Method for rapidly determining relative affinity level of SRBC specific antibody in SRBC-immunized animal model

Abstract

The invention discloses a method for rapidly determining the relative affinity level of an SRBC specific antibody in an SRBC-immunized animal model. The method comprises the following steps: incubating an SRBC antigen and a sample under different elution conditions, and detecting the trend that the binding level of the SRBC antigen and an SRBC specific antibody in the sample is reduced along with the change of the elution conditions by using a flow cytometry, so as to obtain the relative affinity level of the antibody in the sample. The method has the advantages of simple and rapid operation, safe process, low cost, reliable data and accurate result.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and relates to a method for rapidly determining the relative affinity level of SRBC-specific antibodies in an animal model immunized with SRBC. Background Art

[0002] Sheep red blood cells (SRBC) are classic T cell-dependent (TD) antigens with a long history. Due to their low price, easy availability, and ability to trigger strong antibody immune responses, they are still widely used in basic research on humoral immunity, vaccine development, and other related fields. However, it is currently difficult to detect the affinity level of specific antibodies in an animal model immunized with SRBC. The existing methods for detecting the affinity level are the indirect plaque formation method that relies on cell culture, or the hemolysin transfer method based on radiolabeled SRBC with safety risks. These methods are cumbersome and not easy to operate, and have not been widely applied. Therefore, we hope to explore a simple and efficient method for detecting the affinity level of SRBC-specific antibodies. Although the method for detecting the titer level of SRBC-specific antibodies using the enzyme-linked immunosorbent assay (ELISA) method has been relatively mature, there is no affinity detection method based on the ELISA platform. The preparation process of membrane antigens in the ELISA protocol is cumbersome and complex, and the commercial SRBC antibody ELISA detection kit is not designed for detecting antibody affinity and is costly. Therefore, we did not design and explore an affinity detection method based on the ELISA platform. Using flow cytometry can avoid the disadvantages of the above ELISA platform, such as the need for antigen preparation and high cost. By using flow cytometry, we established a new detection system that can relatively quickly and accurately detect the titer and affinity level of SRBC-specific antibodies in body fluid samples (such as serum, tissue fluid) of an animal model immunized with SRBC (such as mice). We further verified this method using affinity maturation-deficient mice, and the results showed that this method can further distinguish the high and low affinity based on the detection of the titer level of SRBC-specific antibodies. This method is simple and efficient, and can provide a new accurate and efficient technical solution for research on humoral immunity, antibody affinity maturation, vaccine response mechanism, etc. Summary of the Invention

[0003] The object of the present invention is to provide a method for rapidly determining the relative affinity level of SRBC-specific antibodies in an animal model immunized with SRBC in view of the above deficiencies of the prior art.

[0004] The object of the present invention can be achieved by the following technical solutions:

[0005] A method for rapidly determining the relative affinity level of SRBC-specific antibodies in an animal model immunized with SRBC, comprising the following steps:

[0006] (1) Detect the titer curves of all samples to be compared using flow cytometry. Then, select a signal intensity value within the range where the dilution factor and signal intensity are linearly correlated among all samples as the common binding signal fluorescence intensity value (CBFI), and obtain the dilution factor (DF) value corresponding to the CBFI of each sample ( Figure 2 ). Among them, the samples are from the body fluids of animals immunized with SRBC.

[0007] (2) Relative affinity value (RAV) detection: Starting from the dilution factor (DF) corresponding to the same CBFI of the sample to be tested, add glycine-HCl incubation solutions with different gradients for dilution and incubation. After incubation, detect using flow cytometry. Plot a graph of the antibody fluorescence intensity against the glycine concentration, and use the negative reciprocal of the slope of the linear fitting line of the antibody fluorescence intensity against the glycine concentration as the relative affinity value (RAV) to characterize the relative affinity level ( Figure 3 ).

[0008] As a preference of the present invention, the body fluids of animals immunized with SRBC are the sera and tissue fluids of animals immunized with SRBC, preferably the sera of mice immunized with SRBC.

[0009] As a preference of the present invention, the method for detecting the titer curve using flow cytometry in step (1) is: Incubate SRBC with the sample to be tested. The sample contains SRBC-specific antibodies that can bind to SRBC. Then, add a fluorescent dye-labeled antibody that specifically recognizes mouse IgG to further label these bound antibodies. The fluorescent dye is excited by the laser of the flow cytometer to generate a fluorescent signal. The intensity of the fluorescent signal can reflect the number of antibodies bound to the surface antigen of the measured SRBC cells, thereby enabling the identification of whether there are SRBC-specific antibodies in the sample and the degree of binding. The flow chart is as Figure 1 shown.

[0010] As a preference of the present invention, the common binding signal fluorescence intensity value (CBFI) is selected as a value in the middle part of the linearly correlated range to avoid errors at both ends. The error at the high signal end may come from the saturation of low-titer samples; the error at the low signal end may come from the too-small reading window caused by too low a signal.

[0011] As a preference of the present invention, the specific steps of step (1) are as follows: Take sheep red blood cells in a centrifuge tube, centrifuge at 4°C to discard the supernatant, resuspend with PBS, wash several times, resuspend according to the required amount of 100 μl per well, and add to a 96-well U-bottom plate to ensure the same cell amount is added to each well; Dilute the sample, add it to the prepared 96-well plate coated with SRBC, mix evenly with SRBC, and incubate at 4°C for 15 - 20 minutes; After incubation, centrifuge, discard the supernatant, resuspend with PBS per well, and centrifuge again. Repeat the above operation once to wash away the excess antibodies in the sample that did not bind to the antigen SRBC; The sample wells are stained with a fluorescent dye-labeled antibody that specifically recognizes a specific antibody subtype of the immunized species (in the examples of the present invention, rabbit anti-mouse IgG(H+L) AF488 flow antibody is used) diluted at a ratio of 1:400 - 600, and incubated in the dark at 4°C for 15 - 20 minutes. After incubation, repeat the above washing operation to wash away the excess antibody, and the cell pellet remaining in the plate is resuspended with flow cytometry buffer, 200 μl per well, and detected using a flow cytometer. Plot the titer curves for all samples to be compared, and then select a signal intensity value within the range where the dilution factor and signal intensity are linearly correlated among all samples as the CBFI, and obtain the corresponding dilution factor DF value for each sample's CBFI. Detect each sample through this step to obtain the respective DF values corresponding to the specific CBFI for each sample.

[0012] As a preference of the present invention, the flow cytometry buffer is FACS buffer, and the PBS contains 2% fetal bovine serum and 1 mM EDTA.

[0013] As a preference of the present invention, the glycine-HCl incubation solution is obtained by mixing physiological saline and 0.1 M glycine-HCl eluent with pH 2.7 at volume ratios of 399:1, 199:1, 132:1, and 99:1 respectively to obtain glycine-HCl incubation solutions with gradually decreasing pH values and glycine concentrations of 0.25 mM, 0.5 mM, 0.75 mM, and 0.1 mM ( Figure 4 ).

[0014] As a preference of the present invention, the specific steps of step (2) are as follows: taking the dilution multiple corresponding to the same CBFI value of the sample as the starting point, adding it to glycine-HCl sample incubation solutions with different gradients for dilution respectively, then transferring it to a prepared 96-well plate coated with SRBC to completely resuspend the cell precipitate, and incubating at 4 °C for 15-20 minutes; after the incubation, centrifuging, discarding the supernatant, adding 200 μl of glycine-HCl incubation solution with the same concentration as that during incubation to each well, resuspending and then centrifuging, repeating the above operation once to wash away the excess antibodies in the sample that cannot bind to the antigen SRBC due to the acidic environment; using a fluorescent dye-labeled antibody that specifically recognizes a specific antibody subtype of the immunized species (in the examples of the present invention, the used is rabbit anti-mouse IgG(H+L) AF488 flow antibody), diluting it according to a ratio of 1:500 for staining, and incubating in the dark at 4 °C for 15-20 minutes. After the staining, repeating the above cleaning operation to wash away the excess antibodies, the difference is that the cleaning solution is replaced by physiological saline from the glycine-HCl incubation solution. Finally, resuspending the cell precipitate with physiological saline, 200 μl per well, and detecting with a flow cytometer. After the detection, plotting a curve of the antibody fluorescence intensity against the glycine concentration, and taking the negative reciprocal of the slope of the linear fitting line of the antibody fluorescence intensity against the glycine concentration as the relative affinity value RAV.

[0015] Principle of the present invention:

[0016] This method relies on flow cytometry. SRBC is incubated with the sample to be tested. The serum sample after immunization with SRBC contains SRBC-specific antibodies, which can bind more to SRBC. Then, a fluorescent dye-labeled antibody that specifically recognizes a specific antibody subtype (such as IgG) of the immunized species (in the examples of the present invention, the used is rabbit anti-mouse IgG(H+L) AF488 antibody) is used to further label these bound antibodies. The fluorescent dye is excited by the laser of the flow cytometer to generate a fluorescent signal, and the intensity of the fluorescent signal can reflect the number of antibodies bound to the antigen on the surface of the measured SRBC cells. Thus, it can be determined whether there are SRBC-specific antibodies in the sample and the degree of binding. For a specific sample, it is serially diluted, and the above flow cytometry detection is performed on each dilution of the sample to obtain Figure 2 the titer curve in; obtaining the dilution factor (DF) corresponding to the common binding signal fluorescence intensity value (CBFI) (which needs to be within the linear range) subjectively selected by an operator from the curve. Performing the detection of step one on each sample to obtain the respective DF values of each sample corresponding to a specific CBFI.

[0017] In the curve range where the dilution degree is linearly corresponding to the fluorescence intensity of the antibody, when different samples reach the same CBFI value, it indicates that the same amount of antibody binds to SRBC. On this basis, the acidity of the incubation solution is gradually increased, making the solution environment gradually less conducive to the binding of antibodies in the samples to SRBC. For samples with strong antibody affinity, the signal decreases slowly with the increase of acidity, which is reflected by a smaller absolute value of the slope of the linear fitting line; for samples with weak antibody affinity, the signal decreases rapidly with the increase of acidity, which is reflected by a larger absolute value of the slope of the linear fitting line. Using the negative reciprocal of the slope as the characterization of the relative affinity level is called the relative affinity value (RAV):

[0018] RAV = -1 / slope

[0019] The present invention utilizes the characteristic of flow cytometry that can identify and detect single cells, uses intact SRBC cells with natural antigen expression on the surface, and detects the titer level of SRBC-specific antibodies in the body fluids of animals immunized with SRBC (in the present invention, mainly the sera of mice immunized with SRBC are used). Based on this, a method for detecting the affinity level of these antibodies is designed.

[0020] The present invention fixes the starting point of affinity detection by setting the common binding signal fluorescence intensity value (CBFI). Different samples reach the same starting point of affinity detection through their respective dilution factors (DF) corresponding to CBFI, that is, at this time, the same number of antibodies bind to SRBC cells, thereby excluding the influence of the number of specific antibodies in the samples and being able to specifically detect the affinity level of these antibodies.

[0021] The present invention has explored the conditions for detecting the affinity level: a series of incubation solutions with gradually decreasing pH values are prepared by using glycine-HCl eluent with gradually increasing concentration and normal saline. In these incubation solutions, each sample uses the same CBFI starting point. Due to different affinities of different samples, the slopes of the signal changes with the concentration of the incubation solution are also different (using the negative reciprocal of the slope to represent the relative affinity RAV), so that the relative levels of affinity can be compared among different samples.

[0022] Beneficial effects:

[0023] (1) Simple, rapid and safe operation. This method only requires basic laboratory conditions (ordinary reagents and consumables) and a flow cytometer that has been widely popularized and used, and can be completed. The operation and analysis time for one experiment is about 2 hours / sample, and high-throughput operation of multiple samples can be achieved through 96-well plates and multi-channel pipettes, realizing an efficiency of 5 hours / 10 samples. At the same time, this method does not involve the use of toxic, harmful and radioactive reagents and instruments. Therefore, it has the advantages of simplicity, high efficiency and safety.

[0024] (2) Low cost. The SRBC used in this method is a commercially available product with long-term stable supply. The cost of SRBC for detecting 96-well samples is only a few yuan; this method only requires a common 96-well U-bottom plate instead of a specially treated adsorption-type 96-well plate, and the consumable cost is also low; flow cytometry experimental platforms are available and widely used in most biomedical research institutions, enterprises, hospitals, etc. This method requires a short operation time and low cost.

[0025] (3) Reliable data and accurate results. In Examples 2 and 3, we verified the accuracy of this detection method using two samples with different known affinity levels. The results showed that the relative affinity data between samples compared by the method of the present invention are reliable and the results are accurate. Description of the Drawings

[0026] Figure 1 Schematic diagram of flow cytometry for detecting SRBC-specific antibodies

[0027] Figure 2 Schematic diagram of using flow cytometry to detect the titer level of SRBC-specific antibodies in a sample, so as to determine the dilution factor (DF) required to reach CBFI

[0028] Figure 3 Schematic diagram of relative affinity value (RAV) detection

[0029] Figure 4 Glycine-HCl gradient incubation solution

[0030] Figure 5 Relative affinity levels of SRBC-immunized wild-type (WT) mouse antiserum. A, SRBC immunization protocol for mice; B, titer curves of antiserum at each time point; C, affinity detection curves of samples at each time point when CBFI is 2000; D, linear fitting lines of sample affinity data at each time point in C; E, relative affinity level values (RAV) of samples at each time point obtained from the lines in D. N (number of samples in each group) = 4 - 8; One-way ANOVA was used for statistical significance of inter-group differences, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

[0031] Figure 6Relative affinity levels of anti - sera from ShipΔB mice immunized with SRBC. A, B, C, D, and E are the test results of serum samples on the 14th, 21st, 28th, 35th, and 42nd days after SRBC immunization, respectively; on the left, middle, and right of each figure are the titer curves of the samples at the corresponding time points, the linear fitting lines of the affinity test data, and the calculation results of the RAV value. N (number of samples in each group) = 3 - 8; the t - test was used for statistical significance of differences between groups, *, p < 0.05; **, p < 0.01 Detailed implementation manners

[0032] Example 1

[0033] Take the total required amount of sheep red blood cells (SRBC) at a standard of 2 * 10^7 per well in a 15 - ml centrifuge tube, centrifuge at 600g for 5 minutes at 4°C, discard the supernatant, add 10 ml of PBS to resuspend thoroughly, wash, centrifuge again under the same conditions, discard the supernatant, resuspend according to the required amount of 100 μl per well, and add to a 96 - well U - bottom plate to ensure the same cell amount is added to each well.

[0034] Dilute the samples according to the experimental requirements, add them to the above - prepared 96 - well plate coated with SRBC, mix well with SRBC, and incubate at 4°C for 20 minutes.

[0035] After 20 minutes, centrifuge at 600g for 5 minutes at 4°C, discard the supernatant, add 200 μl of PBS to each well, gently mix and resuspend, and centrifuge again. Repeat the above operation once. The main purpose is to wash away the excess antibodies in the samples that did not bind to the antigen SRBC.

[0036] Stain the sample wells with rabbit anti - mouse IgG (H + L) AF488 antibody at a ratio of 1:500. Incubate in the dark at 4°C for 20 minutes.

[0037] After incubation, repeat the washing operation to wash away the excess antibodies to remove the excess flow - through antibodies.

[0038] Resuspend the cell pellet remaining in the plate with flow - cytometry buffer (FACS buffer, PBS containing 2% fetal bovine serum, 1 mM EDTA), 200 μl per well, and detect using a flow cytometer.

[0039] Detect the titer curves of all samples to be compared, and then select a signal - intensity value within the range where the dilution factor and signal intensity are linearly correlated in all samples as the CBFI. Meeting this standard is fine; but the optimal CBFI is preferably in the middle part of the linear - correlation range to avoid errors at both ends: the error at the high - signal end may come from the saturation of low - titer samples; the error at the low - signal end may come from too small a reading window due to too low a signal.

[0040] Mix normal saline (0.9% NaCl) with glycine-HCl eluent (0.1 M, pH 2.7) in the following ratios to prepare glycine-HCl sample incubation solutions with different pH gradients: the ratios of normal saline to glycine-HCl eluent are 399:1, 199:1, 132:1, and 99:1 respectively, resulting in glycine concentrations of 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM, and the pH values gradually decrease (as Figure 4 shown).

[0041] Dilute the samples starting from the same point, i.e., the CBFI value, and add them to the prepared different-gradient glycine-HCl sample incubation solutions respectively (the volume of the incubation solution used is 100 μl per well, and the samples are diluted according to the DF value corresponding to CBFI at this volume). Mix well, and then transfer them to a 96-well plate coated with SRBC. Gently pipette to completely resuspend the cell precipitate and incubate at 4°C for 15 minutes.

[0042] After incubation, centrifuge at 600 g for 5 minutes at 4°C, discard the supernatant, add 200 μl of glycine-HCl sample incubation solution with the same concentration as during incubation to each well, gently mix and resuspend, and centrifuge again. Repeat the above operation once. The main purpose is to wash away the excess antibodies in the samples that cannot bind to the antigen SRBC due to the acidic environment.

[0043] Use rabbit anti-mouse IgG (H+L) AF488 flow antibody, dilute it at a ratio of 1:500 for staining, and incubate in the dark at 4°C for 20 minutes.

[0044] After staining, repeat the above washing operation to wash away the excess antibodies. The difference is that the washing solution is all normal saline. The main purpose is to wash away the excess flow antibody to avoid false positive signals.

[0045] Finally, resuspend the cell precipitate with normal saline, 200 μl per well, and use a flow cytometer for detection. After the detection, plot a curve of the antibody fluorescence intensity against the glycine concentration, and use the negative reciprocal of the slope of the linear fitting line of the antibody fluorescence intensity against the glycine concentration as the relative affinity value (RAV).

[0046] Example 2

[0047] After immunizing animals with a known antigen, the affinity of the antibodies produced will gradually increase over time; especially after booster immunization, the affinity level will increase significantly [3]. Based on this, we designed an immunization protocol, as Figure 5As shown in A, the first immunization was carried out on day 0, and the second immunization was carried out on day 28. Blood was taken weekly from the second week (day 14) after immunization to prepare serum until the sixth week after the first immunization (the second week after the second immunization, day 42 in total). The affinity of SRBC-specific antibodies in these sera was detected using the method of Example 1. Figure 5 B is the titer curve. The DF value corresponding to each sample when the CBFI value is 2000 was obtained from the titer curve. Using these DF values, the detection starting point of each sample was set to the same CBFI (2000 in this experiment), and the affinity of each sample was detected. The results are as Figure 5 shown in C. The affinity levels from low to high are days 14, 21, 28, 35, and 42. Figure 5 The detection data of each sample in C were linearly fitted to obtain Figure 5 D; According to the formula in 3.2, the RAV value of each sample was obtained. As Figure 5 shown in E, the affinity level (RAV value) increased significantly with the increase of immunization time, and increased significantly after the second immunization, which was consistent with the previously reported results. These results indicate that the data of this method are reliable and the results are accurate, and it can accurately detect the relative affinity level of SRBC-specific antibodies in samples.

[0048] Example 3

[0049] A mouse model (ShipΔB) with characteristics of affinity maturation defect confirmed in a previous published paper of this laboratory [4] was used to further verify the accuracy of the method of the present invention. The method for measuring RAV was the same as that in Example 1 (since the antiserum titers at the three time points of the primary immunization of ShipΔB mice were relatively low, all samples at these three time points were detected using a CBFI value of 500). As Figure 6 shown, under the same immunization protocol, the mice with affinity maturation defect could not produce antibodies with gradually increasing affinity over time like wild-type mice. From day 28, the affinity of SRBC antibodies in the sera of ShipΔB mice was significantly lower than that of wild-type mice at the same time points.

[0050] These results indicate that the data of this method are reliable and the results are accurate, and it can accurately detect the relative affinity level of SRBC-specific antibodies in samples.

[0051] References:

[0052] 1. DeHeer, D.H. and T.S. Edgington, Estimation of relative antibody affinity at the level of the antibody-secreting cell during maturation of the immune response.

[0053] Cellular Immunology, 1975. 18(2): p. 466 - 475.

[0054] 2. Taliaferro, W.H., L.G. Taliaferro, and A.K. Pizzi, Avidity and intercellular

[0055] transfer of hemolysin. J Infect Dis, 1959. 105: p. 197 - 221.

[0056] 3. Kenneth Murphy, C.W., Janeway's immunobiology. 9th edition ed. 2017.

[0057] 4. Zhang, W., et al., Excessive CD11c(+)Tbet(+)B cells promote aberrant T(FH)

[0058] differentiation and affinity-based germinal center selection in lupus. Proc Natl Acad Sci U S A, 2019. 116(37): p. 18550 - 18560.

Claims

1. A method for rapidly determining the relative affinity level of SRBC-specific antibodies in an animal model of SRBC immunity, characterized in that: The following steps are involved: (1) All samples to be compared are tested for titer curves by flow cytometry, and then a signal intensity value in all samples that is within a range in which the dilution factor is linearly correlated with the signal intensity is selected as the common binding signal fluorescence intensity value CBFI, and the dilution factor DF value corresponding to the CBFI of each sample is obtained; wherein the sample is from the body fluid of an animal immunized with SRBC; (2) Relative affinity value (RAV) detection: The sample to be tested is diluted with glycine-HCl incubation solution of different gradients according to the dilution factor DF corresponding to the same CBFI as the starting point, and incubated. After the incubation, it is detected by flow cytometry, and the antibody fluorescence intensity and glycine concentration are plotted. The negative reciprocal of the slope of the linear fitting line between the antibody fluorescence intensity and the glycine concentration is taken as the relative affinity value (RAV), which is used to characterize the relative affinity level.

2. The rapid determination method according to claim 1, characterized in that The SRBC-immunized animal body fluid is the serum or tissue fluid of the SRBC-immunized animal, preferably the serum of the SRBC-immunized mouse.

3. The rapid determination method according to claim 1, characterized in that The method for titer curve detection using flow cytometry in step (1) is as follows: SRBC is incubated with a sample to be tested, the sample contains SRBC-specific antibodies that can bind to SRBC, and then fluorescent dye-labeled antibodies that specifically recognize specific antibody subtypes of the immunized species are added to further label these bound antibodies. The fluorescent dye is excited by the laser of the flow cytometer to generate a fluorescent signal. The intensity of the fluorescent signal can reflect the number of antibodies bound to the surface antigens of the SRBC cell membrane being tested, thereby determining whether SRBC-specific antibodies exist in the sample and the degree of binding.

4. The rapid determination method according to claim 1, characterized in that The common binding signal fluorescence intensity value CBFI selects a value in the middle part of the linear correlation range to avoid errors at both ends.

5. The rapid determination method according to claim 1, characterized in that: Step (1) The specific steps are as follows: take sheep red blood cells in a centrifuge tube, centrifuge at 4°C, discard the supernatant, resuspend in PBS, wash several times, resuspend in the required amount of 100 μl per well, add to a 96-well U-bottom plate, and ensure that the same amount of cells is added to each well; dilute the sample, add to the above-prepared 96-well plate with SRBC, mix evenly with SRBC, and incubate at 4°C for 15-20 minutes; centrifuge after the incubation, discard the supernatant, resuspend in PBS for each well, centrifuge again, repeat the above operation once, and wash away the excess antibodies in the sample that are not bound to the antigen SRBC; the sample wells use a specific recognition of the specific immunized species The fluorescent dye-labeled antibodies of antibody subtypes were stained at a dilution ratio of 1:400-600 and incubated at 4°C in the dark for 15-20 minutes. After the incubation, the above-mentioned washing operation to wash away the excess antibodies was repeated to wash away the excess flow antibodies. The cell pellets remaining in the plate were resuspended with flow cytometry buffer, 200 μl per well, and detected using a flow cytometer. Titer curves were drawn for all samples to be compared, and then a signal intensity value that was within the range of linear correlation between dilution factor and signal intensity in all samples was selected as the CBFI, and the dilution factor DF value corresponding to the CBFI of each sample was obtained.

6. The rapid determination method according to claim 5, characterized in that: The flow cell buffer is FACS buffer, and PBS contains 2% fetal bovine serum and 1 mM EDTA.

7. The rapid determination method according to claim 1, characterized in that: The glycine-HCl incubation solution is prepared by mixing physiological saline and 0.1M, pH 2.7 glycine-HCl eluent in volume ratios of 399:1, 199:1, 132:1, and 99:1 to obtain a glycine-HCl incubation solution with a gradually decreasing pH value and a glycine concentration of 0.25mM, 0.5mM, 0.75mM, and 0.1mM.

8. The rapid determination method according to claim 1, characterized in that: The specific steps of step (2) are as follows: the sample is diluted by adding different gradients of glycine-HCl sample incubation solution at the dilution multiple corresponding to the same CBFI value as the starting point, and then transferred to the prepared 96-well plate with SRBC, the cell pellet is completely resuspended, and incubated at 4°C for 15-20 minutes; after the incubation, centrifuge, discard the supernatant, add 200ul of glycine-HCl incubation solution of the same concentration as in the incubation to each well, resuspend and centrifuge, repeat the above operation once, and wash away the excess antibodies in the sample that cannot bind to the antigen SRBC due to the acidic environment; use specific recognition The fluorescent dye-labeled antibody of the specific antibody subtype of the immunized species was diluted at a ratio of 1:500 for staining, and incubated in the dark at 4°C for 15-20 minutes. After the staining, the above-mentioned washing operation to wash away the excess antibody was repeated. The difference was that the washing solution was replaced by normal saline instead of glycine-HCl incubation solution. Finally, the cell pellet was resuspended with normal saline, 200 μl per well, and detected by flow cytometry. At the end of the detection, the antibody fluorescence intensity and glycine concentration were plotted, and the negative reciprocal of the slope of the linear fitting line between the antibody fluorescence intensity and the glycine concentration was taken as the relative affinity value RAV.

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