HK1092529A - Gastrin hormone immunoassays - Google Patents

Description

Gastrin hormone immunoassay

Related patent

This application claims priority to U.S. provisional application 60/458,244 (application date 2003, 3/28) entitled "Gastrin hormone immunoassays", the specification of which is incorporated herein by reference in its entirety. Also, a divisional application of this application entitled "Monoclonal Antibodies to Gastrin Hormone" (filed 3/29/2004, U.S. provisional application Ser. No. 60 /), the specification of which is also incorporated herein by reference in its entirety.

Technical Field

The present invention relates to an ELISA assay for the detection and/or quantification of biologically active peptides, in particular gastrin peptide, in a biological fluid sample. The invention particularly relates to methods for detecting and/or quantifying free and total peptides (including peptides bound to antibodies) in a biological fluid sample.

Background

Although the gastrin hormone was first discovered as early as a hundred years ago and first isolated and purified in the 60's of the twentieth century, its efficacy in different tissues, normal or diseased tissues, has not yet been fully appreciated. One of the major reasons for the gap in the knowledge of the gastrin system is the difficulty in separately detecting and quantifying each of the various gastrin hormone forms.

In mammals, gastrin, a peptide hormone exists in a variety of forms, and can be divided into two major classes, depending on the number of amino acid residues in the peptide chain: "Small" gastrin and "Large" gastrin. "Small" gastrins include mature gastrin 17(G17) and glycine-G17 (G17-Gly); "Large" gastrins include gastrin 34(G34) and glycine-G34 (G34-Gly). The mature form of G17 is a major effector of gastric acid secretion and is estimated to be 6-fold more potent in this regard than G34. Various forms of gastrin are produced in vivo by cleavage of a precursor peptide known as "progastrin", and in some cases, by modification of the cleaved peptide. The C-terminus of human G34 has the complete 17 amino acid sequence of G17, and thus it is presumed that it immunologically cross-reacts with G17.

Mature G17 modified both the amino-terminal and carboxy-terminal residues: cyclization of the N-terminal glutamic acid to pyroglutamic acid (pGlu); the free carboxyl group of the C-terminal phenylalanine is amidated with peptidylglycine alpha-amidating monooxygenase (PAM) to form C-terminal amidated phenylalanine Phe-NH 2. (see Dockray et al, Ann. Rev. Physiol. (2001) 63: 119-.

Mature G17 is the major form of "small" gastrin in humans, having the amino acid sequence: pEGPWLEEEAYGWMDF-NH 2(SEQ ID NO: 1). G17-Gly is an incompletely processed form of gastrin, a minor component of "small" gastrin in healthy humans, and has the amino acid sequence: pEGPWLEEEAYGWMDFG (SEQ ID NO: 2).

Gastrin 34 is the major form of "big" gastrin in humans, having the amino acid sequence pEGPQGPPHLVADPSKKEGPWLEEEEEAEYGWMDF-NH 2(SEQ ID NO: 3), glutamate-gastrin 34(G34-Gly), with an additional C-terminal glycine residue, and the amino acid sequence pELGPQGPLVADPSKKEGPWLEEEAEYGWMDFG (SEQ ID NO: 4).

Gastrin is secreted by the antral G cells of the stomach under stimulation by gastrin-releasing peptide (GRP). Gastrin secretion is inhibited by the paracrine effects of gastric acid and various polypeptide hormones, particularly somatostatin. Gastrin peptides have long been thought to function to stimulate gastric acid secretion in healthy individuals, and until recently, studies have shown that these peptides also control the proliferation, differentiation and maturation of different cell types in the gastrointestinal system (GI system).

In addition to their regional role in the gastrointestinal system, G17 and G17-Gly (the latter being somewhat less) are also released into the blood; it has been found that gastrin concentrations are elevated in the serum of patients with gastrointestinal disorders and diseases such as gastric, colon and pancreatic cancer. It has also recently been found that these gastrins are also associated with other diseases unrelated to the gastrointestinal tract, including Small Cell Lung Cancer (SCLC) and metastatic liver tumors. . See example "Gastrin and Colon Cancer" in the following literature: a differentiating simplificating simplifications "S.N.Joshi et al, digest Diseases (1996) 14: 334-344; and "Gastrin and colorimetric cancer" Smith, A.M. and Watson, S.A. Alimentary Pharmacology and Therapeutics (2000)14 (10): 1231-1247.

Antibodies are key reagents in many detection techniques for use in medicine, veterinary medicine and other fields. . Such assays include many commonly used immunoassay techniques, such as enzyme-linked immunosorbent assays (ELISAs), Radioimmunoassays (RIA), Immunohistochemistry (IHC), and Immunofluorescence (IF) assays.

When used in many of the above assays, monoclonal antibodies (MAbs) have unique properties that are superior in many respects to polyclonal antisera, and antibodies purified from polyclonal antisera. These include: specificity for a single antigenic determinant of a target antigen (i.e. specificity for a single epitope); the antibodies obtained by different antibody preparation methods have invariable specificity for a long time and constant affinity and chemical components. In addition, monoclonal antibodies can be produced indefinitely and indefinitely by in vitro methods. These properties are in sharp contrast to those of polyclonal antibodies. The latter requires the use of in vivo immunization methods which are inevitably affected by biological differences and have a limited ability to produce antibodies during the life cycle of the immunized animal.

Despite these advantages, differences still exist between individual monoclonal antibodies, even if they are specific for the same epitope. For example, by immunizing with a single epitope segment, it induces differences in any or all of the following characteristics between the various monoclonal antibodies obtained: 1) good specificity for molecular composition and epitope tertiary structure; 2) an antibody idiotype; 3) (ii) antibody affinity; 4) antibody allotypes; and 5) antibody isotypes. These differences in properties affect the efficacy of monoclonal antibodies (mabs) in a particular immunoassay, such that different Mab strains against the same antigen segment exhibit different efficacy in a particular assay. Thus, some mabs are preferred over others that bind to the same epitope when used as reagents in a particular immunoassay.

The immunoassay may be enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), and immunodetection assays such as ELISPOT (enzyme-linked immunospot assay), slot blot, and western blot. General guidance for these techniques is found in Ausubel et al (eds) (1987) "Current protocols in Molecular Biology" John Wiley and Sons, New York, N.Y.. Alternatively, the immunoassay may be an Immunohistochemical (IHC) staining or Immunofluorescence (IF) method for visualizing gastrin hormone in a tissue sample. See "Principles and Practice of immunology" (1991) Christopher p.price and David j.neoman (eds), Stockton Press, New York, n.y.

Monoclonal antibodies specific for the N-terminal region and the C-terminal region of G17 have also been reported. . See Azuma et al, Gastroenterologica Japnica (1986)21(4): 319-324; ohning et al, Peptides (1994)15(3): 417-; fuerle et al, Pancreas (1995)10(3): 281 and 286; kovacs et al, Peptides (1996)17(4): 583-; ohning et al, Am J. physiol. (1996)271(3 Pt 1): g470-476; sipponen et al, (2002) Scand. J. gastroenterol.37(7): 785-791. However, these antibodies, either alone or in combination, do not have gastric secretion for each form of biological fluid in normal or pathological statesAbility of the elements to be distinguished and quantified.

Polyclonal anti-gastrin antibodies have been reported to effectively inhibit gastrin activity ("Inhibition of gastrin activity by digestion with antibodies to the C-terminal polypeptide of gastrin" Jaffe et al, (1969)65(4): 633-639); polyclonal antibodies against gastrin of non-human origin have been used to treat patients with zee-erger syndrome (mixed non-islet cell secreting adenomas) in which excess gastrin is produced without ingestion of a stimulus. Reference is made to Hughes et al, "Therapy with stomach Antibody in the Zollinger-Ellison Syndrome"; hughes et al, digest Diseases (1976)21(3): 201-204. However, these rabbit-derived anti-gastrin antibodies "achieved short-term efficacy in patients at best" (Hugh at p.204).

Recently, it has been suggested that the ratio of acylated/non-acylated gastrin hormones in serum can provide an indicator useful as a predictor of an individual's risk of developing duodenal ulcers or gastric atrophy. Reference is made to published U.S. patent application 2003/0049689 entitled "Diagnosis and Treatment of organic diagnostic disease", inventor t.c. wang.

To date, mabs that sensitively detect and accurately distinguish various forms of gastrin hormones, such as G17, G17-Gly, G34, and G34-Gly, have not been available. Furthermore, until the present invention, it has not been possible to accurately measure the amount of each form of gastrin in a biological fluid sample. Each gastrin form in a biological fluid sample has not been accurately measured. The use of the monoclonal antibodies (mabs) of the invention in assays in clinical trials enables more accurate determination of the biological activity of gastrin under normal or pathological conditions, and provides Mab compositions for use in pharmaceutical applications and methods for the prevention and treatment of gastrin-related diseases and conditions.

Disclosure of Invention

The present invention provides a method for determining the total amount of gastrin hormone (both antibody-bound and free) in a biological fluid sample. The method comprises the following steps: (a) obtaining a gastrin-containing biological fluid sample from a patient; (b) providing an immobilized antibody that selectively binds to a C-terminal epitope of gastrin; (c) incubating the sample with an N-terminal sequence of gastrin peptide under suitable conditions for binding of the gastrin hormone in the sample to said antibody, thereby producing an immobilized complex of said antibody bound to gastrin; (d) washing the immobilized complex to remove the gastrin peptide having the N-terminal sequence, and incubating the complex with a suitable detectable label-conjugated antibody that selectively binds to an N-terminal epitope of gastrin hormone to form an immobilized detectable label-conjugated antibody complex; (e) washing the immobilized detectable label-conjugated antibody complex and incubating it with a chromogenic agent; and (f) measuring the developed reagent to determine the total amount of gastrin hormone in the biological fluid sample.

The invention also provides a method for determining the amount of free gastrin in a biological fluid sample. The method comprises the following steps: (a) obtaining a gastrin-containing biological fluid sample from a patient; (b) providing an immobilized antibody that selectively binds to an N-terminal epitope of gastrin; (c) incubating the sample under suitable conditions for binding of gastrin in the sample to the antibody to produce an immobilized complex of the antibody bound to gastrin; (d) washing the immobilized complex to remove unbound components, and reacting the complex with a suitable detectable label conjugated antibody capable of selectively binding to a C-terminal epitope of gastrin hormone; (e) washing the immobilized detectable label-conjugated antibody complex and incubating it with a chromogenic agent; and (f) measuring the developed reagent to determine the amount of free gastrin hormone in the biological fluid sample.

The invention also provides a method for determining the total amount of bound and free polypeptide in a biological fluid, wherein at least a portion of the polypeptide reversibly binds to a first binding sequence. The method comprises the following steps: (a) obtaining a biological fluid sample containing the polypeptide from the patient; (b) providing a solid phase substrate coated with an antibody that selectively binds to a primary epitope of a polypeptide, which primary epitope is not present in the first binding sequence; (c) incubating the sample with a peptide fragment having a first binding sequence and no prime epitope under suitable conditions for binding of the polypeptide to said antibody, thereby producing a complex of said antibody and polypeptide; (d) washing the reaction well to remove unbound antibody and peptide fragments, and reacting the complex with an appropriate detectable label conjugated antibody that selectively binds to a minor epitope of the polypeptide; (e) washing the reaction hole, and adding a color developing agent into the reaction hole; and (f) measuring the developed reagent to determine the total amount of bound and free polypeptide in the biological fluid sample.

The invention also provides a method of assessing the condition of a gastrin hormone-blocking treatment in a patient suffering from a gastrin hormone-induced disease or condition. The method comprises the following steps: (a) obtaining a first gastrin-containing biological fluid sample from the patient prior to or at an early stage of treatment; (b) determining the level of gastrin hormone in the first sample by immunoassay; (c) diagnosing based on the disease or condition to be treated and the level of gastrin hormone in the first sample; (d) administering a treatment to a patient comprising: a first agent or a substance capable of producing a first agent, which first agent is capable of binding to a gastrin hormone and thereby modulating the binding of gastrin to its target receptor in vivo; (e) after a suitable period of time, during which the treatment should have had some effect, a second biological fluid sample is taken from the patient; (f) determining total gastrin levels (including bound and free gastrin) in a first portion of a second sample using an immunoassay, wherein the first portion of the second sample is incubated with: (i) a second agent capable of replacing any gastrin bound by the first agent; and (ii) an immobilized anti-gastrin hormone antibody, wherein the immobilized antibody does not bind to a second agent; washing to remove the second reagent and adding a detectable antibody that binds to the gastrin hormone and does not compete with the immobilized antibody to form an immune complex in which the immobilized antibody binds to the gastrin hormone which is in turn bound by the detectable antibody; (g) detecting the amount of detectable antibody in the immune complex, thereby allowing determination of the amount of total gastrin hormone in the second sample; (h) determining the level of free gastrin hormone in a second portion of the sample from the second sample by repeating steps (f) and (g), wherein the incubating of step (f) is performed in the absence of the second agent; and (j) comparing the determined amount of free gastrin hormone in the first sample with the amount of free gastrin and total gastrin in the second sample, thereby determining the efficacy of the gastrin hormone blockade therapy for the patient.

Drawings

FIG. l shows the gastrin concentration (picomolar, 10) after undergoing an enzymatic chromogenic reaction with tetramethylbenzidine sulfonate (TMBS) as chromogenic substrate^-12Molal) to 450nm (A)₄₅₀) A schematic of the resulting calibration curve for total gastrin 17 plotted against absorbance.

FIG. 2 shows the same method as above, with gastrin concentration (picomolar, 10)^-12Molal) to 450nm (A)₄₅₀) A schematic of the resulting calibration curve for free gastrin 17 plotted against absorbance. .

Detailed Description

The following provides definitions of terms and phrases used in the present invention:

"gastrin hormone" or "gastrin hormone form" as used interchangeably herein, refers to any biologically active and/or immunologically cross-reactive gastrin hormone peptide. Major forms of gastrin hormones include, but are not limited to: gastrin 17(G17), whether C-terminally amidated or having a free C-terminus; glycine-gastrin 17 (G17-Gly); gastrin 34(G34), including C-terminally amidated forms or forms with a free C-terminus; and glycine-gastrin 34 (G34-Gly).

As used herein, "selective" for a particular form of a gastrin hormone refers to the antibody having specificity for a particular target epitope on a particular form of gastrin and being capable of binding to any one of the gastrin forms that contains the target epitope. For example, the C-terminus of mature (amidated) G17 is identical to mature G17 and G34. Therefore, MAbs specific for the C-terminal target epitope on the C-terminus of mature G17 also selectively recognize G17 (and G34).

As used herein, "total amount" of one gastrin form in a sample refers to the sum of the amount of free (unbound) gastrin hormone form plus the amount of complexed (bound) gastrin hormone form. The complex gastrin may be bound by an antibody or other gastrin-binding component of the sample.

As used herein, "biological fluid" refers to any fluid containing material of biological origin. Preferred biological fluids for use in the present invention include animal (especially mammalian) body fluids, preferably human. The body fluid may be any body fluid including, but not limited to, plasma, serum, lymph fluid, cerebrospinal fluid (CSF), and other similar substances.

As used herein, "preservative" refers to any reactant, supplement, or additive capable of reducing the time-dependent degradation of gastrin in a biological fluid sample or a fluid sample with a biological component. Preservatives useful in the practice of the present invention include any of a wide variety of preservatives well known in the art, including, but not limited to, common chemical preservatives such as sodium azide, EDTA, and the like, and protease inhibitors such as PMSF (phenylmethylsulfonyl fluoride), aprotinin (e.g., aprotinin), or biological preservatives such as heparin.

As used herein, "test plate" refers to any solid substrate on which a plurality of liquid samples can be individually tested using the methods of the present invention. As used herein, the "reaction well" of a test plate refers to the area on the test plate that serves as the sample receiving area of the plate. The reaction wells of a typical assay plate are obtained by forming depressions in the surface of the plate sufficient to receive and hold the sample volume and the volume of buffer or wash solution added at any step in the assay process.

As used herein, "measuring" with respect to a target molecule refers to detecting, quantifying, or determining the amount of an analyte or target molecule.

The present invention discloses in detail mabs particularly suitable for use in immunoenzymatic assays (commonly referred to as "ELISA" or enzyme-linked immunosorbent assays) which are intended to measure a specific form of gastrin in biological fluids.

The mabs used in the assays of the invention include those that bind selectively to the N-terminus of gastrin 17(G17) at an epitope of the amino acid sequence pEGPWLE (SEQ ID NO: 5).

Mabs used in the assays of the invention also include mabs that bind to gastrin 17(G17) or gastrin 34(G34) selectively at an epitope of amino acid sequence EEAYGWMDF-NH2(SEQ ID NO: 6).

On the other hand, the Mab used in the assay of the present invention comprises a Mab that selectively binds to the N-terminus of human gastrin 34(G34) at the epitope of amino acid sequence pELGPQG (SEQ ID NO: 7).

In another aspect, the MAb used in the assay of the present invention comprises a MAb that selectively binds to the epitope of amino acid sequence YGWMDFG (SEQ ID NO: 8) with glycine-gastrin 17(G17-Gly) and glycine-gastrin 34 (G34-Gly).

The mabs used in the assays of the invention preferably bind to the gastrin hormone form with a binding constant (Ka) for selective binding of about 10⁶-10⁷LM^-1Mab of (a); preferably capable of binding to the gastrin hormone form and having a Ka of about 10⁷-10⁸LM^-1And more preferably Ka of about 10⁹-10¹⁰LM^-1And further preferably Ka is 10¹⁰-10¹¹LM^-1Most preferably, Ka is 10¹¹-10¹²LM^-1。

The sample for analysis according to the method of the invention is preferably a sample from a mammalian biological fluid. The sample comprises, or is suspected of containing, an amount of the peptide to be detected, quantified or determined. Preferably, the sample contains a gastrin hormone in the form of at least one gastrin. More preferably, the sample has added thereto a preservative to form a sample mixture, and the sample mixture is frozen within about 1-15 minutes after sampling from the mammalian body.

As used herein, "suitable conditions" for binding are conditions of temperature, pH and ionic strength that allow the antibody to bind to its cognate antigen and allow the performance of an enzyme labeling reaction in which an enzyme label is coupled to the antibody as a detection agent. Such conditions suitable for antibody-antigen binding and various labeling enzyme reactions are well known to those skilled in the art, and the specific suitable conditions for each reaction can be determined by routine means without undue experimentation.

As used herein, "detectable label conjugated antibody" refers to any labeled antibody wherein the label provides a detectable signal, such as an enzymatic label; or may be detected by another reagent, such as a labeled secondary antibody, which itself may be detected by providing a detectable signal such as a radioactive label, an enzymatic label, a fluorescent or luminescent label, or a separately detectable moiety such as a biotin label detectable by a moiety conjugated to avidin.

As used herein, a "detectable label-conjugated antibody complex" refers to a complex having an antibody conjugated to a detectable label, which antibody binds to its cognate antigen, which may be gastrin hormone. This gastrin hormone-antibody complex provides a detectable signal that can be measured and is directly related to the concentration of antibody measured. In a preferred concentration range, the signal is proportional to the concentration of the antibody complex conjugated to the detectable label.

As used herein, "color-developing agent" refers to an agent capable of being developed by an antibody-conjugated enzyme. For example, the developer for alkaline phosphatase may be p-nitrophenyl phosphate (pNPP).

The present invention proposes an assay method for measuring the amount of total gastrin (both bound and free) and a method for evaluating a gastrin-blocking treatment. The measurement method is as follows. Methods of evaluating a patient's gastrin-blocking treatment are particularly valuable in clinical practice to determine when one or the other treatment regimen is to be undertaken, and are critical to the therapeutic outcome of the patient. The method of the present invention provides information upon which these critical decisions are made. The present method provides for the measurement of gastrin either before or early in treatment (as described in us patent 5622702, shortly after immunization of a patient with a gastrin hormone peptide-conjugated vaccine) and provides for the measurement of one or more of the total and/or free gastrin amount after a period of time in which the effect has begun to be produced according to the intended treatment.

Analytical method

The following is a detailed description of the assay method (immunoenzymatic assay) of the present invention for determining the amount of total (uncomplexed plus complexed with antibody) or free (uncomplexed) human gastrin hormone (G17, G17-Gly, G34 or G34-Gly) in a biological fluid such as human plasma by using monoclonal and/or polyclonal antibodies specific for the C-terminus or N-terminus of the specific molecular form of gastrin hormone to be tested. In addition, polyclonal antibody compositions directed against the C-terminus of the molecule or against the N-terminus of the molecule may also be used in combination with monoclonal antibodies directed against the N-terminus of the molecule or against the C-terminus of the molecule, respectively.

In the assay described below, NUNC Maxisorp, F96 ELISA plate (catalog No. 439454) assay plates were used, and the antibody coating solution was a boric acid buffer solution (20mM, pH 8.0, containing 0.1% sodium azide).

1. Coating with a flat plate: the surfaces of the reaction wells of the assay plates are coated with an antibody that selectively recognizes the particular form of the human gastrin molecule to be tested, at an optimal concentration.

The optimal antibody concentration is determined by plotting a standard curve of known concentrations of the gastrin form to be tested, with the required sensitivity and accuracy over the required effective concentration range. For G17, the effective G17 concentration range tested was generally from background concentration (about 4pM or less) to 25pM, or to 50 pM. However, in patients with gastrin tumours, plasma gastrin levels can be as high as 800pM or even 1000pM (1.0 micromolar). One of ordinary skill in the art can readily determine whether appropriate sensitivity and accuracy are within the desired range without undue experimentation.

2. Washing a flat plate: the coating solution was poured off, and wash buffer (approximately 400. mu.l per well) was added and then poured off. This wash cycle is repeated as many times as desired. The wash buffer was 0.010M phosphate buffer (0.0027M potassium chloride, 0.137M sodium chloride, pH7.4, containing 0.01% w/v Triton X-100). Plate washing can be automated (the instrument used in the assays described below is Labsystems Wellwash4Mk 2, Life Sciences International (UK) Ltd, Basingstoke, UK).

3. Sealing the flat plate: protein and detergent blocking buffer (coating buffer containing 1% BSA/0.1% Triton X-100) was added to the reaction wells. The plates may be stored in this form.

4. Addition of samples and standards: the plate was washed as described above. Assay buffer (made with 1% BSA, 0.1% bovine gamma globulin and 200 KIU/ml aprotinin in wash buffer) containing rabbit IgG (400. mu.g/ml) and EDTA (3.4mM) was added to each well (100. mu.l/well). Test standards (e.g., increasing amounts of gastrin hormone in gastrin depleted plasma) and test plasma samples were added to the reaction wells (20 μ l/well). The reaction was carried out at 4 ℃ overnight. Removal of gastrin from serum samples can be conveniently achieved by leaving the sample at room temperature overnight.

In these assays for the detection of total gastrin hormone, the dissociation peptide G17(1-9) (100. mu.g/ml) was included in the assay buffer, rabbit IgG and EDTA mixture.

5. Addition of conjugate

After washing, each well is filled with a detection buffer containing an enzyme-labeled monoclonal or polyclonal antibody specific for the N-terminus of the gastrin hormone form to be detected and conjugated with rabbit IgG (100 μ g/ml). The reaction was carried out at room temperature (+22 ℃ C.), shaking with a microplate shaker. Examples of suitable enzyme substrates for detecting the color development of the complex are: nitro-benzene phosphate for alkaline phosphatase, or tetramethylbenzidine sulfonate (TMBS) for horseradish peroxide. The degree of colour development can be read in absorbance units (AU, absorbance at 405nm in the case of p-nitrophenol; absorbance at 450nm in the case of TMBS) and can be used as an indicator of the amount of G17 in the test sample, the exact concentration of which can be converted by reading the absorbance of the test sample and then referring to a standard curve prepared from known concentrations of gastrin hormone.

7. Reading when sufficient signal is detected, the signal is measured using an instrument such as a full wavelength microplate reader or a fluorescence microplate reader.

8. Data processing the detected signals obtained with a standard solution of known concentration of gastrin to establish a calibration curve (signal-concentration). The calibration curve is used to convert the concentration of the gastrin form in the test sample.

Detailed assays for determining the amount of total and free gastrin hormone forms are described below:

determination of Total G17

In this assay, an antibody specific for the C-terminus of human gastrin 17And coating the surface of the reaction hole of the test plate. Plate washing and plate blocking were carried out according to the conventional methods described above. The plates were washed using the method described. Each well was loaded with a solution containing rabbit IgG (400. mu.g/ml), the dissociation peptide G17_(1-9)Detection buffer (100. mu.l/ml) and EDTA (3.4mM) (100. mu.l/well). Then the detection standard (0-4.1-10.2-26.6-64-160G 17 of 400-1000pM in the plasma without gastrin) and the test plasma sample were added into the wells (20. mu.l/well). The reaction was carried out in a refrigerator at 4 ℃ overnight. After washing, a detection buffer containing an alkaline phosphatase-conjugated monoclonal antibody specific for the N-terminus of G17 and rabbit IgG (100. mu.g/ml) was added to each well. After washing, the chromogenic substrate pNPP was added, the plate was incubated and allowed to develop color, and the absorbance was read with the above microplate reader. A calibration curve (signal-concentration) was established using the detection signal obtained with a standard G17 solution of known concentration. This calibration curve was used to convert the concentration of G17 in the test sample. Fig. 1 shows a schematic diagram of a calibration curve.

Determination of free G17

An antibody specific for the N-terminus of human gastrin 17 was coated on the surface of the reaction well of the test plate. Plate washing and plate blocking were carried out according to the conventional methods described above. The plates were washed using the method described. Assay buffer containing rabbit IgG (400. mu.g/ml) (prepared by adding 1% BSA, 0.1% bovine gamma globulin, and 200 KIU/ml aprotinin to the wash buffer) was added to each well, and the reaction was performed at room temperature (+22 ℃) with shaking on a microplate shaker. After washing, a detection buffer containing a monoclonal antibody specific for the C-terminus of G17, conjugated with alkaline phosphatase as an enzyme label, and rabbit IgG (100 μ G/ml) was added to each well. The reaction was carried out at room temperature (+22 ℃) and shaken on a microplate shaker. After washing, the chromogenic substrate pNPP was added, the plate was incubated and allowed to develop color, and the absorbance was read with the above microplate reader. The detection signal obtained with a standard G17 solution of known concentration was used to establish a calibration curve (signal-concentration) as described in the total G17 assay. This calibration curve was used to convert the concentration of G17 in the test sample. Fig. 2 shows a schematic diagram of a calibration curve.

Determination of Total G17-Gly

Antibodies specific for the C-terminus of the human glycine-gastrin 17 molecule were coated onto the surface of the wells of the assay plate as described above. Plate washing and plate blocking were carried out according to the conventional methods described above. The plates were washed using the method described. Each well was loaded with a solution containing rabbit IgG (400. mu.g/ml), the dissociation peptide G17_(1-9)(100. mu.g/ml) and EDTA (3.4mM) assay buffer (made up of 1% BSA, 0.1% bovine gamma globulin, and 200 KIU/ml aprotinin in wash buffer) (100. mu.l/well). Then, the assay standard (0-4.1-10.2-26.6-64-160-400-pM G17-Gly in the gastrin-removed plasma) and the test plasma sample were added to the wells (e.g., 20. mu.l/well). The reaction was carried out at 4 ℃ overnight. The subsequent steps were as described above for the determination of total G17.

Determination of free G17-Gly

Antibodies specific for the N-terminus of the G17-Gly molecule were coated onto the surface of the wells of the assay plate. Plate washing and plate blocking were carried out according to the conventional methods described above. The plates were washed using the method described. Assay buffer containing rabbit IgG (400. mu.g/ml) (prepared by adding 1% BSA, 0.1% bovine gamma globulin, and 200 KIU/ml aprotinin to the wash buffer) (e.g., 100. mu.l/well) was added to each well, followed by addition of sample and standard (e.g., 50. mu.l/well), and the reaction was performed at room temperature (+22 ℃) with shaking on a microplate shaker. After washing, a detection buffer containing an alkaline phosphatase-conjugated monoclonal antibody specific to the C-terminus of G17-Gly and rabbit IgG (100. mu.g/ml) was added to each well. The reaction was carried out at room temperature (+22 ℃) and shaken on a microplate shaker. The subsequent steps were as described in the determination of free G17 above.

Determination of G34

An antibody specific for the N-terminus of human gastrin 34 was coated on the surface of the reaction well of the test plate. Plate washing and plate blocking were carried out according to the conventional methods described above. The plates were washed using the method described. Assay buffer containing rabbit IgG (400. mu.g/ml) (prepared by adding 1% BSA, 0.1% bovine gamma globulin, and 200 KIU/ml aprotinin to the wash buffer) (e.g., 100. mu.l/well) was added to each well, followed by addition of sample and standard (e.g., 50. mu.l/well), and the reaction was performed at room temperature (+22 ℃) with shaking on a microplate shaker. After washing, a detection buffer containing an alkaline phosphatase-conjugated monoclonal antibody specific to the C-terminus of G34 and rabbit IgG (100. mu.g/ml) was added to each well, and the reaction was carried out at room temperature (+22 ℃) with shaking using a microplate shaker. Adding chromogenic substrate pNPP, reading sample signals in the reaction holes of the plate by using a microplate reader, and processing subsequent data as described above. A calibration curve (signal-concentration) was established using the detection signal obtained with a standard G34 solution of known concentration. The calibration curve was used to convert the concentration of G34 in the test sample

Examples

Example 1: measurement of total gastrin 17 in a serum sample depleted of gastrin to which a known amount of gastrin 17 had been added.

The gastrin hormone in serum was removed by allowing endogenous proteases to completely degrade existing gastrin by allowing the serum to stand overnight at room temperature.

To determine the precision and accuracy of the in-batch analysis, a known amount of gastrin 17(G17) was added to replicate samples of the gastrin-depleted serum sample to achieve the nominal concentrations shown in table 1. The measurement of total G17 was performed using N-terminal gastrin peptide in the same manner as in the detection of serum samples containing anti-gastrin hormone antibodies. In the above sample/standard addition procedure, N-terminal gastrin peptide G17(1-9) was added at a concentration of 100 μ G/ml. The results presented in table 1 indicate that the assay accurately quantifies G17 within the accepted limits of the ELISA method, which are ± 20% relative error. More importantly, the assay is extremely accurate at concentrations of G17 equal to and less than 100pM (the concentrations described above being those typically measured in patients).

TABLE 1

Total gastrin 17 assay

Gastrin 17 concentration (pM)
							7.50	15.00	100.0	600.0	720.0
Mean value sdcB (%) RE (%)	7.50.811.20.0	14.30.96.5-4.7	99.31.71.7-0.7	717.116.22.319.5	814.17.70.913.1

6 duplicate samples taken with n-6 measurements

sd standard deviation

Coefficient of variation of CV (calculation before rounding)

RE relative error (after rounding off)

Example 2: measurement of free gastrin 17 in a gastrin depleted serum sample to which a known amount of gastrin 17 has been added.

The present assay was carried out in accordance with the method for the determination of free gastrin 17(G17) described in the "method of determination" above. The results presented in table 2 indicate that the assay accurately quantifies free G17 within the accepted limits of the ELISA method. More importantly, the assay is extremely accurate at concentrations of G17 equal to and less than 100pM (the concentrations described above being those typically measured in patients).

TABLE 2

Determination of free gastrin 17

Precision and accuracy data for in-batch analysis

Gastrin 17 concentration (pM)
							7.50	15.00	100.0	600.0	720.0
Mean value sdcB (%) RE (%)	7.551.6722.20.7	13.411.269.4-10.6	94.015.475.8-6.0	556.643.017.7-7.2	892.8112.112.6-0.8

Measurement of 9 replicate samples n-9

sd standard deviation

Coefficient of variation of CV (calculation before rounding)

RE relative error (after rounding off)

Example 3: gastrin 17 stability

The stability of gastrin at room temperature (about 22 ℃) can be assessed by using the above-described assay for total gastrin, i.e. measuring the total amount of G17 in samples with known G17 concentrations of 15, 100 and 600pM, both at the time the sample is ready (0 hour sample) and at room temperature after 2 hours of standing on the bench. The results are shown in table 3 below, which shows that the concentration of G17 in each sample was greatly reduced. This demonstrates that proper sample processing techniques, including as little exposure as possible to elevated temperatures in the preparation of plasma from a patient's blood sample, are important for obtaining accurate gastrin values when using the detection methods of the present invention to determine the amount of gastrin hormone in a sample.

TABLE 3

Determination of total gastrin 17

Gastrin 17 stability at Room temperature (ca 22 ℃ C.)

		Measurement of Gastrin G17 concentration (pM)
							15	100	600
0a hour	Mean value sdcB (%) RE (%)	11.62.823.8-22.7	89.44.34.8-10.6	605.525.04.10.9
					2 hours	Mean value sdcB (%) RE (%)	5.53.155.2-63.3	59.12.03.5-40.9	400.519.74.9-33.3

A takes the mean result as a benchmark

sd standard deviation

Coefficient of variation of CV (calculation before rounding)

RE relative error (after rounding off)

Reference to

All patents and publications mentioned in this specification are herein incorporated by reference in their entirety.

Preservation of hybridoma cell lines

The following hybridomas used to produce the specific MAbs of the present invention were deposited at the American type culture Collection (ATCC, Manassas, Va.) from 3/25 of 2004.

1. Hybridoma 400-1, which produces MAb 400-1, is designated as accession number _.

2. Hybridoma 400-2, which produces MAb 400-2, is designated as accession number _.

3. Hybridoma 400-3, which produces MAb 400-3, is designated as accession number _.

4. Hybridoma 400-4, which produces MAb 400-4, is assigned accession number _.

5. Hybridoma 401-2, which produces MAb 401-2, is designated as accession number _.

6. Hybridoma 445-1, which produces MAb 445-1, is designated as accession number.

7. Hybridoma 445-2, which produces MAb 445-2, is designated as accession number.

8. Hybridoma 458-1, which produces MAb 458-1, is assigned accession number _.

Claims (10)

1. A method for determining the total amount of gastrin hormone in a biological fluid sample, the method comprising the steps of:

(a) obtaining a gastrin-containing biological fluid sample from a patient;

(b) providing an immobilized antibody that selectively binds to a C-terminal epitope of gastrin;

(c) incubating the sample with an N-terminal sequence of gastrin peptide under suitable conditions for binding of the gastrin hormone in the sample to said antibody, thereby producing an immobilized complex of said antibody bound to gastrin;

(d) washing the immobilized complex to remove unbound antibody and N-terminal sequence gastrin peptide, and incubating the complex with a suitable detectable label-conjugated antibody that selectively binds to an N-terminal epitope of gastrin hormone to form an immobilized detectable label-conjugated antibody complex;

(e) washing the immobilized detectable label-conjugated antibody complex and incubating it with a chromogenic agent;

(f) measuring the developed reagent to determine the total amount of gastrin hormone in the biological fluid sample.

2. A method for determining the amount of free gastrin in a biological fluid sample, the method comprising the steps of:

(a) obtaining a gastrin-containing biological fluid sample from a patient;

(b) providing an immobilized antibody that selectively binds to a C-terminal epitope of gastrin;

(c) incubating the sample under suitable conditions for binding of gastrin in the sample to said antibody to produce an immobilized complex of said antibody bound to gastrin;

(d) washing the immobilized complex to remove unbound antibody, reacting the complex with an antibody conjugated to a suitable detectable label, said antibody being capable of selectively binding to an epitope bound to the N-terminus of the gastrin hormone;

(e) washing the immobilized detectable label-conjugated antibody complex and incubating it with a chromogenic agent; and

(f) measuring the developed reagent to determine the amount of free gastrin hormone in the biological fluid sample.

3. The method of claim 1 or 2, wherein the body fluid is serum

4. The method of any one of claims 1 to 3, wherein the C-terminal selective antibody and/or the N-terminal selective antibody is a monoclonal antibody.

5. The method of any one of claims 1 to 4, wherein the gastrin hormone is G17 or G17-Gly and the C-terminal selective antibody and N-terminal selective antibody bind to G17.

6. The method of claim 5, wherein said C-terminal selective antibody is a monoclonal antibody having the characteristics of the monoclonal antibody produced by hybridoma 458-1; or a monoclonal antibody having the characteristics of the monoclonal antibody produced by hybridoma 445-1(ATCC accession # s) or hybridoma 445-2(ATCC accession # s); and/or the N-terminal selective antibody is a monoclonal antibody having the characteristics of a monoclonal antibody produced by any one of the hybridomas 400-1, 400-2, 400-3, or 400-4.

7. The method of claim 6, wherein said C-terminal selective antibody is a monoclonal antibody produced by hybridoma 458-1; and/or the N-terminal selective antibody is a monoclonal antibody produced by any one of the hybridomas 400-1, 400-2, 400-3, or 400-4.

8. The method of any one of claims 1 to 4, wherein the gastrin hormone is G34 or G34-Gly and the C-terminal selective antibody and N-terminal selective antibody are conjugated to G34 or G34-Gly.

9. The method of claim 8, wherein said C-terminal selective antibody is a monoclonal antibody having the characteristics of the monoclonal antibody produced by hybridoma 458-1; or a monoclonal antibody having the characteristics of the monoclonal antibody produced by hybridoma 445-1(ATCC accession # s) or hybridoma 445-2(ATCC accession # s); and/or the N-terminal selective antibody is a monoclonal antibody having the characteristics of the monoclonal antibody produced by hybridoma 401-2.

10. The method of claim 8, wherein said C-terminal selective antibody is a monoclonal antibody produced by hybridoma 458-1; and/or the N-terminal selective antibody is a monoclonal antibody produced by hybridoma 401-2.