A method for screening monoclonal antibody pairs that are not interfered by complement

Abstract

The present invention provides a method for screening a monoclonal antibody pair that is not interfered by complement, comprising the following steps: S10: obtaining a negative sample mixture; S20: adding an antigen to the negative sample mixture to obtain a test sample one; S30: heating the negative sample mixture and adding an antigen to obtain a test sample two; S40: adding complement to the test sample two to obtain a test sample three; S50: using a chemiluminescent reagent prepared by the monoclonal antibody pair to be selected, respectively performing immunochemiluminescence detection on the test sample one, the test sample two and the test sample three to obtain a test result one, a test result two and a test result three; S60: obtaining a monoclonal antibody pair that is not interfered by complement according to the test result one, the test result two and the test result three. The present invention solves the technical problem that immunodiagnosis is often interfered by complement, which affects the accuracy of immunodiagnosis, and achieves the technical effect that immunodiagnosis is not easily interfered by complement and improves the accuracy of immunodiagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, in particular to a method for screening monoclonal antibody pairs that are not interfered by complement. Background Art

[0002] Immunodiagnosis, especially the diagnostic technology based on double antibody sandwich, has developed rapidly in recent years due to its high sensitivity and strong specificity, such as enzyme-linked immunosorbent assay, immunochromatography, chemiluminescence detection technology, etc. In particular, immunochemiluminescence detection technology has gradually become the main clinical diagnostic technology due to its advantages such as high sensitivity, fast speed and automation. However, in clinical applications, immunodiagnosis is often affected by interferences in samples.

[0003] Interfering substances in the sample include rheumatoid factor (RF), heterophilic antibodies, autoantibodies, complement, biotin, blood lipids, bilirubin, etc. Among them, complement is a very important type of interfering substance.

[0004] At present, there is no easy and universal method to solve complement interference. Common methods, such as heating samples at 53°C for 10 minutes to inactivate complement, are difficult to implement clinically. Blocking the complement activation cascade reaction with metal chelators such as ethylenediaminetetraacetic acid (EDTA) and aminotriacetic acid (NTA) is limited by its own interference with antigen-antibody reaction or environmental issues. There are also recommendations to use poultry such as chicken IgY antibodies to replace antibodies from mouse, rabbit and other sources, which are not affected by complement interference, but poultry antibodies are currently rarely used and cannot solve the complement interference problem faced by a large number of immune diagnosis needs. Summary of the invention

[0005] The present invention solves the technical problem that immunodiagnosis is often interfered by complement, which affects the accuracy of immunodetection, reduces the probability of complement interference from the selection of diagnostic raw materials, and achieves the technical effect that immunodiagnosis is not easily interfered by complement, thereby improving the accuracy of immunodiagnosis.

[0006] To solve the above problems, the present invention provides a method for screening monoclonal antibody pairs that are not interfered by complement, comprising the following steps: S10: obtaining a negative sample mixture; S20: adding an antigen to the negative sample mixture to obtain a test sample one; S30: heating the negative sample mixture and adding an antigen to obtain a test sample two; S40: adding complement C1q to the test sample two to obtain a test sample three; S50: using a chemiluminescent reagent prepared from the monoclonal antibody pair to be selected, performing immunochemiluminescence detection on the test sample one, the test sample two and the test sample three, respectively, to obtain a test result one, a test result two and a test result three; S60: obtaining a monoclonal antibody pair that is not interfered by complement according to the test result one, the test result two and the test result three.

[0007] Compared with the prior art, the technical effect achieved by adopting this technical solution is as follows: the present invention provides a method for screening monoclonal antibody pairs that are not interfered with by complement, by heating the negative sample mixture, the complement thereof can be inactivated, and then the detection sample one is a non-inactivated sample, the detection sample two is an inactivated sample, an equal amount of the analyte is added to the inactivated and non-inactivated samples, the above samples are tested simultaneously with the selected antibody pairs, the changes in the test results after complement inactivation are calculated, and then human C1q is added to the inactivated and added analyte sample two, that is, the detection sample three, and the effect of C1q on the test results is tested again, thereby confirming the antibody pairs that are susceptible to complement interference, and at the same time screening out the antibody pairs that are not susceptible to complement interference. This method can screen out antibody pairs that are not susceptible to complement interference in the raw material selection stage of immunodiagnosis, and reduce the probability of complement interference in the development stage of immunodiagnosis products.

[0008] In one example of the present invention, S10 includes the following steps: S11: obtaining a negative sample; S12: determining the C1q content of the negative sample to obtain a test result; S13: classifying the negative sample according to the test result and obtaining a negative sample mixture.

[0009] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: by finely classifying negative samples according to their C1q content, a mixture of negative samples with different complement contents can be obtained, making the screening results more accurate and more meaningful.

[0010] In one example of the present invention, negative samples are classified and a negative sample mixture is obtained, which includes the following steps: S14: negative samples with a C1q content greater than 220 μg / mL are mixed and labeled as a high complement negative sample mixture, negative samples with a C1q content greater than 170 μg / mL and less than 220 μg / mL are mixed and labeled as a medium complement negative sample mixture, and negative samples with a C1q content less than 170 μg / mL are mixed and labeled as a low complement negative sample mixture.

[0011] Compared with the prior art, the technical effect achieved by adopting the technical scheme is as follows: a portion of the high complement negative sample mixture, the medium complement negative sample mixture, and the low complement negative sample mixture are each taken for experimental operation, so that the screening method of the present invention has higher accuracy and stronger contrast, and can obtain the detection results of each sample under different complement contents, and then screen out the antibody pairs that are not interfered by complement from the monoclonal antibody pairs to be selected for subsequent immunoassays.

[0012] In one embodiment of the present invention, the negative sample mixture is heated at a temperature of 50-55° C. for a heating time of 10-15 min.

[0013] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: by heating the negative sample mixture, its complement can be inactivated, and then the inactivated sample and the non-inactivated sample can be used as a control group to determine the antibody pairs susceptible to complement interference.

[0014] In one example of the present invention, S60 includes the following steps: S61: Calculate the change ratio based on test result one, test result two, and test result three. When the difference in the change ratio between test result one and test result two is less than 10%, and the difference in the change ratio between test result two and test result three is less than 10%, the monoclonal antibody pair to be selected is not affected by complement; S62: Obtain a monoclonal antibody pair that is not interfered by complement.

[0015] Compared with the prior art, the technical effect achieved by adopting the technical scheme is as follows: by comparing the measured values, when the difference in the change ratio between the test result one and the test result two is less than 10%, the monoclonal antibody pairs susceptible to complement interference can be confirmed; when the difference in the change ratio between the test result two and the test result three is less than 10%, the monoclonal antibody pairs not susceptible to complement interference can be further screened out. The method is simple, effective and highly accurate.

[0016] In one embodiment of the present invention, the added amounts of the negative sample mixture and the antigen in the test sample 1 and the test sample 2 are the same.

[0017] Compared with the prior art, the technical effect achieved by adopting this technical solution is as follows: test sample one is the control group of test sample two. To ensure the reliability of the experimental data, according to the control variable method, the two differ only in whether they are heated or not, and the negative sample mixture and the amount of antigen added are controlled to be the same, or the antigen is added to test sample one and test sample two in the same proportion.

[0018] In one example of the present invention, the following steps are included before S40: S31: collecting serum; S32: purifying C1q in the serum to obtain a crude pure product; S33: further purifying the crude pure product to obtain complement C1q.

[0019] Compared with the prior art, the technical effects achieved by this technical solution are: the coating of the capture antibody in the enzyme-linked immunosorbent assay, the coupling of the capture antibody with magnetic beads in the chemiluminescence assay, antibody labeling such as biotin labeling, acridinium ester labeling, fluorescent microsphere labeling, and antigen-antibody binding can all cause changes in the antibody conformation, thereby exposing the binding site of the FC segment complement C1q. When C1q binds to the sites on two or more antibody FCs, the complement system in the sample can be activated. C1q is an 18-mer complex with 6 antibody binding sites. When the C1q molecule binds to the capture antibody and the detection antibody at the same time, it leads to a false positive or false increase; C3b and C4b formed after the complement system is activated are both bound to the Fab of the antibody, thereby affecting the binding of the capture antibody or the detection antibody to the antigen, resulting in a false negative or false decrease, which will affect the accuracy of the immunoassay. Therefore, human serum C1q is purified to facilitate subsequent addition to the inactivated and negative sample mixture added with the analyte for detection to verify whether the selected monoclonal antibody will be interfered with by complement.

[0020] In one example of the present invention, S40 includes the following steps: S41: adding complement C1q to the test sample two to obtain the test sample three.

[0021] Compared with the prior art, the technical effect achieved by adopting this technical solution is as follows: by adding human C1q to the inactivated and tested samples, and again testing the effect of C1q on the test results, it is possible to further screen out antibody pairs that are not easily interfered by complement, thereby improving the accuracy of the screening results.

[0022] In one example of the present invention, the immunochemiluminescence detection includes the following steps: S44: preparing a luminescent reagent; S45: preparing a solid phase reagent; S46: mixing the luminescent reagent and the solid phase reagent with the test sample one, the test sample two, and the test sample three respectively, and detecting using a chemiluminescence immunoassay analyzer.

[0023] Compared with the existing technology, the technical effect achieved by adopting this technical scheme is as follows: using immunochemiluminescence to detect sample one, sample two, and sample three, recording their brightness values ​​and calculating the change ratio. If the measured value changes not significantly, the monoclonal antibody pair is not susceptible to complement interference. By comparing the data, the monoclonal antibody pairs that are not susceptible to complement interference are screened out, with higher accuracy, and the operation process is simple and easy to implement.

[0024] In one embodiment of the present invention, the C1q content of the negative sample is determined using a complement C1q diagnostic kit.

[0025] Compared with the existing technology, the technical effect achieved by adopting this technical solution is as follows: the complement C1q diagnostic kit has the advantages of being sensitive, rapid and simple in quantitative method, and improving screening efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the electrophoresis band diagram of the second round of purified human serum C1q provided in the embodiment of the present invention, wherein Lane 1 is 10ul of sample loading; Lane 2 is 5ul of sample loading; Lane 3 is the Marker.

[0027] Figure 2 This is a comparison chart of the detection results of the antibody pair 260-60 provided in the embodiment of the present invention.

[0028] Figure 3 This is a comparison chart of the detection results of the antibody pair 260-72 provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0030] [Example 1]

[0031] This embodiment takes the screening of anti-Mullerian hormone (AMH) monoclonal antibody pairs that are not interfered by complement as an example, and reduces complement interference in diagnostic raw materials, making the antibody pairs more suitable for chemiluminescent immunoassay.

[0032] Therefore, the present invention provides a method for screening monoclonal antibody pairs that are not interfered by complement, comprising the following steps:

[0033] S10: Obtain negative sample mixture;

[0034] S20: adding antigen to the negative sample mixture to obtain test sample 1;

[0035] S30: heating the negative sample mixture and adding antigen to obtain test sample 2;

[0036] S40: adding complement C1q to the test sample 2 to obtain the test sample 3;

[0037] S50: using the chemiluminescent reagent prepared by the selected monoclonal antibody pair, performing immunochemiluminescent detection on the test sample 1, the test sample 2, and the test sample 3, respectively, to obtain the test result 1, the test result 2, and the test result 3;

[0038] S60: According to the first test result, the second test result and the third test result, a monoclonal antibody pair that is not interfered by complement is obtained.

[0039] Specifically, the antigen is AMH antigen, and the preparation of AMH antigen includes the following steps: synthesizing the full-length (1-560AA) gene sequence of AMH, adding an 8×His tag to the C-terminus, inserting the pcDNA3.3 plasmid with 5'NheI and 3'EcoRI restriction sites to construct an expression vector. Conventional technical methods are used to transform 293T cells, and the full-length AMH antigen is obtained by affinity purification with a nickel column, and the concentration is determined by a Roche AMH electrochemiluminescence detection kit.

[0040] The present invention provides a method for screening monoclonal antibody pairs that are not interfered with by complement. By heating a negative sample mixture, its complement can be inactivated, and then the detection sample one is a non-inactivated sample, the detection sample two is an inactivated sample, an equal amount of the analyte is added to the inactivated and non-inactivated samples, and the above samples are tested simultaneously with the selected antibody pair, and the change in the test result after complement inactivation is calculated, and then human C1q is added to the inactivated and added sample to be tested, that is, the detection sample three, and the effect of C1q on the test result is tested again, thereby confirming the antibody pair that is susceptible to complement interference, and at the same time screening out the antibody pair that is not susceptible to complement interference. This method can screen out antibody pairs that are not susceptible to complement interference in the raw material selection stage of immunodiagnosis, and reduce the probability of complement interference in the development stage of immunodiagnosis products.

[0041] Furthermore, S10 includes the following steps:

[0042] S11: Obtain negative samples;

[0043] S12: Determine the C1q content of the negative sample and obtain the test result;

[0044] S13: According to the test results, the negative samples are classified and a negative sample mixture is obtained.

[0045] Specifically, samples of anti-Mullerian hormone-negative (females older than 55 years old) were collected, and the C1q content was measured using the complement C1q diagnostic kit (immunoenhanced turbidimetry) sold by Mecan Biotechnology Co., Ltd.

[0046] By finely classifying negative samples according to their C1q content, a mixture of negative samples with different complement contents can be obtained, making the screening results more accurate and more meaningful.

[0047] Further, classifying the negative samples and obtaining a negative sample mixture comprises the following steps:

[0048] S14: negative samples with C1q content greater than 220 μg / mL are mixed and labeled as a high complement negative sample mixture, negative samples with C1q content greater than 170 μg / mL and less than 220 μg / mL are mixed and labeled as a medium complement negative sample mixture, and negative samples with C1q content less than 170 μg / mL are mixed and labeled as a low complement negative sample mixture.

[0049] A portion of the high complement negative sample mixture, the medium complement negative sample mixture, and the low complement negative sample mixture are each taken for experimental operation, so that the screening method of the present invention has higher accuracy and stronger contrast, and can obtain the detection results of each sample under different complement contents, and then screen out the antibody pairs that are not interfered by complement from the monoclonal antibody pairs to be selected for subsequent immunoassays.

[0050] Further, the negative sample mixture is heated at a temperature of 50-55° C. for a heating time of 10-15 min.

[0051] By heating the negative sample mixture, its complement can be inactivated, and then the inactivated sample and the non-inactivated sample can be used as a control group to determine the antibody pairs that are susceptible to complement interference.

[0052] Specifically, half of the high complement negative sample mixture, the medium complement negative sample mixture, and the low complement negative sample mixture were heated at 53°C for 10 minutes to inactivate complement, that is, test sample one contained non-inactivated samples, and test sample two contained inactivated samples. The C1q content of the non-inactivated samples and the inactivated samples was re-measured. The results are shown in Table 1. The complement inactivation ratio was greater than 90%.

[0053] Table 1

[0054]

[0055] Furthermore, S60 includes the following steps:

[0056] S61: Calculate the change ratio according to the first test result, the second test result, and the third test result. When the difference between the change ratios of the first test result and the second test result is less than 10%, and the difference between the change ratios of the second test result and the third test result is less than 10%, the monoclonal antibody pair to be selected is not affected by complement.

[0057] S62: Obtain a monoclonal antibody pair that is not interfered by complement.

[0058] By comparing the measured values, when the difference in the change ratio between test result one and test result two is less than 10%, the monoclonal antibody pairs that are susceptible to complement interference can be confirmed; when the difference in the change ratio between test result two and test result three is less than 10%, the monoclonal antibody pairs that are not susceptible to complement interference can be further screened out. This method is simple, effective and highly accurate.

[0059] Furthermore, the added amounts of the negative sample mixture and the antigen in the test sample 1 and the test sample 2 are the same.

[0060] Specifically, AMH antigen prepared at a final concentration of 10 ng / ml (diluted about 60,000 times) was added to test sample 1 and test sample 2 at the same ratio.

[0061] Test sample one is the control group of test sample two. To ensure the reliability of the experimental data, according to the control variable method, the two samples differ only in whether they are heated or not. The negative sample mixture and the amount of antigen added are controlled to be the same, or the antigen is added to test sample one and test sample two in the same proportion.

[0062] Furthermore, before S40, the steps include:

[0063] S31: serum collection;

[0064] S32: Purify C1q in serum to obtain a crude pure product;

[0065] S33: Further purify the crude product to obtain complement C1q.

[0066] Specifically, the complement added in this example is derived from human serum. The first round of purification of human serum C1q includes the following steps: collecting 1L human serum, adding 0.3% TNBP + 1% Triton X-100, and inactivating at 24°C-30°C for 4-6 hours; preparing 40ml Cys-C affinity filler at a coupling ratio of 5mg / ml, and balancing with 20mM PB (pH=7.4), 1M NaCl solution; loading 200mg of rabbit anti-Cys-C polyclonal antibody purified (ammonium sulfate precipitation and DEAE column (pH=6.5, collecting flow-through)), balancing with 20mM PB (pH=7.4), 1M NaCl solution; balancing again with 20mM Tris (pH=7.4) buffer; mixing the inactivated human serum with 20mMTris (pH=7.4), 20mM EDTA at a ratio of 1:1, loading, and balancing with 20mM Tris (pH=7.4), 5mM EDTA buffer; Tris (pH = 7.4), 1M NaCl elution; the eluate was dialyzed 3-5 times with 20mM Tris (pH: 7.4), 20mM EDTA. After dialysis, centrifuge at 12000rpm for 40min, discard the supernatant, and dissolve the precipitate in 20mM Tris (pH: 7.4), 1M NaCl; the dissolved material was centrifuged at 16000rpm for 40min, and the supernatant was collected. The absorbance was detected by UV280, and the concentration was calculated by the formula C = UV280 / 0.6727.

[0067] Specifically, the second round of purification of human serum C1q includes the following steps: taking 10 ml of Protein A filler, balancing 5-10 column volumes with pure water, balancing 3-5 column volumes with 20 mM Tris (pH: 7.4) and 2 M NaCl, adding NaCl to the first round of purification product to a final concentration of 2 M, loading the sample, and collecting the flow-through; taking 10 ml of Protein G filler, balancing 5-10 column volumes with pure water, balancing 3-5 column volumes with 20 mM Tris (pH: 7.4) and 2 M NaCl, mixing the flow-through collected from the Protein A column with goat anti-human IgM antibody at a mass concentration of 7:3 and combining at room temperature for 1 hour, loading the mixed solution, collecting the flow-through, detecting the absorbance by UV280, calculating the concentration by the formula C = UV280 / 0.6727, and running SDS gel to detect the purity. The results are shown in FIG. Figure 1 .

[0068] The coating of capture antibodies in enzyme-linked immunosorbent assays, the coupling of capture antibodies to magnetic beads in chemiluminescent assays, antibody labeling such as biotin labeling, acridinium ester labeling, fluorescent microsphere labeling, and antigen-antibody binding can all cause changes in antibody conformation, thereby exposing the binding site of complement C1q in the FC segment. When C1q binds to sites on two or more antibody FCs, the complement system in the sample can be activated. C1q is an 18-mer complex with 6 antibody binding sites. When C1q molecules bind to capture antibodies and detection antibodies at the same time, false positives or false increases will occur; C3b and C4b formed after complement system activation are bound to the Fab of the antibody, thereby affecting the binding of the capture antibody or detection antibody to the antigen, resulting in false negatives or false decreases, which will affect the accuracy of immunoassays. Therefore, human serum C1q is purified to facilitate subsequent addition to the inactivated and negative sample mixtures added with the analyte for testing to verify whether the selected monoclonal antibodies will be interfered with by complement.

[0069] Further, S40 includes the following steps:

[0070] S41: Add complement C1q to test sample two to obtain test sample three.

[0071] By adding human C1q to the inactivated and tested samples, and testing the effect of C1q on the test results again, it is possible to further screen out antibody pairs that are not easily interfered by complement, thereby improving the accuracy of the screening results.

[0072] Furthermore, the immunochemiluminescence detection comprises the following steps:

[0073] S44: preparing a luminescent reagent;

[0074] S45; preparing solid phase reagent;

[0075] S46: Mix the luminescent reagent and the solid phase reagent with the test sample 1, the test sample 2, and the test sample 3 respectively, and perform detection using a chemiluminescent immunoassay analyzer.

[0076] Use immunochemiluminescence detection to detect sample one, sample two, and sample three, record their brightness values ​​and calculate the change ratio. If the measured value changes not significantly, the monoclonal antibody pair is not easily affected by complement interference. By comparing the data, the monoclonal antibody pairs that are not easily affected by complement interference are screened out, which has higher accuracy, simple operation process, and is easy to implement.

[0077] Specifically, the preparation of the luminescent reagent includes the following steps: taking 0.1 mg of the detection antibody, adding acridinium ester label at a molar ratio of 1:5-1:15, mixing and reacting for 2 hours at room temperature in the dark, dialyzing and desalting in PB buffer in the dark, the dialysate volume is 20 times larger than the labeling volume, changing the solution once every 4 hours, at least 3 times, and diluting with PBS-BSA to 1-10 μg / mL for use after measuring the concentration.

[0078] Specifically, the preparation of the solid phase reagent includes the following steps: taking 0.1 mg of the capture antibody, adding biotin label at a molar ratio of 1:5-1:15, mixing and reacting at room temperature for 2 hours, dialyzing and desalting with 20 mM PB buffer, the dialysate volume is 20 times larger than the label volume, changing the liquid once every 4 hours, changing the liquid at least 3 times, and diluting with PBS-BSA to 0.1-1 mg / mL for use after measuring the concentration. Take about 0.5 mL of magnetic beads, wash with PB three times, dilute to 1-10 mg / mL, add an equal volume of biotin-labeled antibody, mix and react at room temperature for 2 hours, wash with PBS-BSA 3 times, and dilute to 0.1-1 mg / mL for use.

[0079] Specifically, a paired AMH antibody pair is selected and a chemiluminescent detection reagent is configured to detect the test sample one and the test sample two.

[0080] In summary, further, the chemiluminescent immunoassay analyzer MS120 produced by Meikang Bio was selected, and the one-step method was used to mix 100 μL of luminescent reagent and 50 μL of solid phase reagent with 30 μL of test sample 1, and 100 μL of luminescent reagent and 50 μL of solid phase reagent with 30 μL of test sample 2, and incubated at 37°C for 15 minutes. The brightness value was recorded and the change ratio was calculated (with test sample 1 as a reference, the measured value was 100%), and test results 1 and 2 were obtained. The results are shown in Table 2. Antibody pair 260-60 (see Figure 2 ), 260-67, 260-66, and 260-49 values ​​decreased significantly (>10%), with the largest decrease of nearly 30%, indicating that complement interference caused a false increase in the values ​​of the four antibody pairs; the antibody pair 260-72 (see Figure 3 ), 260-295, 260-71, 260-73, 260-346 measured values ​​changed insignificantly (<10%).

[0081] Further, a final concentration of 300 μg / mL C1q was added to the test sample 2 to obtain the test sample 3, and the test sample 3 was tested again under the same conditions to obtain the test result 3, as shown in Table 2. Compared with the test results of the test sample 2, the measured values ​​of the antibody pairs 260-60, 260-67, 260-66, and 260-49 all increased by more than 150%, while the measured values ​​of the antibody pairs 260-72, 260-295, 260-71, 260-73, and 260-346 did not change significantly (<10%). Therefore, it can be determined that the antibody pairs 260-60, 260-67, 260-66, and 260-49 are interfered by complement for AMH immunoassay, while the antibody pairs 260-72, 260-295, 260-71, 260-73, and 260-346 are not significantly interfered by complement.

[0082] Table 2

[0083]

[0084]

[0085] Note: Table 2 is for antibody pair screening a : Test sample 2 measurement value / test sample 1 measurement value; b : Test sample three measurement value / test sample two measurement value.

[0086] Furthermore, the C1q content of negative samples was determined using a complement C1q diagnostic kit.

[0087] Specifically, the complement C1q diagnostic kit (immunoenhanced turbidimetry) sold by Mecan Biotechnology Co., Ltd. is used to measure the C1q content, which has the advantages of being sensitive, rapid and simple in quantitative methods, and improves screening efficiency.

[0088] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A method for screening monoclonal antibody pairs that are not interfered with by complement, characterized in that: The following steps are involved: S10: Obtain negative sample mixture; S20: adding antigen to the negative sample mixture to obtain a test sample 1; S30: heating the negative sample mixture and adding the antigen to obtain a second test sample; S40: adding complement C1q to the test sample 2 to obtain a test sample 3; S50: using the chemiluminescent reagent prepared by the selected monoclonal antibody pair, respectively performing immunochemiluminescent detection on the test sample 1, the test sample 2, and the test sample 3 to obtain the test result 1, the test result 2, and the test result 3; S60: obtaining the monoclonal antibody pair that is not interfered by complement according to the first test result, the second test result, and the third test result; Wherein, the S10 comprises the following steps: S11: Obtain negative samples; S12: Determine the C1q content of the negative sample to obtain a test result; S13: Classifying the negative samples according to the detection results, and obtaining the negative sample mixture.

2. The method according to claim 1, characterized in that The method of classifying the negative samples and obtaining a negative sample mixture comprises the following steps: S14: The negative samples with C1q content greater than 220 μg / mL are mixed and labeled as a high complement negative sample mixture, the negative samples with C1q content greater than 170 μg / mL and less than 220 μg / mL are mixed and labeled as a medium complement negative sample mixture, and the negative samples with C1q content less than 170 μg / mL are mixed and labeled as a low complement negative sample mixture.

3. The method according to claim 1, characterized in that The negative sample mixture is heated at a temperature of 50-55° C. for a heating time of 10-15 min.

4. The method according to claim 1, characterized in that The S60 includes the following steps: S61: calculating the change ratio according to the first test result, the second test result, and the third test result, and when the difference between the change ratios of the first test result and the second test result is less than 10%, and the difference between the change ratios of the second test result and the third test result is less than 10%, the monoclonal antibody pair to be selected is not affected by the complement; S62: Obtaining the monoclonal antibody pair that is not interfered by complement.

5. The method according to claim 1, characterized in that The added amounts of the negative sample mixture and the antigen in the test sample 1 and the test sample 2 are the same.

6. The method according to claim 1, characterized in that The S40 includes the following steps: S31: serum collection; S32: purifying the C1q in the serum to obtain a crude pure product; S33: further purifying the crude product to obtain complement C1q.

7. The method according to claim 6, characterized in that The S40 comprises the following steps: S41: adding the complement C1q to the test sample two to obtain the test sample three.

8. The method according to claim 1, characterized in that The immunochemiluminescence detection comprises the following steps: S44: preparing a luminescent reagent; S45; preparing solid phase reagent; S46: The luminescent reagent and the solid phase reagent are mixed with the detection sample 1, the detection sample 2, and the detection sample 3 respectively, and detection is performed using a chemiluminescent immunoassay analyzer.

9. The method according to claim 1, characterized in that: The determination of the C1q content of the negative sample uses a complement C1q diagnostic kit.

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