CN116136491A - Device and method for detecting anti-CD47 antibody - Google Patents
Device and method for detecting anti-CD47 antibody Download PDFInfo
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Abstract
The invention belongs to the field of detection, relates to a device and a method for detecting an anti-CD 47 antibody, and in particular relates to a device and a method for detecting affinity of the anti-CD 47 antibody and CD47 based on a biomembrane interference technology. More specifically, the present invention relates to a device for detecting an anti-CD 47 antibody, comprising: the sensor and the CD47 molecules are combined on the surface of the sensor to form a biological film layer, and the solidifying signal height of the biological film is 0.2-0.4nm. The method is simple to operate, good in detection repeatability and short in detection time; the device can be repeatedly used in a regeneration process, continuously detects 10 or even 15 samples, has an affinity CV value within 5%, has a detection time of only about 75 minutes, and is suitable for rapid detection of multiple samples.
Description
Technical Field
The invention belongs to the field of detection, relates to a device and a method for detecting an anti-CD 47 antibody, and in particular relates to a device and a method for detecting affinity of the anti-CD 47 antibody and CD47 based on a biomembrane interference technology.
Background
CD47 is also known as integrin-associated protein (integrin associated protein, IAP). CD47 has a molecular weight of about 50kDa and belongs to the immunoglobulin superfamily. CD47 is involved in a variety of physiological functions, such as cell transfer, T cell and Dendritic Cell (DC) activation, axon development, and the like. Studies have shown that under pathological conditions, CD47 is highly expressed on the surfaces of many hematological malignancies and solid tumor cells, and by binding to sirpa receptors on phagocytes (macrophages, dendritic cells, etc.), a series of inhibitory signals are initiated, which can be transduced into the interior of phagocytes, eventually causing the phagocytes' phagocytes to disappear and failing to phagocyte to eliminate malignant proliferation.
In recent years, in a plurality of immunotherapy schemes, CD47 is a very hot target, and the anti-CD 47 monoclonal antibody medicament has wide application prospect and good effect of treating tumors, and can be used for treating various tumors, such as multiple myeloma, breast cancer, non-small cell lung cancer, hepatocellular carcinoma, melanoma, ovarian cancer, childhood brain tumor and the like. Therefore, development of antibody drugs with high affinity to CD47 for the treatment of tumors is of great importance.
The ability of therapeutic monoclonal antibodies to specifically bind to a target antigen directly affects the effectiveness of antibody therapy, a key quality attribute of monoclonal antibody products. At present, the antigen-antibody activity detection method mainly adopts an ELISA method, although the finished product is low, the detection is long in time consumption, complicated in steps and poor in detection repeatability, but the current Chinese biopharmaceutical enterprises develop rapidly and compete increasingly, and the method can help research and development production personnel judge the quality of the product in a shorter time by adopting a quicker and reliable detection means, so that the method makes a next decision, and has important significance in shortening the research and development and production cycle of the product and enhancing the development activity of the enterprises.
The biological film interference technique (Bio-layer interferometry, BLI) is a real-time, label-free, rapid detection technique that relies on the principle that when biomolecules bind to the sensor surface, a layer of biological film is formed that interferes with the waveform of light transmitted through the sensor. The interference phenomenon is detected in a phase shifted manner, and a change in the number of molecules bound to the sensor can be detected. The BLI technique has been successfully applied to the detection of protein intermolecular interactions.
The BLI has two main classes of sensors: one is a capture type sensor, which can directly immobilize a protein and then detect affinity to another protein, such as an AHC sensor and his1k sensor, where the immobilized protein can be removed by a regeneration process, but the reproducibility of such a sensor is poor, typically 10 times of experiments are repeated, the CV value of the affinity is greater than 5%, even greater than 10% or 20%, and it takes time to immobilize the protein again for each detection. Another type of sensor, where proteins are immobilized on the sensor and cannot be removed by a regeneration process, such as SA sensors, AR2G sensors, APS sensors, is generally rarely used because AR2G and APS immobilized proteins take a long time to operate. For SA sensors, since the protein cannot be removed by regeneration after the biotinylated protein is immobilized on the SA sensor, generally, after one row of SA sensor is immobilized with the protein, only one detection can be performed, and if a second detection is performed, the protein immobilization is repeated by using a new row of sensors. The economic cost of an array of sensors is not low and it takes time to fix an array of sensors, including for example pre-wetting, balancing, fixing and operating time.
Therefore, there is still a need to develop a sensor capable of detecting an anti-CD 47 antibody with good reproducibility.
Disclosure of Invention
The present inventors have made intensive studies and creative efforts to produce a sensor for detecting an anti-CD 47 antibody. The inventors have also explored the regeneration conditions of CD 47. The inventors have surprisingly found that: CD47 can be effectively regenerated without re-fixing, so that the time required for fixing protein (CD 47) is saved while the disposable use of the sensor is reduced; the sensor of the present invention has good reproducibility, and even if it is repeatedly used for 10 times or even 15 times, the CV value of the affinity can be less than 5%, whereas the conventional capture type sensor is generally repeated for 10 times, and the CV value of the affinity is more than 5%, or even more than 10% or 20%. The following invention is thus provided:
one aspect of the invention relates to a device for detecting an anti-CD 47 antibody, comprising:
the sensor and the CD47 molecule are used,
the CD47 molecules are combined to the surface of the sensor to form a biological film layer, and the solidifying signal height of the biological film is 0.2-0.4nm, preferably 0.3nm.
In some embodiments of the invention, the device, wherein the CD47 molecule is bound to the sensor surface via a biotin-streptomycin affinity system, an amino-amino coupling system, or a his tag-anti-his tag antibody system.
In some embodiments of the invention, the device is capable of regeneration by 1-5M magnesium chloride solution; preferably, it is capable of regeneration by 2-3M magnesium chloride solution; preferably, the regeneration time is 2-20 seconds, preferably 2-10 seconds, 3-8 seconds or 5 seconds.
In some embodiments of the invention, the device, wherein the sensor is one selected from the group consisting of SA sensor, AR2G sensor, and APS sensor.
In some embodiments of the invention, the device, wherein,
the device is capable of being used at least 10 times in succession, and the CV value of the affinity for 10 successive uses is less than 5%;
preferably, the device is capable of being used at least 15 times in succession, and the CV value of the affinity for 15 successive uses is less than 5%.
In some embodiments of the invention, the device is made by a method comprising:
(1) Obtaining (e.g., preparing or purchasing) biotinylated CD47;
(2) Solidifying the biotinylated CD47 onto a sensor (preferably a SA sensor);
preferably, the method also comprises the step (3),
(3) Immersing the sensor in 1-5M magnesium chloride solution; preferably, 2-3M magnesium chloride solution is used; preferably, the time of immersion is maintained for 2-20 seconds, preferably 2-10 seconds, 3-8 seconds or 5 seconds;
preferably, the method also comprises the step (4),
(4) Pre-combining the sample to be detected, and regenerating the device by using 1-5M magnesium chloride solution; preferably, 2-3M magnesium chloride solution is used for regeneration; preferably, the regeneration time is 2-20 seconds, preferably 2-10 seconds, 3-8 seconds or 5 seconds.
In some embodiments of the invention, the device, wherein, in step (1),
biotin reagent and CD47 at 1:2 to 2:1 (preferably 1:1.5 to 1.5:1 or 1:1), and reacting at room temperature for 10-60 minutes (preferably 20-50 minutes or 30-40 minutes);
preferably, desalination is performed using a desalination column;
preferably, the concentration of the sample to be measured is detected;
preferably, the concentration of the sample to be measured is detected using a micro-spectrophotometer.
In some embodiments of the invention, the device, wherein, in step (1),
mixing biotin reagent and CD47 in a molar ratio of 1:1, and reacting for 30min at room temperature; desalting was performed using a desalting column, and the concentration of the sample was measured using a micro spectrophotometer.
In some embodiments of the invention, the device, wherein, in step (2),
the sensor was pre-wetted with PBS,0.02% Tween-20, pH7.4 (pre-wet) for 5-15 min (preferably 8-12 min or 10 min); preferably, the sensor is immersed in PBS,0.02% Tween-20, pH7.4 for equilibration; preferably, the equilibration time is 30-120 seconds, preferably 30-90 seconds, 40-80 seconds, 50-70 seconds or 60 seconds;
immersing the sensor in biotinylated CD47, allowing the biotinylated CD47 to solidify on the sensor with a solidification signal height of 0.2-0.4nm (preferably 0.3 nm); preferably, the sensor is immersed in PBS,0.02% Tween-20, pH7.4 for equilibration; preferably, the equilibration time is 30-120 seconds, preferably 30-90 seconds, 40-80 seconds, 50-70 seconds or 60 seconds;
preferably, the method further comprises the following steps:
the pre-wet was replaced with PBS,0.02% Tween-20,0.1% BSA, pH7.4, and pre-wet for 5-15 minutes (preferably 8-12 minutes or 10 minutes).
In some embodiments of the invention, the device, wherein step (2) is performed by a molecular interaction device, preferably an Octet Red96e molecular interaction device.
In some embodiments of the invention, the device, wherein, in step (2),
taking a SA (streptavidin) sensor, prewetting with PBS (phosphate buffered saline), 0.02% Tween-20 and pH7.4 for 10min; using Octet Red96e, the sensor was immersed in biotinylated CD47, curing the biotinylated CD47 onto the SA sensor with a curing signal height of 0.3nm.
In some embodiments of the invention, the device, wherein step (3) is performed by a molecular interaction device, preferably an Octet Red96e molecular interaction device.
In some embodiments of the invention, the device, wherein step (3), further comprises the step of immersing the device in PBS,0.02% Tween-20,0.1% BSA, pH7.4, preferably for a period of 2-20 seconds, preferably 2-10 seconds, 3-8 seconds, or 5 seconds. Preferably, step (3) is repeated at least 1 time, e.g. 1-5 times, 1 time, 2 times or 3 times.
In some embodiments of the invention, the device, wherein in step (3), further comprising the step of immersing the device in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for equilibration; preferably, the time is 30-120 seconds, preferably 30-90 seconds, 40-80 seconds, 50-70 seconds or 60 seconds.
In some embodiments of the invention, the device, wherein step (4) is performed by a molecular interaction device, preferably an Octet Red96e molecular interaction device.
In some embodiments of the invention, the device, wherein, in step (4),
CD47 immobilized on the sensor surface was pre-bound with 0-30nM of anti-CD 47 antibody and the sensor was regenerated using 2M magnesium chloride (to remove pre-bound anti-CD 47 antibody).
In some embodiments of the invention, the device, wherein step (2) and step (3) are performed by a molecular interaction device, preferably an Octet Red96e molecular interaction device.
In some embodiments of the invention, the device, wherein step (2) and step (4) are performed by a molecular interaction meter, preferably an Octet Red96e molecular interaction meter.
In some embodiments of the invention, the device, wherein step (3) and step (4) are performed by a molecular interaction meter, preferably an Octet Red96e molecular interaction meter.
In some embodiments of the invention, the device, wherein step (2), step (3) and step (4) are performed by a molecular interaction device, preferably an Octet Red96e molecular interaction device.
Preferably, step (4) is repeated at least 1 time, e.g. 1-5 times, 1 time, 2 times, 3 times, 4 times or 5 times.
Another aspect of the invention relates to a method for detecting the affinity of an anti-CD 47 antibody for a CD47 molecule (using K D Represented) by using the device of any one of the present invention.
In some embodiments of the invention, the method comprises the steps of:
1) Immersing the device in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for equilibration for 30-120 seconds, preferably 30-90 seconds, 40-80 seconds, 50-70 seconds or 60 seconds;
2) Immersing the device in a sample to be tested (preferably at a concentration of 0-30 nM) to bind the CD47 immobilized on the sensor surface to the anti-CD 47 antibody for 30-120 seconds, preferably 30-90 seconds, 40-80 seconds, 50-70 seconds or 60 seconds;
3) Immersing the device in PBS,0.02% Tween-20,0.1% BSA, pH7.4, allowing the anti-CD 47 antibody bound to CD47 to dissociate in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 90-240 seconds, preferably 90-150 seconds, 100-140 seconds, 110-130 seconds or 120 seconds;
preferably, the method also comprises the step 4),
4) Regenerating the device with 1-5M magnesium chloride solution; preferably, 2-3M magnesium chloride solution is used for regeneration; preferably, the regeneration time is 2-20 seconds, preferably 2-10 seconds, 3-8 seconds or 5 seconds.
In some embodiments of the invention, the method, wherein any 1 step, any 2 steps, any 3 steps, or all 4 steps of step 1), step 2), step 3), and step 4) are performed by a molecular interaction device, preferably an Octet Red96e molecular interaction device.
In some embodiments of the invention, the method comprises the steps of:
I. experiments were performed using Octet Red96e, 1) the sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 60s for equilibration;
immersing the sensor in 0-30nM of anti-CD 47 antibody, and combining the CD47 solidified on the surface of the sensor with the anti-CD 47 antibody for 60s;
III. the sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4, and the anti-CD 47 antibody bound to CD47 was dissociated in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 120s;
the sensor was regenerated using 2M magnesium chloride.
Preferably, step 4) is repeated at least 1 time, e.g. 1-5 times, 1 time, 2 times, 3 times, 4 times or 5 times.
In the present invention, when referring to the amino acid sequence of the CD47 protein or CD47 (NCBI GenBank: NP 942088.1), it includes the full length of the CD47 protein, or the V region of CD47, i.e., the region that binds to an anti-CD 47 antibody or a fragment comprising the V region; also included are full length fusion proteins of the CD47 protein or fusion proteins of the V region, e.g., fragments fused to His tags. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced in the amino acid sequence of the CD47 protein without affecting its biological function. Thus, in the present invention, the term CD47 protein shall include all such sequences, including natural or artificial variants thereof. And, when describing a sequence fragment of the CD47 protein, it also includes the corresponding sequence fragment in its natural or artificial variant.
In the present invention, the term "regeneration" refers to the process of removing bound analyte from a ligand after the end of a sample analysis. Efficient regeneration, i.e. removal of bound analyte without affecting ligand activity. Incomplete regeneration or reduced binding activity of the sensor surface ligands after regeneration, and the assay process is compromised.
In the present invention, the term "K D "refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. K can be determined using methods known to those skilled in the art D Determination of K, e.g. using an Octet molecular interaction apparatus D 。
In the present invention, the term "continuous use", "10 times continuous use", "15 times continuous use", and the like, wherein "continuous" means that the time interval between two uses does not cause failure or degradation of the device, for example, the time interval between two uses is not more than 48 hours, not more than 36 hours, not more than 24 hours, not more than 12 hours, not more than 6 hours, not more than 3 hours, not more than 2 hours, not more than 1 hour, not more than 30 minutes, not more than 20 minutes, not more than 15 minutes, not more than 10 minutes, or not more than 5 minutes.
Advantageous effects of the invention
The invention achieves any one or more of the following technical effects (1) - (3):
(1) The CD47 can be effectively regenerated for multiple times after being solidified to the SA sensor, the re-fixation is not needed, the disposable use of the sensor is reduced, and the time for fixing the protein (CD 47) is saved;
(2) The sensor has good repeatability, and even if the sensor is repeatedly used for 10 times or even 15 times, the CV value of affinity can be less than 5 percent, so that the sensor is suitable for rapid detection of multiple samples; whereas conventional capture-type sensors typically repeat 10 times with affinities having CV values greater than 5%, even greater than 10% or 20%.
(3) The detection time is effectively shortened, and only about 5 minutes is needed in a single detection process.
Drawings
Fig. 1: affinity detection of anti-CD 47 antibodies to CD 47. The curves in the figure represent the binding curves of AK117 at different concentrations to CD47 immobilized on the sensor surface, and the curves represent the concentrations of AK117 from 30nM, 10nM, 3.33nM, 1.11nM, 0.37nM, respectively, from top to bottom. The light curve in the figure represents the measured curve, the dark curve represents the fitted curve, and the two curves substantially coincide.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Major instrument and device information:
molecular interaction instrument (manufacturer: sartorius, model: octet Red96 e);
a micro-spectrophotometer (manufacturer: thermo, model: nanodrop 2000);
centrifuge (manufacturer: eppendorf, model: 5418);
analytical balance (manufacturer: METTER TOLEDO, model: AL 104).
Major reagent, consumable information:
SA sensor (manufacturer: sartorius, cat# 18-5019);
the anti-CD 47 antibody AK117 (manufacturer: kang Fang organism) AK117 sequences can be found in the heavy and light chain sequences of 6F7H1L1 (hG 4) of China patent CN 112442123A.
CD47 IgV TEV-His, which includes genbank id: NP942088.1 position: the fusion protein of the 19-141 human CD47 mature peptide and TEV (ENLYFQG) -his label is synthesized by the biological medicine company of Zhongshan Kang Fang;
2M magnesium chloride (self-formulated) solution;
PBS,0.02% Tween-20,0.1% BSA pH7.4 (self-formulated);
PBS,0.02% Tween-20, pH7.4 (self-formulated);
desalting column (manufacturer: thermo, cat# 89882);
biotin (manufacturer: thermo, cat. No. 21335).
Example 1
Experimental methods
CD47 biotinylation
1.1. 1mM biotin was prepared by dissolving biotin in PBS,0.02% Tween-20, pH 7.4.
1.2. The molar ratio of CD47 to biotin is 1:1. The mixture was left at room temperature for half an hour.
1.3. The sample was desalted using a desalting column.
1.4. The samples were tested for concentration using a micro-spectrophotometer.
CD47 sensor preparation
2.1. A series of 6 SA sensors were immersed in PBS,0.02% Tween-20, pH7.4 (prewetted solution) for 10min.
2.2. Experiments were performed using Octet Red96 e:
2.2.1. the sensor was immersed in PBS,0.02% Tween-20, pH7.4 for 60s for equilibration.
2.2.2. The sensor was immersed in 0.5mg/mL biotinylated CD47, and the biotinylated CD47 was immobilized onto the SA sensor with an immobilized signal height of 0.3nm.
2.2.3. The sensor was immersed in PBS,0.02% Tween-20, pH7.4 for 60s for equilibration.
2.3. The sensor pre-wet solution was replaced with PBS,0.02% Tween-20,0.1% BSA, pH7.4, and pre-wet for 10min.
2.4. Experiments were performed using Octet Red96 e:
2.4.1. the sensor was immersed in 2M magnesium chloride for 5s; the sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 5s. This step was repeated 3 times.
2.4.2. The sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 60s for equilibration.
2.4.3. The sensor was immersed in AK117 at 30nM and CD47 cured on the sensor surface was pre-bound to AK117 for 60s.
2.4.4. The sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 120s for equilibration.
2.4.5. The sensor was immersed in 2M magnesium chloride for regeneration for 5s and the sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for equilibration for 5s. This step was repeated 4 times.
3. Affinity detection
3.1. A row of 6 SA sensors of cured CD47 was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for prewetting.
3.2. Experiments were performed using Octet Red96 e:
3.2.1. the sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 60s for equilibration.
3.2.2. The sensor was immersed in AK117 solution (zero concentration: PBS,0.02% Tween-20,0.1% BSA, pH 7.4) of 30nM, 10nM, 3.33nM, 1.11nM, 0.37nM, 0nM, respectively, and the CD47 immobilized on the sensor surface was allowed to bind to AK117 for 60s.
3.2.3. The sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4, and AK117 bound to CD47 was dissociated in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 120 seconds.
3.2.4. The sensor was immersed in 2M magnesium chloride for regeneration for 5s and the sensor was immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for equilibration for 5s. This step was repeated 4 times.
(II) results of experiments
The results were analyzed using a 1:1 fitting model to obtain affinity results. The experimental results are shown in table 1 and fig. 1.
Table 1: AK117 and CD47 affinity constant detection results
| K D (M) | kon(1/Ms) | kdis(1/s) |
| 3.82E-10 | 1.65E+06 | 6.31E-04 |
AK117 has an affinity for CD47 of 3.82E-10M. It was demonstrated that AK117 can be detected by the present invention with a stronger affinity for CD 47.
Example 2
Experimental methods
CD47 sensors were prepared according to the methods of "1" section and "2" section in example 1, AK117 antibodies were repeatedly tested for affinity constant for CD47 15 times according to the method of "3" section in example 1, with fresh PBS,0.02% tween-20,0.1% bsa, ph7.4 equilibration/dissociation solution and AK117 antibody samples each time 5 changes were made.
(II) results of experiments
The results were analyzed using a 1:1 fitting model to obtain affinity results. The experimental results are shown in Table 2.
Table 2: AK117 and CD47 affinity constant detection reproducibility results
The experimental results show that the affinity K of 15 experiments D CV values for kon, kdis and highest concentration binding signal values were all within 5%. The CD47 sensor prepared by the invention can be repeatedly regenerated and repeatedly used by adopting 2M magnesium chloride, 15 samples are repeatedly detected, and the CD47 solidified on the sensor can always keep good activity, so that good repeatability of an affinity result is ensured. In addition, the inventionThe method has the characteristics of simple and convenient operation and short detection time, and is suitable for rapid detection of multiple samples.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (10)
1. An apparatus for detecting an anti-CD 47 antibody, comprising:
the sensor and the CD47 molecule are used,
the CD47 molecules are combined to the surface of the sensor to form a biological film layer, and the solidifying signal height of the biological film is 0.2-0.4nm, preferably 0.3nm.
2. The device of claim 1, wherein the CD47 molecule is bound to the sensor surface by a biotin-streptomycin affinity system, an amino-amino coupling system, or a his tag-anti-his tag antibody system.
3. The device according to any one of claims 1 to 2, which is capable of regeneration by a 1-5M magnesium chloride solution;
preferably, the device is capable of regeneration by 2-3M magnesium chloride solution;
preferably, the regeneration time is 2-20 seconds, preferably 5 seconds.
4. A device according to any one of claims 1 to 3, wherein the sensor is one selected from SA sensors, AR2G sensors and APS sensors.
5. The apparatus according to any one of claims 1 to 4, wherein,
the device is capable of being used at least 10 times in succession, and the CV value of the affinity for 10 successive uses is less than 5%;
preferably, the device is capable of being used at least 15 times in succession, and the CV value of the affinity for 15 successive uses is less than 5%.
6. The device according to any one of claims 1 to 5, wherein the device is manufactured by a method comprising the steps of:
(1) Obtaining biotinylated CD47;
(2) Solidifying the biotinylated CD47 onto a sensor (preferably a SA sensor);
preferably, the method also comprises the step (3),
(3) Immersing the product of step (2) in 1-5M magnesium chloride solution; preferably 2-3M magnesium chloride solution; preferably, the time of immersion is kept between 2 and 20 seconds, preferably 5 seconds;
preferably, the method also comprises the step (4),
(4) Pre-combining with a sample to be detected, and regenerating by using 1-5M magnesium chloride solution; preferably, 2-3M magnesium chloride solution is used for regeneration; preferably, the regeneration time is 2-20 seconds, preferably 5 seconds.
7. The apparatus of claim 6, wherein, in the step (1),
biotin reagent and CD47 at 1:2 to 2:1 (preferably 1:1 molar ratio), and reacting at room temperature for 10-60 minutes (preferably 30-40 minutes);
preferably, desalination is performed using a desalination column;
preferably, the concentration of the sample to be measured is detected using a micro-spectrophotometer.
8. The apparatus according to any one of claims 6 to 7, wherein in step (2),
taking a sensor, using PBS (phosphate buffered saline), 0.02% Tween-20, pH7.4 as a pre-wetting solution, and pre-wetting for 5-15 minutes (preferably 10 minutes);
immersing the sensor in biotinylated CD47, allowing the biotinylated CD47 to solidify on the sensor with a solidification signal height of 0.2-0.4nm (preferably 0.3 nm);
preferably, the pre-wet is replaced with PBS,0.02% Tween-20,0.1% BSA, pH7.4, pre-wet for 5-15 minutes (preferably 10 minutes).
9. A method of detecting the affinity of an anti-CD 47 antibody to a CD47 molecule by measuring the affinity using the device of any one of claims 1 to 8.
10. The method of claim 9, comprising the steps of:
1) The device is immersed in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for equilibration for 30-120 seconds, preferably 60 seconds;
2) Immersing the device in a sample to be tested (preferably at a concentration of 0-30 nM) to bind the immobilized CD47 on the sensor surface to the anti-CD 47 antibody for 30-120 seconds, preferably 60 seconds;
3) Immersing the device in PBS,0.02% Tween-20,0.1% BSA, pH7.4, allowing the anti-CD 47 antibody bound to CD47 to dissociate in PBS,0.02% Tween-20,0.1% BSA, pH7.4 for 90-240 seconds, preferably 120 seconds;
preferably, the method also comprises the step 4),
4) Regenerating the device with 1-5M magnesium chloride solution; preferably, 2-3M magnesium chloride solution is used for regeneration; preferably, the regeneration time is 2-20 seconds, preferably 5 seconds.
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