP-40S.

Condition 6 is 17mm Ahlstrom 8964 glass fiber treated with SB containing 1% PVP and 0.25% Tergitol NP-40S. The Limit of Detection (LoD) is calculated from the measurement of a blank (solution of a buffer containing no CCL21). Table 22 summarizes the measurements from figure 19.

Table 22

Figure 20 illustrates results of relative fluorescent intensity of buffer solutions containing concentration of 1 x 10³ pg/mL of CCL21 and 5 x 10³ pg/mL of CCL21 as measured by point of care devices made with different sample pad conditions. Condition 7 is Ahlstrom 8964 glass fiber treated with Stabilization Buffer (SB) containing 0.25% Tergitol NP-40S. Condition 8 is Ahlstrom 8964 glass fiber treated with CDB containing 0.25% Tergitol NP-40S and 1% polyvinylpyrrolidone. The Limit of Detection (LoD) is calculated from the measurement of a blank (solution of a buffer containing no CCL21).

Table 23 summarizes the measurements from figure 20.

Table 23

The addition of 1% polyvinylpyrrolidone to Ahlstrom 8964 glass fiber treated with Stabilization Buffer (SB) containing 0.25% Tergitol NP-40S provided slightly higher fluorescent intensity of CCL21 readings.

Figure 21 illustrates results of relative fluorescent intensity of CCL21 in a buffer solution containing concentration of 1.6 x 10⁴ pg/mL of CCL21 as measured by point of care devices made with different sample pad conditions. Condition 8 is Ahlstrom 8964 glass fiber treated with Stabilization Buffer (SB) containing 0.75% Tergitol NP-40S. Condition 9 is Ahlstrom 8964 glass fiber treated with CDB containing 0.75% Tergitol NP-40S and 0.5 mg/mL anti-Red Blood Cell rabbit antibody. Condition 10 is Ahlstrom 8964 glass fiber treated with SB containing 0.75% Tergitol NP-40S and 1 .0 mg/mL anti-Red Blood Cell rabbit antibody. The Limit of Detection (LoD) is calculated from the measurement of a blank (solution of a buffer containing no CCL21).

Table 24 summarizes the measurements from figure 21 .

Table 24

These results demonstrate that the preferred sample pad condition is Ahlstrom 8964 glass fiber treated with SB containing 0.75% Tergitol NP-40S and 0.5 mg/mL anti-Red Blood Cell rabbit antibody. That is, better results were obtained with the solution containing 1.0 mg/mL anti-Red Blood Cell rabbit antibody than with the solution containing 0.5 mg/mL anti-Red Blood Cell rabbit antibody.

Figure 22 illustrates results of relative fluorescent intensity of CCL21 in buffer solutions containing concentration of 2 x 10⁴ pg/mL of CCL21 and 4 x 10⁴ pg/mL of CCL21 as measured by point of care devices made with different sample pad conditions. Condition 11 is Ahlstrom 8964 glass fiber treated with Stabilization Buffer (SB) containing 0.75% Tergitol NP-40S and 0.5 mg/mL anti-Red Blood Cell rabbit antibody. Condition 12 is Ahlstrom 8980 glass fiber treated with SB containing 0.75% Tergitol NP-40S and 0.5 mg/mL anti-Red Blood Cell rabbit antibody. The Limit of Detection (LoD) is calculated from the measurement of a blank (solution of the buffer containing no CCL21). Table 25 summarizes the measurements from figure 22.

Table 25

These results demonstrate that the preferred sample pad condition is Ahlstrom 8980 glass fiber treated with SB containing 0.75% Tergitol NP-40S and 0.5 mg/mL anti-Red Blood Cell rabbit antibody. That is, better results were obtained with Ahlstrom 8980 glass fiber than with Ahlstrom 8964 glass fiber.

For the results referred to in tables 26-28, Conjugate Dilution Buffer (CDB) is 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5.

Figure 23 illustrates results of relative fluorescent intensity of CCL21 in buffer solutions containing concentrations of 5 x 10² pg/mL of CCL21 , 1 x 10³ pg/mL of CCL21 and 5 x 10³ pg/mL of CCL21 as measured by point of care devices made with different conjugate release pad conditions. Condition 1: Treated with Conjugate Dilution Buffer (CDB) being 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5. Condition 2: Treated with CDB with 0.25% Tergitol NP-40S. Condition 3: Treated with CDB with 0.50% Tergitol NP-40S. Condition 4: Treated with CDB with 1 .00% Tergitol NP-40S. The Limit of Detection (LoD) is calculated from the measurement of a blank (solution of the buffer containing no CCL21).

Table 26 summarizes the measurements from figure 23.

Table 26

These results demonstrate that the preferred sample pad condition occurs where the solution includes 1.00% Tergitol NP-40S. The fluorescent intensity readings increased progressively with increasing levels of Tergitol NP-40S. Acceptable results are achieved with content of Tergitol NP-40S of from about 0.5% to 1 .00%.

Figure 24 illustrates results of relative fluorescent intensity of CCL21 in buffer solutions containing concentration of 2 x 10⁴ pg/mL of CCL21 and 4 x 10⁴ pg/mL of CCL21 as measured by point of care devices made with different conjugate release pad materials. Condition 5 is Ahlstrom 8964 glass fiber treated with Conjugate Dilution Buffer (CDB). Condition 6 is Ahlstrom 8980 glass fiber treated with CDB. The Limit of Detection (LoD) is calculated from the measurement of a blank (solution of the buffer containing no CCL21).

Table 27 summarizes the measurements from figure 24.

Table 27

Better results were obtained with Ahlstrom 8980 glass fiber than with Ahlstrom 8964 glass fiber.

Figure 25 shows the CXCL13 and CCL21 signal intensities observed from a buffer solution measured with a test cartridge with a single test line containing CXCL13 antibodies and CCL21 antibodies, each loaded at a concentration of 1 mg/mL. The test cartridges have a conjugate release pad loaded with a CXCL13 antibody conjugated to Eu bead and CCL21 antibody conjugated to Eu bead. The loading level for both conjugated antibodies is 0.006% in the presence of 0.5% Tergitol NP-40S. The two conditions represented in figure 25 are for antibody to Eu bead coupling ratios of 1 :20 and 1:10 (ratio of mass of antibody:mass of Eu bead). The test buffer of 0.05M Tris with 0.5% Tergitol NP-40S, 0.5M urea at pH 8.0 was used to create test solutions of: 0 ng/mL CXCL13 and 0 ng/mL CCL21 ; 0 ng/mL CXCL13 and 11.5 ng/mL CCL21 ; and 28.5 ng/mL CXCL13 and 0 ng/mL CCL21.

The values observed in figure 25 are summarized in table 28.

Table 28

The data show that conjugation coupling ratio of the antibody to the 0.3 pm Europium fluorescent bead at the two coupling ratios gives equivalent signal intensity at a CXCL13 concentration level of 28.5 ng/mL, whereas the signal intensity at a concentration level of 11.5 ng/mL CCL21 is higher for the 1 :20 conjugation ratio. The data shows that a treated conjugation release pad containing either 1 :10 or 1 :20 conjugated antibodies show stabilization of both CXCL13 and CCL21 in solution.

The Stabilization Buffer and Conjugate Dilution Buffer described above is a preferred buffer for application to the sample pad and conjugate release pad respectively. Other buffers can be used as the stabilization buffer and conjugate dilution buffer in order to stabilize both CXCL13 and CCL21 in solution. These buffers can be applied to sample pad and conjugate release pad respectively in order to obtain acceptable results. Examples of other acceptable stabilization buffers and conjugate dilution buffers include the following:

1. BSA-Based Conjugate Diluent

Tris-HCI: 20 mM (pH 7.4)

NaCI: 150 mM

BSA: 1% (w/v)

Tween-20: 0.05% (v/v)

Sucrose: 5% (w/v)

Sodium Azide: 0.02% (preservative)

Purpose: Stabilizes gold or latex conjugates and prevents aggregation.

2. Casein-Based Conjugate Diluent

PBS (Phosphate-Buffered Saline, pH 7.4): 10 mM

Casein: 0.5% (w/v)

Sucrose: 3% (w/v)

Tween-20: 0.05% (v/v)

Proclin 300: 0.05% (preservative)

Purpose: Casein blocks non-specific binding and stabilizes conjugates. 3. PVP-Based Conjugate Diluent

HEPES Buffer: 10 mM (pH 7.4)

NaCI: 150 mM

PVP-40 (Polyvinylpyrrolidone, MW 40,000): 0.5% (w/v)

Tween-20: 0.1% (v/v)

Trehalose: 2% (w/v)

Thimerosal: 0.01% (preservative)

Purpose: PVP enhances colloidal stability and prevents aggregation.

4. Sucrose-Trehalose Conjugate Diluent

Tris-HCI: 50 mM (pH 8.0)

NaCI: 100 mM

Sucrose: 2% (w/v)

Trehalose: 2% (w/v)

Tween-20: 0.05% (v/v)

Gentamicin: 0.02% (antimicrobial agent)

Purpose: Protects conjugates during drying and storage by acting as a cryoprotectant.

5. HSA-Based Conjugate Diluent

MES Buffer: 25 mM (pH 6.5)

KCI: 100 mM

Human Serum Albumin (HSA): 0.5% (w/v)

Trehalose: 3% (w/v)

Pluronic F-127: 0.05% (v/v)

Proclin 150: 0.02% (preservative)

Purpose: HSA prevents non-specific binding and stabilizes proteins in the conjugate formulation.

As will be apparent to those skilled in the art, other chemokine proteins containing same C-C and C-X- C structural motifs and present in lung lymph nodes can be stabilized using the methods described herein.

As will also be apparent to those skilled in the art, other cytokine proteins and present in lung lymph nodes can be stabilized using the methods described herein.

As will also be apparent to those skilled in the art, cytokine proteins obtained from lymph nodes of other organs including thyroid, breast, pancreas, stomach, liver and kidney can be stabilized using the methods described herein for the purpose of diagnosis and staging of lymphoma cancer.

As will also be apparent to those skilled in the art, proteins present in lung lymph nodes used for malignancy diagnoses and cancer staging can be stabilized using the methods described herein.

As will also be apparent to those skilled in the art, biocides such as ProCiin™, sodium azide, Microcide I, Microcide II, Microcide III, Thimerosal, etc. can be added to the stabilizing solution for preservation again microbial degradation.

As will also be apparent to those skilled in the art, zwitterionic surfactants such as CHAPS (3-((3- cholamidopropyl) dimethylammonio)-1 -propanesulfonate) and CHAPSO (3-([3- cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate) can be added to the stabilizing solution.

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The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

Claims

1. A method of stabilizing a chemokine protein with a C-X-C or a C-C structural motif comprising the step of introducing the chemokine protein into a solution comprising a buffer; a non-ionic or amphoteric surfactant; and an aqueous chaotropic agent.

2. The method of claim 1 wherein the chemokine protein is selected from the group consisting of CCL21, CXCL 13 and CCL19.

3. The method of claim 1 wherein the chemokine protein is CCL21 and/or CXCL 13.

4. The method of claim 1 wherein the chemokine protein is CCL21 .

5. The method according to claim 1 wherein the buffer is selected from the group consisting of 1 X phosphate buffer, 1 X phosphate buffer saline ( 1X PBS), 0.05M tris(hydroxymethyl)aminomethane (Tris) and 0.01M 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

6. The method according to claim 5 wherein the non-ionic or amphoteric surfactant selected from 1% polyvinylpyrrolidone (PVP), 0.25% Tween 20, 0.25% Tween 80, 0.5% Tween 80, 1% poly(ethylene glycol) 8000 PEG 8000, 0.5% IGEPAL CA-630,0.5% toO.75% Tergitol NP-40S and 0.5% CHAPSO.

7. The method according to claim 6 wherein the aqueous chaotropic agent selected from the group consisting of 30 mM ethylenediaminetetraacetic acid (EDTA), 1% glycine, 1% ethanol and 0.5M urea.

8. The method of claim 7 further comprising an osmolyte.

9. The method of claim 8 where the osmolyte is selected from the group consisting of trehalose, mannose, raffinose, sorbitol, mannitol, xylitol, glycerol, TMAO, TMAO Dihydrate, proline, betaine, DMSO.

10. The method of claim 9 where the osmolyte is selected from the group consisting of 0.1 M betaine, 0.1 M TMAO, 0.075M TMAO Dihydrate, 0.3M sorbitol, 0.3M mannitol, 0.3M D-Mannose and 0.3M xylitol.

11 . The method of claim 1 wherein the solution comprises 0.01% to 2% by volume of the non-ionic or amphoteric surfactant, and 0.03M to 2.0M of the aqueous chaotropic agent.

12. The method of claim 1 wherein the pH of the solution is in the range of 7.2 to 9.0.

13. The method of claim 1 wherein the buffer is 1 X Phosphate, pH 7.4, the surfactant is 0.5% to.75% Tergitol NP-40S, and the chaotropic agent is 0.5M urea.

14. The method of claim 13 wherein the solution further comprises 0.3M sorbitol or 0.3M mannitol.

15. The method of claim 14 wherein the solution further comprises 0.03% ProCiin 300 and/or 0.09% sodium azide.

16. A solution for stabilizing a chemokine protein with a C-X-C or a C-C structural motif comprising a buffer; a non-ionic or amphoteric surfactant; and an aqueous chaotropic agent.

17. The solution according to claim 16 wherein the chemokine protein is selected from the group consisting of CCL21 , CXCL13 and CCL19.

18. The solution according to claim 16 wherein the chemokine protein is CCL21 and/or CXCL13.

19. The solution according to claim 16 wherein the chemokine protein is CCL21.

20. The solution according to claim 16 wherein the buffer is selected from the group consisting of 1 X

Phosphate buffer, 1 X phosphate buffer saline ( 1X PBS), 0.05M tris(hydroxymethyl)aminomethane (Tris) and 0.01M 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).

21 . The solution according to claim 20 wherein the non-ionic or amphoteric surfactant selected from the group consisting of 1% polyvinylpyrrolidone (PVP), 0.25% Tween 20, 0.25% Tween 80, 0.5% Tween 80, 1% polyethylene glycol) 8000 PEG 8000, 0.5% IGEPAL CA-630,0.5% to 0.75% Tergitol NP-40S and 0.5% CHAPSO.

22. The solution according to claim 21 wherein the aqueous chaotropic agent selected from the group consisting of 30 mM ethylenediaminetetraacetic acid (EDTA), 1% glycine, 1% ethanol and 0.5M urea.

23. The solution of claim 16 further comprising an osmolyte.

24. The solution of claim 23 where the osmolyte is selected from the group consisting of trehalose, mannose, raffinose, sorbitol, mannitol, xylitol, glycerol, TMAO, TMAO Dihydrate, proline, betaine, DMSO.

25. The solution of claim 23 where the osmolyte is selected from the group consisting of 0.1 M betaine, 0.1 M TMAO, 0.075M TMAO Dihydrate, 0.3M sorbitol, 0.3M mannitol, 0.3M D-Mannose and 0.3M xylitol.

26. The solution of claim 16 wherein the solution comprises 0.01 % to 2% by volume of the surfactant, and 0.03M to 2.0M of the chaotropic agent.

27. The solution of claim 16 wherein the pH of the solution is in the range of 7.2 to 9.0.

28. The solution of claim 16 wherein the buffer is 1 X Phosphate, pH 7.4, the surfactant is 0.5% to.75% Tergitol NP-40S, and the chaotropic agent is 0.5M urea.

29. The solution of claim 28 wherein the solution further comprises 0.3M sorbitol or 0.3M mannnitol.

30. The solution of claim 28 wherein the solution further comprises 0.03% ProCiin 300 and/or 0.09% sodium azide.

31 . A method of detecting at least one chemokine protein with a C-X-C or a C-C structural motif conjugated to antibodies thereto labelled with a fluorescent label in a solution comprising the following steps: providing a test substrate comprising an elongate nitrocellulose membrane, the nitrocellulose membrane having a first end for receiving the solution and a second end whereby the solution flows from the first end to the second end by means of wicking, the substrate including a sample pad located at the first end of the nitrocellulose membrane and an absorbent pad located on the second end of the nitrocellulose membrane, at least one of a test line for the at least one chemokine protein being printed on the nitrocellulose membrane between the sample pad and the absorbent pad, the nitrocellulose membrane further including a control line printed thereon between the sample pad and the absorbent pad; treating the sample pad with a stabilization buffer; treating the conjugate release pad with conjugate dilution buffer; applying the solution to the first end of the nitrocellulose membrane; reading the level of fluorescent intensity of the at least one of a test line and the control line with a reader; and determining the level of the at least one chemokine protein present in the solution based on the reading.

32. The method according to claim 31 wherein the at least one chemokine protein to be detected is selected from the group consisting of CCL21 , CXCL13 and CCL19.

33. The method according to claim 31 wherein the at least one chemokine protein to be detected is CCL21 or CXCL13.

34. The method according to claim 31 wherein the at least one chemokine protein to be detected is CCL21.

35. The method according to claim 31 wherein the stabilization buffer is 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5 and the conjugate dilution buffer is 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5.

36. The method according to claim 31 wherein the step of treating the sample pad with stabilization buffer includes treating the sample pad with stabilization buffer including 0.75% Tergitol NP-40S and 0.5 mg/mL anti-Red Blood Cell rabbit antibody.

37. The method according to claim 31 wherein the step of treating the sample pad with stabilization buffer includes treating the sample pad with stabilization buffer including 01.00% Tergitol NP-40S.

38. The method according to claim 31 wherein antibodies to red blood cells are added to the sample pad to immobilize any red blood cells and keep them from moving into the nitrocellulose.

39. The method according to claim 31 wherein the stabilization buffer includes 0.25% Tergitol NP- 40S and 1 % polyvinylpyrrolidone.

40. The method according to claim 31 wherein the stabilization buffer includes 0.75% Tergitol NP- 40S and 0.5 mg/mL anti-Red Blood Cell rabbit antibody.

41 . The method according to claim 31 wherein the nitrocellulose membrane is Ahlstrom 8964 glass fiber.

42. The method according to claim 40 where a single test line for CXCL13 and CCL21 is printed on the nitrocellulose membrane.

43. A test substrate comprising an elongate nitrocellulose membrane, the nitrocellulose membrane having a first end for receiving the solution and a second end whereby the solution flows from the first end to the second end by means of wicking, the substrate including a sample pad located at the first end of the nitrocellulose membrane and an absorbent pad located on the second end of the nitrocellulose membrane, at least one of a test line for detecting at least one chemokine protein with a C-X-C or a C-C structural motif being printed on the nitrocellulose membrane between the sample pad and the absorbent pad, the nitrocellulose membrane further including a control line printed thereon between the sample pad and the absorbent pad; wherein the sample pad is treated with a Stabilization Buffer (SB), wherein the conjugate release pad is treated with Conjugate Dilution Buffer (CDB) buffer.

44. The test substrate according to claim 43 wherein the at least one chemokine protein to be detected is selected from the group consisting of CCL21 , CXCL13 and CCL19.

45. The test substrate according to claim 43 wherein the at least one chemokine protein to be detected is CCL21 or CXCL13.

46. The test substrate according to claim 43 wherein the at least one chemokine protein to be detected is CCL21.

47. The test substrate according to claim 43, wherein the stabilization buffer is 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5 and the conjugate dilution buffer is 0.05M, tris(hydroxymethyl)aminomethane buffer (Tris), pH 8.5.

48. The test substrate according to claim 43 wherein the sample pad is treated with stabilization buffer including 0.75% Tergitol NP-40S and 0.5 mg/mL anti-Red Blood Cell rabbit antibody.

49. The test substrate according to claim 43 wherein antibodies to red blood cells are added to the sample pad to immobilize any red blood cells and keep them from moving into the nitrocellulose.

50. The test substrate according to claim 43 where a single test line for CXCL13 and CCL21 is printed on the nitrocellulose membrane.