WO2023124108A1 - 基于抗原检测试剂的抗原中和当量的确定方法 - Google Patents

基于抗原检测试剂的抗原中和当量的确定方法 Download PDF

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WO2023124108A1
WO2023124108A1 PCT/CN2022/112642 CN2022112642W WO2023124108A1 WO 2023124108 A1 WO2023124108 A1 WO 2023124108A1 CN 2022112642 W CN2022112642 W CN 2022112642W WO 2023124108 A1 WO2023124108 A1 WO 2023124108A1
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antigen
antibody
neutralization equivalent
detection reagent
dilution
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French (fr)
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许行尚
陈杰弗瑞
王鹏
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南京岚煜生物科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard

Definitions

  • the invention relates to the technical field of medical detection, in particular to a method for determining the neutralization equivalent of an antigen based on an antigen-based detection reagent.
  • In vitro diagnostic In vitro diagnostic (In Vitro Diagnosis, IVD) medical devices are products that obtain clinical diagnostic information by testing human samples (blood, body fluids, tissues, etc.), including reagents, calibrators, control substances, equipment or systems. Diagnostic reagents are one of the basic tools to detect whether a patient is sick or not, and the accuracy of the results directly affects the doctor's diagnosis and the patient's health and life safety. With the advancement of modern medical technology, more precise requirements are put forward for the results of medical tests, and thus the quality requirements for in vitro diagnostic products are also getting higher and higher.
  • the detection limit refers to the biological sample is processed and tested according to the requirements of the analytical method, and the lowest value that can be distinguished from the noise and reported with a certain confidence level is the lowest value that is measured. It is also used to refer to the minimum detectable concentration.
  • the visible detection limit is also a quantitative indicator. Due to the lack of antibody standards, the minimum detection limit of antibody detection reagents cannot be accurately determined.
  • a method for assigning an antibody standard and determining the antigen neutralization equivalent is disclosed, which specifically includes the following steps: Determination of the antigen neutralization equivalent of the S1 antibody standard or sample: the antigen of known purity and concentration Perform gradient dilution with the matrix, add an equal amount of antibody standard or antibody-containing sample to each gradient dilution of different concentrations, and after the reaction, use the primary antibody detection reagent to perform antibody detection on each mixture to determine the antibody standard
  • the antigen neutralization equivalent of the product or sample; the measurement unit of the antigen neutralization equivalent of the antibody standard or sample in a certain volume is expressed by the mass-volume concentration of the antibody that can neutralize the antigen, in order to specifically clarify the mass-volume unit
  • the mass number is the amount of the corresponding neutralized antigen, so add "NAE" after the mass unit, such as: 1ng NAE/mL, 1 ⁇ g NAE/mL, 1mg NAE/mL, etc.
  • antibody detection reagents are used to determine the neutralization equivalent of antigens, but some antibody detection reagents will change their potency after a period of time during use, which will affect the detection results, while the antigens are relatively stable and will not appear. This problem therefore requires the development of a method for the determination of antigen neutralizing equivalents based on antigen detection reagents.
  • the technical problem to be solved by the present invention is to provide a method for determining the antigen neutralization equivalent based on the antigen detection reagent.
  • This method uses the antigen detection reagent to determine the antigen neutralization equivalent. Quantification can also solve the problem that the minimum detection limit of antibody detection reagents currently on the market cannot be determined or the value is inaccurate.
  • the technical solution adopted in the present invention is: the method for determining the neutralization equivalent of an antigen based on an antigen detection reagent, specifically comprising the following steps:
  • S1 diluted antigen the antigen of known purity and concentration is serially diluted with matrix to obtain a series of serial dilutions of different concentrations;
  • S2 antigen and antibody incubation reaction add an equal amount of antibody standard or antibody-containing sample to each of the gradient dilutions of different concentrations obtained in the step S1, and react to generate a mixed solution;
  • step S3 Determination of antigen neutralization equivalent: adding a specific antigen detection reagent corresponding to the antigen in step S1 to each mixture obtained in step S2 for detection, and recording data, thereby determining antigen neutralization equivalent.
  • the method for determining the antigen neutralization equivalent is the method of assigning the amount of antibody in a certain volume, wherein the unit of measurement of the antigen neutralization equivalent of antibody in a certain volume of antibody standard or sample is the mass-volume concentration of the antibody that can neutralize the antigen
  • NAE is added after the mass unit, such as: 1ng NAE/mL, 1 ⁇ g NAE/mL, 1mg NAE/mL, etc.
  • the specific antigen detection reagent is used as the detection reagent for detection.
  • the dilution with a negative reaction The amount of antigen is the antigen neutralization equivalent of the antibody standard or sample. If the sample is a diluted sample, the dilution factor needs to be considered.
  • the antigen in the step S1 is the original antigen or has the same antigenic determinant as the original antigen and can be identified Different antigens specifically recognized by antibodies.
  • step S3 when a specific antigen detection reagent is added to each mixed solution for detection, when a negative reaction occurs in a certain dilution of the mixed solution in the test result, it is determined that the mixed solution corresponds to The amount of antigen in the dilution is the antigen neutralizing equivalent of the antibody contained in the standard or sample.
  • the antigens in the step S1 are the same or different antigens that have the same antigenic determinant and can be specifically recognized by the same antibody when used for different times.
  • the amount of the antibody may change after a period of time.
  • the original antigen can be used to re-quantify the antibody (recalculate its antigen neutralization equivalent), or the antigen that has been used before (Antigen 1)
  • Different antigen 2 is used to calculate the antigen neutralization equivalent of the antibody contained in the sample.
  • Antigen 1 and antigen 2 have the same epitope and can be specifically recognized by the same antibody.
  • step S2 an equal amount of antibody standard or sample containing specific antibody is added to each antigen diluent of different concentration, and a mixed solution is generated after a constant temperature reaction at 37°C for 30 minutes. .
  • the dilution ratios of the gradient dilution of the antigen in the step S1 are respectively: 1:10, 1:20, 1:80, 1:160 and 1:320.
  • the further improvement of the present invention is that it also includes the determination of the antibody titer of the sample in step S4: the sample containing the antibody in the step S3 is gradiently diluted with a matrix to obtain gradient dilutions of different dilutions; antibody detection reagents are used for each Antibody detection was performed on serial dilutions of dilutions to determine the antibody titer of the sample.
  • the dilution ratios of the samples containing the antibody are serially diluted with matrix: 1:2, 1:4, 1:8, 1:16, 1:32, 1: 64, 1:128.
  • the antibody detection reagent is used to detect the antibody of each dilution of the dilution, and when a negative reaction occurs in the dilution of a certain dilution in the test result, the dilution is determined
  • the dilution of the previous higher concentration dilution is the antibody titer.
  • the antibody titer of the sample in step S4 is detected after gradient dilution with the same antibody-containing sample as in step S2. In all gradient dilutions, there will be a negative reaction at one dilution, while a negative reaction will appear.
  • the dilution of the previous higher concentration dilution of the reaction is the antibody titer of the sample.
  • the minimum detection limit of the antibody detection reagent obtained above is used, and the minimum detection limit of the antibody detection reagent is confirmed and verified through steps S6 and S7.
  • the minimum detection limit of the above antibody detection reagents can be confirmed and verified as follows:
  • step S4 select the last dilution of the antibody titer determined now as the new antibody titer, and then perform multiple repeated detections, Until the positive rate is ⁇ 90%; then the minimum detection limit for confirmation at this time is recalculated based on the new antibody titer;
  • the method for verifying the minimum detection limit of the antibody detection reagent in step S7 is: dilute 3 clinical samples to the concentration of the minimum detection limit, perform 3 tests first, and pass the verification if they are all positive; If one test is negative, then do 7 more tests, and if they are all positive, the verification will be passed; if two of the 3 tests are negative, then do 18 more tests, if all are positive, then the verification will be passed; if all the 3 tests are negative, the test will be passed. Then the verification fails (when 20 or more times of detection are used at one time, it will take more time, and this progressive method can be used to verify the minimum detection limit of the antibody detection reagent).
  • the method for determining the neutralization equivalent of the antigen based on the antigen detection reagent of the present invention has the beneficial effects of:
  • the antigen detection reagent uses the antigen detection reagent to determine the antigen neutralization equivalent. After the antigen neutralization equivalent is determined, after a period of time, the original antigen can be used to re-quantify the antibody (recalculate the antigen neutralization equivalent), or the original antigen (Antigen 1) different antigen 2 to calculate the antigen neutralization equivalent of the antibody contained in the sample;
  • the method for determining the antigen neutralization equivalent based on the antigen detection reagent specifically includes the following steps:
  • step S1 the antigen with known purity and concentration is serially diluted with the matrix to obtain a series of serial dilutions of different concentrations; when the antibody standard or the sample containing the antibody has been determined to neutralize the antigen, the antigen needs to be re-determined
  • the steps S1-S3 are re-performed to obtain the antigen neutralization equivalent of the antibody standard or the antibody-containing sample; when the antigen neutralization equivalent of the antibody standard or the antibody-containing sample is re-determined, the The antigen in step S1 is the original antigen or a different antigen that has the same antigenic determinant as the original antigen and can be specifically recognized by the same antibody; wherein the antigens used for different times can have the same antigenic determinant and can be recognized by the same antibody.
  • the same or different antigens specifically recognized by the antibody that is, after the antigen neutralization equivalent of the antibody is determined, after a period of time, the original antigen can be used to re-quantify the antibody (recalculate the antigen neutralization equivalent), or the original antigen can be used (Antigen 1)
  • Different antigen 2 is used to calculate the antigen neutralization equivalent of the antibody contained in the sample.
  • Antigen 1 and antigen 2 have the same epitope and can be specifically recognized by the same antibody; the gradient of the antigen in the step S1
  • the dilution ratios are: 1:10, 1:20, 1:80, 1:160 and 1:320, each dilution will get 50 ⁇ L of dilution;
  • Antigen and antibody incubation reaction add an equal amount of antibody standard or antibody-containing sample to each of the gradient dilutions of different concentrations obtained in the step S1, and react to generate a mixed solution; in the step S2, each Add the same amount of antibody standard or samples containing specific antibodies to the antigen diluents of different concentrations, and react at a constant temperature of 37°C for 30 minutes to generate a mixed solution;
  • Determination of the antibody titer of the S4 sample gradiently dilute the sample containing the antibody in the step S3 with a matrix to obtain gradient dilutions of different dilutions; use antibody detection reagents to perform antibody detection on the gradient dilutions of each dilution , so as to determine the antibody titer of the sample; the dilutions of the sample containing the antibody in the step S4 are serially diluted with matrix: 1:2, 1:4, 1:8, 1:16, 1:32, 1 : 64, 1: 128; the antibody titer of the sample in step S4 is detected by gradient dilution using the same antibody-containing sample as in step S2, and there will be a negative reaction in all gradient dilutions, while The titer of the antibody of the sample is the dilution of the higher concentration dilution before the negative reaction occurs;
  • the antigen neutralization equivalent of the sample determined in the step S3 is multiplied by the antibody titer of the sample determined in the step S4 to obtain the minimum detection limit of the antibody detection reagent Out of limit.
  • the lowest detection limit of the antibody detection reagent can be obtained A*B.
  • step S4 select the last dilution of the antibody titer determined now as the new antibody titer, and then perform multiple repeated detections, Until the positive rate is ⁇ 90%; then the minimum detection limit for confirmation at this time is recalculated based on the new antibody titer;
  • the method for verifying the minimum detection limit of the antibody detection reagent in step S7 is: dilute 3 clinical samples to the concentration of the minimum detection limit, perform 3 tests first, and pass the verification if they are all positive; If one test is negative, then do 7 more tests, and if they are all positive, the verification will be passed; if two of the 3 tests are negative, then do 18 more tests, if all are positive, then the verification will be passed; if all the 3 tests are negative, the test will be passed. Then the verification fails (when 20 or more times of detection are used at one time, it will take more time, and this progressive method can be used to verify the minimum detection limit of the antibody detection reagent).
  • antigen 1 is hepatitis B virus surface antigen (HbsAg), 1000 ⁇ g/mL, purchased from absolute antibody; the detection kit used: hepatitis B virus surface antigen, surface antibody , e antigen, e antibody, core antibody detection reagent (colloidal gold method), purchased from Guangzhou Wanfu Biotechnology Co., Ltd.; negative serum used: purchased from Beijing Jingke Hongda Biotechnology Co., Ltd.
  • HbsAg hepatitis B virus surface antigen
  • the detection kit used hepatitis B virus surface antigen, surface antibody , e antigen, e antibody, core antibody detection reagent (colloidal gold method), purchased from Guangzhou Wanfu Biotechnology Co., Ltd.
  • negative serum used purchased from Beijing Jingke Hongda Biotechnology Co., Ltd.
  • the determination of the antigen neutralization equivalent based on the antigen detection reagent specifically includes the following steps:
  • Antibody incubation reaction add 50 ⁇ L of samples containing hepatitis B virus surface antibody (HBsAb) to each serial dilution, and make a water bath at 37°C for 30 minutes;
  • HBsAb hepatitis B virus surface antibody
  • Table 1 is the test result data after antigen 1 and antibody incubation reaction
  • the method for determining the minimum detection limit of an antibody detection reagent specifically includes the following steps:
  • Determination of the antibody titer of the S4 sample Dilute the sample containing HBsAb with the negative serum according to the gradient of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, Obtain gradient dilutions of different dilutions, and each dilution obtains 50 ⁇ L of dilutions; each gradient is detected with hepatitis B virus surface antigen, surface antibody, e antigen, e antibody, core antibody detection reagent (colloidal gold method) (During the detection, the hepatitis B virus surface antibody detection reagent in the kit is utilized to detect HBsAb); the record data is as follows in Table 2;
  • Table 2 is the sample (antibody) detection result data
  • the test results show a negative reaction to a dilution of a certain dilution
  • the antibody titer so as to determine the antibody titer; that is, the sample containing HBsAb
  • the antibody titer was 1:32.
  • step S4 select the last dilution of the antibody titer determined now as the new antibody titer, and then perform multiple repeated detections, Until the positive rate is ⁇ 90%; then the minimum detection limit for confirmation at this time is recalculated based on the new antibody titer;
  • the method for verifying the minimum detection limit of the antibody detection reagent in step S7 is: dilute 3 clinical samples to the concentration of the minimum detection limit, perform 3 tests first, and pass the verification if they are all positive; If one test is negative, then do 7 more tests, and if they are all positive, the verification will be passed; if two of the 3 tests are negative, then do 18 more tests, if all are positive, then the verification will be passed; if all the 3 tests are negative, the test will be passed. Then the verification fails (when 20 or more times of detection are used at one time, it will take more time, and this progressive method can be used to verify the minimum detection limit of the antibody detection reagent).
  • antigen 2 when it is necessary to re-determine the antigen neutralization equivalent of the HBsAb, antigen 2 can also be used.
  • Antigen 2 is Hepatitis B Surface Antigen (HBsAg-subtype Ad), 2000 ⁇ g/mL, purchased from EastCoast Bio; detection kit: Hepatitis B virus surface antigen, surface antibody, e antigen, e antibody, core antibody detection reagent (colloidal gold method), purchased from Guangzhou Wanfu Biotechnology Co., Ltd.; negative serum: purchased from Beijing Jingke Hongda Biotechnology Co., Ltd. company.
  • HBsAg-subtype Ad Hepatitis B Surface Antigen
  • detection kit Hepatitis B virus surface antigen, surface antibody, e antigen, e antibody, core antibody detection reagent (colloidal gold method), purchased from Guangzhou Wanfu Biotechnology Co., Ltd.
  • negative serum purchased from Beijing Jingke Hongda Biotechnology Co., Ltd. company.
  • the determination of the antigen neutralization equivalent based on the antigen detection reagent specifically includes the following steps:
  • S1 Dilution of Antigen 2 Dilute antigen 2 of known concentration with negative serum according to the gradient of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and obtain 50 ⁇ L dilution for each dilution liquid;
  • Antibody incubation reaction add 50 ⁇ L of samples containing hepatitis B virus surface antibody (HBsAb) to each serial dilution solution, and make a water bath at 37°C for 30 minutes;
  • HBsAb hepatitis B virus surface antibody
  • S3 antigen neutralization equivalent to each mixed solution obtained in said step S2, detect (detection) with hepatitis B virus surface antigen, surface antibody, e antigen, e antibody, core antibody detection reagent (colloidal gold method) respectively
  • the record data is as follows in Table 3;
  • Table 3 is the test result data after antigen 2 and antibody incubation reaction
  • the method for determining the minimum detection limit of an antibody detection reagent specifically includes the following steps:
  • Determination of the antibody titer of the S4 sample Dilute the sample containing HBsAb with the negative serum according to the gradient of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, Obtain gradient dilutions of different dilutions, and each dilution obtains 50 ⁇ L of dilutions; each gradient is detected with hepatitis B virus surface antigen, surface antibody, e antigen, e antibody, core antibody detection reagent (colloidal gold method) (During the detection, the hepatitis B virus surface antibody detection reagent in the kit is utilized to detect HBsAb); the record data is as follows in Table 4;
  • Table 4 is the sample (antibody) detection result data
  • the test results show a negative reaction to a dilution of a certain dilution
  • the antibody titer so as to determine the antibody titer; that is, the sample containing HBsAb
  • the antibody titer was 1:32.
  • step S4 select the last dilution of the antibody titer determined now as the new antibody titer, and then perform multiple repeated detections, Until the positive rate is ⁇ 90%; then the minimum detection limit for confirmation at this time is recalculated based on the new antibody titer;
  • the method for verifying the minimum detection limit of the antibody detection reagent in step S7 is: dilute 3 clinical samples to the concentration of the minimum detection limit, perform 3 tests first, and pass the verification if they are all positive; If one test is negative, then do 7 more tests, and if they are all positive, the verification will be passed; if two of the 3 tests are negative, then do 18 more tests, if all are positive, then the verification will be passed; if all the 3 tests are negative, the test will be passed. Then the verification fails (when 20 or more times of detection are used at one time, it will take more time, and this progressive method can be used to verify the minimum detection limit of the antibody detection reagent).

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Abstract

一种基于抗原检测试剂的抗原中和当量的确定方法，具体包括以下步骤：S1稀释抗原：将已知纯度和浓度的抗原用基质进行梯度稀释，获得一系列不同浓度的梯度稀释液；S2抗原、抗体孵育反应：向步骤S1获得的每个不同浓度的梯度稀释液中分别加入等量的抗体标准品或含有抗体的样品，反应生成混合液；S3抗原中和当量的确定：向步骤S2获得的每个混合液中加入与步骤S1中抗原相对应的特异性抗原检测试剂进行检测，记录数据，从而确定抗原中和当量。利用抗原检测试剂确定抗原中和当量，并通过抗原中和当量和抗体的滴度来给抗体赋值，可以用于企业标准品甚至国家标准品的制备。

Description

基于抗原检测试剂的抗原中和当量的确定方法 技术领域
本发明涉及医学检测技术领域，尤其涉及一种抗基于抗原检测试剂的抗原中和当量的确定方法。
背景技术
体外诊断(In Vitro Diagnosis,IVD)医疗器械是通过对人体的样品(血液、体液、组织等)进行检测而获取临床诊断信息的产品，包括试剂、校准品、控制物质、设备或系统。诊断试剂是检测患者是否患病和病情程度的基本工具之一，其结果是否准确直接影响到医生的诊断以及患者的身体健康和生命安全。而随着现代医学科技的进步，对于医学检验的结果提出了更加精准的要求，从而对体外诊断产品的质量要求也越来越高。
标准物质(如国际标准、国家标准、企业标准和临床应用的校准品等)的制备及其量值溯源是获得精准测定结果的前提条件。由于抗体的储存稳定性差，即使深低温保存亦会随着保存时间的延长，而活性逐渐降低；而且，抗体的活性多用相对的稀释滴度表示，很难客观精准量化。因此，各种抗体的标准品或参考物质的制备十分困难。目前国内外均缺乏有关抗体标准品的商品供应，给抗体检测试剂的生产和应用带来了许多难以克服的困难。
另一方面，抗体检测试剂的一个重要的性能参数是检出限。检出限是指生物样品按照分析方法的要求进行处理并检测，能区分于噪声的以特定置信水平报告被测量存在的最低值，它也被用来指最小可检测浓度，可见检出限也是一个量化指标。由于缺乏抗体的标准品，抗体检测试剂的最低检出限也无从精准确定。
中国专利文献CN113125756A中，公开了一种抗体标准品赋值和抗原中和当量确定的方法，具体包括以下步骤：S1抗体标准品或样品的抗原中和当量的确定：将已知纯度和浓度的抗原用基质进行梯度稀释，向每个不同浓度的梯度稀释液中分别加入等量的抗体标准品或含有抗体的样品，反应后，用第一抗体检测试剂对每个混合液进行抗体检测确定抗体标准品或样品的抗原中和当量；其中一定体积中抗体标准品或样品中抗体的抗原中和当量的计量单位以其中抗体可中和抗原的质量-体积浓度表示，为了特别明确质量-体积单位中的质量数是相应中和抗原的量，故而在质量单位后面加上“NAE”如：1ng NAE/mL、1μg NAE/mL、 1mg NAE/mL等。该技术方案中采用的是抗体检测试剂进行抗原中和当量的确定，但有的抗体检测试剂在使用过程中经过一段时间其效价会改变，影响检测结果，而抗原相对比较稳定，不会出现这个问题，因此需要开发一种基于抗原检测试剂的抗原中和当量的确定方法。
发明内容
本发明要解决的技术问题是，提供一种基于抗原检测试剂的抗原中和当量的确定方法，此方法使用抗原检测试剂确定抗原中和当量，抗原中和当量确定后，还可以再次使用对抗体进行定量，同时能解决目前市面上抗体检测试剂最低检出限不能定值或定值不准确的问题。
为了解决上述技术问题，本发明采用的技术方案为：该基于抗原检测试剂的抗原中和当量的确定方法，具体包括以下步骤：
S1稀释抗原：将已知纯度和浓度的抗原用基质进行梯度稀释，获得一系列不同浓度的梯度稀释液；
S2抗原、抗体孵育反应：向所述步骤S1获得的每个不同浓度的梯度稀释液中分别加入等量的抗体标准品或含有抗体的样品，反应生成混合液；
S3抗原中和当量的确定：向所述步骤S2获得的每个混合液中加入与所述步骤S1中抗原相对应的特异性抗原检测试剂进行检测，记录数据，从而确定抗原中和当量。
抗原中和当量的确定的方法即是一定体积中抗体量的赋值方法，其中一定体积中抗体标准品或样品中抗体的抗原中和当量的计量单位以其中抗体可中和抗原的质量-体积浓度表示，为了特别明确质量-体积单位中的质量数是相应中和抗原的量，故而在质量单位后面加上“NAE”如：1ng NAE/mL、1μg NAE/mL、1mg NAE/mL等。抗体标准品赋值和抗体检测试剂最低检出限确定的方法首先确定抗体标准品或样品的抗原中和当量(A)；再确定样品的抗体的滴度(B)；从而抗体检测试剂最低检出限＝A*B；因为抗原通常是稳定的，可定量的，可以和抗体发生中和反应，而中和反应是抗原决定簇和抗体识别端Fab的反应，再使用特异性抗体检测试剂来测定其抗体的最低检出限；其中步骤S1中的采用的抗原是已知纯度和浓度的，进行梯度稀释后，步骤S2中加入等量的抗体标准品或含有抗体的样品进行混合反应，样品可以是混合样品，可以用原样品，可适当准确稀 释，反应后采用特异性抗原检测试剂作为检测试剂进行检测，在所有梯度稀释度中会有一个稀释度出现阴性反应，而出现阴性反应的稀释度的抗原量即为抗体标准品或样品的抗原中和当量，若样品为稀释后的样品，则需考虑稀释因素。
作为本发明的优选技术方案，当确定过抗原中和当量的抗体标准品或含有抗体的样品，需重新确定其抗原中和当量时，则重新进行所述步骤S1～S3获得该抗体标准品或含有抗体的样品的抗原中和当量。
作为本发明的优选技术方案，当重新确定该抗体标准品或含有抗体的样品的抗原中和当量时，所述步骤S1中的抗原为原抗原或与原抗原具有相同的抗原决定簇能被同一种抗体特异性识别的不同抗原。
作为本发明的优选技术方案，所述步骤S3中对每个混合液中加入特异性抗原检测试剂进行检测时，当检测结果中某个稀释度的混合液出现阴性反应，则确定该混合液对应的稀释度的抗原量为该标准品或样品所含抗体的抗原中和当量。
作为本发明的优选技术方案，所述步骤S1中的抗原在不同次数使用时为具有相同的抗原决定簇，能被同一种抗体特异性识别的相同的或不同的抗原。抗体的抗原中和当量确定后，经过一段时间后，抗体的量可能发生变化，可以用原抗原重新对抗体定量(重新计算其抗原中和当量)，也可以选用与原已使用过的抗原(抗原1)不同的抗原2来计算样品中所含抗体的抗原中和当量，抗原1与抗原2具有相同的抗原决定簇，能够被同一种抗体特异性识别。
作为本发明的优选技术方案，所述步骤S2中向每个不同浓度的抗原稀释液中分别加入等量的抗体标准品或含有特异性抗体的样品，经37℃恒温反应30min后，生成混合液。
作为本发明的优选技术方案，所述步骤S1中抗原的梯度稀释的稀释度分别为：1：10、1：20、1：80、1：160和1：320。
本发明进一步改进在于，还包括步骤S4样品的抗体滴度的确定：将所述步骤S3中含有抗体的样品用基质进行梯度稀释，获得不同稀释度的梯度稀释液；采用抗体检测试剂对每个稀释度的梯度稀释液进行抗体检测，从而确定样品的抗体滴度。
本发明进一步改进在于，还包括步骤S5抗体检测试剂最低检出限的获得：将所述步骤S3中确定的样品的抗原中和当量与所述步骤S4中确定的样品的抗 体滴度进行相乘计算，获得抗体检测试剂的最低检出限。根据确定抗原中和当量(A)和确定抗体的滴度(B)；可以获得抗体检测试剂最低检出限＝A*B。
作为本发明的优选技术方案，所述步骤S4中含有抗体的样品用基质进行梯度稀释的稀释度分别为：1：2，1：4，1：8，1：16，1：32，1：64，1：128。
作为本发明的优选技术方案，所述步骤S4中用抗体检测试剂对每个稀释度的稀释液进行抗体检测，当检测结果中某个稀释度的稀释液出现了阴性反应，则确定该稀释液的前一个较高浓度的稀释液的稀释度为抗体滴度。采用上述技术方案，步骤S4中的样品的抗体滴度采用与步骤S2中相同的含有抗体的样品通过梯度稀释后进行检测，在所有梯度稀释度中会有一个稀释度出现阴性反应，而出现阴性反应的前一个较高浓度的稀释液的稀释度即为该样品的抗体的滴度。
利用上述获得抗体检测试剂的最低检出限，并通过步骤S6，S7来对抗体检测试剂的最低检出限进行确认和验证。对以上抗体检测试剂的最低检出限可以进行如下确认与验证：
S6抗体检测试剂的最低检出限的确认：分别选择不同来源具有代表性的至少3个临床含待测抗体的样品，将3个临床含待测抗体的样品按照步骤S1～S5获得抗体检测试剂的最低检出限，若获得的含有抗体的样品的最低检出限和步骤S5中测得的最低检出限一致，则将含有抗体的样品分别稀释至最低检出限的浓度，进行20次重复检测，若阳性率均≥90％，则抗体检测试剂的最低检出限通过确认；
当多次重复检测后，阳性率低于90％，则调整步骤S4中的抗体滴度，选取现确定的抗体滴度的上一稀释度为新的抗体滴度，再进行多次重复检测，直至阳性率≥90％；则此时的确认的最低检出限是根据新的抗体滴度重新进行计算获得的；
S7抗体检测试剂的最低检出限的验证：选择具有时间和区域特征性的3个临床样品(且与步骤S6中最低检出限确定所使用的样品不同)，将3个临床样品稀释至最低检出限的浓度进行20次检测，若达到90％～95％阳性检出率，则最低检出限通过验证；
或者步骤S7抗体检测试剂的最低检出限的验证的方法为：将3个临床样品稀释至最低检出限的浓度，先做3次检测，若均阳性则通过验证；若3次检测中 出现一次阴性，则再做7次检测，若均阳性则通过验证；若3次检测中出现两次阴性，则再做18次检测，若均阳性则通过验证；若3次检测中均为阴性，则验证不通过(当一次性采用20次及20次以上进行检测时，会耗费更多的时间，则可以采用该递进方式进行抗体检测试剂的最低检出限的验证)。
与现有技术相比，本发明的基于抗原检测试剂的抗原中和当量的确定方法具有的有益效果为：
(1)利用抗原检测试剂确定抗原中和当量，并通过抗原中和当量和抗体的滴度来给抗体标准品赋值，可以用于企业标准品甚至国家标准品的制备；
(2)采用抗原检测试剂确定抗原中和当量，在抗原中和当量确定后，经过一段时间后，可以用原抗原重新对抗体定量(重新计算抗原中和当量)，也可以选用与原抗原(抗原1)不同的抗原2来计算样品中所含抗体的抗原中和当量；
(3)同时能精准确定抗体检测试剂的最低检出限，解决了市面上抗体检测试剂最低检出限不能定值或定值不准确的问题，是目前对抗体检测试剂最低检出限精准确定的创新性解决方法；
(4)通过对抗体检测试剂最低检出限的精准确定，能够保证试剂质量稳定，提高检测准确性，提高临床效率。
具体实施方式
为了加深对本发明的理解，下面将结合附图和实施例对本发明做进一步详细描述，该实施例仅用于解释本发明，并不对本发明的保护范围构成限定。
实施例：该基于抗原检测试剂的抗原中和当量的确定方法，具体包括以下步骤：
S1稀释抗原：将已知纯度和浓度的抗原用基质进行梯度稀释，获得一系列不同浓度的梯度稀释液；当确定过抗原中和当量的抗体标准品或含有抗体的样品，需重新确定其抗原中和当量时，则重新进行所述步骤S1～S3获得该抗体标准品或含有抗体的样品的抗原中和当量；当重新确定该抗体标准品或含有抗体的样品的抗原中和当量时，所述步骤S1中的抗原为原抗原或与原抗原具有相同的抗原决定簇能被同一种抗体特异性识别的不同抗原；其中不同次数使用的抗原可以是具有相同的抗原决定簇，能被同一种抗体特异性识别的相同的或不同的抗原；即抗体的抗原中和当量确定以后，经过一段时间后，可以用原抗原重新对抗 体定量(重新计算抗原中和当量)，也可以选用与原抗原(抗原1)不同的抗原2来计算样品中所含抗体的抗原中和当量，抗原1与抗原2具有相同的抗原决定簇，能够被同一种抗体特异性识别；所述步骤S1中抗原的梯度稀释的稀释度分别为：1：10、1：20、1：80、1：160和1：320，每份稀释均得到50μL稀释液；
S2抗原、抗体孵育反应：向所述步骤S1获得的每个不同浓度的梯度稀释液中分别加入等量的抗体标准品或含有抗体的样品，反应生成混合液；所述步骤S2中向每个不同浓度的抗原稀释液中分别加入等量的抗体标准品或含有特异性抗体的样品，经37℃恒温反应30min后，生成混合液；
S3抗原中和当量的确定：向所述步骤S2获得的每个混合液中加入与所述步骤S1中抗原相对应的特异性抗原检测试剂进行检测，记录数据，从而确定抗原中和当量；
S4样品的抗体滴度的确定：将所述步骤S3中含有抗体的样品用基质进行梯度稀释，获得不同稀释度的梯度稀释液；采用抗体检测试剂对每个稀释度的梯度稀释液进行抗体检测，从而确定样品的抗体滴度；所述步骤S4中含有抗体的样品用基质进行梯度稀释的稀释度分别为：1：2，1：4，1：8，1：16，1：32，1：64，1：128；步骤S4中的样品的抗体滴度采用与步骤S2中相同的含有抗体的样品通过梯度稀释后进行检测，在所有梯度稀释度中会有一个稀释度出现阴性反应，而出现阴性反应的前一个较高浓度的稀释液的稀释度即为该样品的抗体的滴度；
S5抗体检测试剂最低检出限的获得：将所述步骤S3中确定的样品的抗原中和当量与所述步骤S4中确定的样品的抗体滴度进行相乘计算，获得抗体检测试剂的最低检出限。根据确定抗原中和当量(A)和确定抗体的滴度(B)；可以获得抗体检测试剂最低检出限＝A*B。
S6抗体检测试剂的最低检出限的确认：分别选择不同来源具有代表性的至少3个临床含待测抗体的样品，将3个临床含待测抗体的样品按照步骤S1～S5获得抗体检测试剂的最低检出限，若获得的含有抗体的样品的最低检出限和步骤S5中测得的最低检出限一致，则将含有抗体的样品分别稀释至最低检出限的浓度，进行20次重复检测，若阳性率均≥90％，则抗体检测试剂的最低检出限通过确认；
当多次重复检测后，阳性率低于90％，则调整步骤S4中的抗体滴度，选取现确定的抗体滴度的上一稀释度为新的抗体滴度，再进行多次重复检测，直至阳性率≥90％；则此时的确认的最低检出限是根据新的抗体滴度重新进行计算获得的；
S7抗体检测试剂的最低检出限的验证：选择具有时间和区域特征性的3个临床样品(且与步骤S6中最低检出限确定所使用的样品不同)，将3个临床样品稀释至最低检出限的浓度进行20次检测，若达到90％～95％阳性检出率，则最低检出限通过验证；
或者步骤S7抗体检测试剂的最低检出限的验证的方法为：将3个临床样品稀释至最低检出限的浓度，先做3次检测，若均阳性则通过验证；若3次检测中出现一次阴性，则再做7次检测，若均阳性则通过验证；若3次检测中出现两次阴性，则再做18次检测，若均阳性则通过验证；若3次检测中均为阴性，则验证不通过(当一次性采用20次及20次以上进行检测时，会耗费更多的时间，则可以采用该递进方式进行抗体检测试剂的最低检出限的验证)。
具体地，当该实施例中使用抗原1时，抗原1为乙型肝炎病毒表面抗原(HbsAg)，1000μg/mL，购自absolute antibody；使用的检测试剂盒：乙型肝炎病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体检测试剂(胶体金法)，购自广州万孚生物技术股份有限公司；使用的阴性血清：购自北京经科宏达生物技术有限公司。
基于抗原检测试剂的抗原中和当量的确定，具体包括以下步骤：
S1稀释抗原1：用阴性血清将已知浓度的抗原1按照1：10，1：20，1：40，1：80，1：160，1：320的梯度进行稀释，每份稀释得到50μL稀释液；
S2抗原1、抗体孵育反应：向每个梯度稀释液分别加入50μL含乙型肝炎病毒表面抗体(HBsAb)的样品，37℃作水浴30min；
S3抗原中和当量的确定：向所述步骤S2获得的每个混合液分别用乙型肝炎病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体检测试剂(胶体金法)进行检测(检测时是利用试剂盒中的乙型肝炎病毒表面抗原检测试剂检测HbsAg)，记录数据如下表1；
表1 为抗原1、抗体孵育反应后检测结果数据
抗原稀释梯度 1：10 1：20 1：40 1：80 1：160 1：320
阴阳性 + + - - - -
从记录数据中可以确定，1：40稀释度的混合液中的抗原为50μL抗体标准品的抗原中和当量，经计算得到此含HBsAb的样品的抗原中和当量1为1000μg/mL÷40＝25μg/mL。
将抗体血清分装、冻存；使用时，将分装冻存的抗体血清取出，恢复室温后，用上述方法重新确定抗体的抗原中和当量，即重新对抗体赋值，保证抗体的量值精准确定，避免抗体因活性下降而造成临床诊断误差。
抗体检测试剂最低检出限的确定方法，具体包括以下步骤：
S4样品的抗体滴度的确定：用阴性血清将含HBsAb的样品按照1：2，1：4，1：8，1：16，1：32，1：64，1：128的梯度进行稀释，获得不同稀释度的梯度稀释液，每份稀释得到50μL稀释液；每个梯度液分别用乙型肝炎病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体检测试剂(胶体金法)进行检测(检测时是利用试剂盒中的乙型肝炎病毒表面抗体检测试剂检测HBsAb)；记录数据如下表2；
表2 为样品(抗体)检测结果数据
抗体稀释梯度 1：2 1：4 1：8 1：16 1：32 1：64 1：128
阴阳性 + + + + + - -
当检测结果中某个稀释度的稀释液出现了阴性反应，则确定该稀释液的前一个较高浓度的稀释液的稀释度为抗体滴度，从而确定抗体滴度；即此含HBsAb的样品的抗体滴度为1：32。
S5抗体检测试剂最低检出限的获得：将所述步骤S3中获得的样品中抗体的抗原中和当量(25μg/mL)与所述步骤S4中确定的样品的抗体滴度(1：32)进行相乘计算，获得抗体检测试剂的最低检出限；即乙型肝炎病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体检测试剂(胶体金法)的最低检出限为25μg/mL÷32＝0.78μg/mL。
对以上抗体检测试剂的最低检出限可以进行如下确认与验证：
S6抗体检测试剂的最低检出限的确认：分别选择不同来源具有代表性的至少3个临床含待测抗体的样品，将3个临床含待测抗体的样品按照步骤S1～S5获得抗体检测试剂的最低检出限，若获得的含有抗体的样品的最低检出限和步骤 S5中测得的最低检出限一致，则将含有抗体的样品分别稀释至最低检出限的浓度，进行20次重复检测，若阳性率均≥90％，则抗体检测试剂的最低检出限通过确认；
当多次重复检测后，阳性率低于90％，则调整步骤S4中的抗体滴度，选取现确定的抗体滴度的上一稀释度为新的抗体滴度，再进行多次重复检测，直至阳性率≥90％；则此时的确认的最低检出限是根据新的抗体滴度重新进行计算获得的；
S7抗体检测试剂的最低检出限的验证：选择具有时间和区域特征性的3个临床样品(且与步骤S6中最低检出限确定所使用的样品不同)，将3个临床样品稀释至最低检出限的浓度进行20次检测，若达到90％～95％阳性检出率，则最低检出限通过验证；
或者步骤S7抗体检测试剂的最低检出限的验证的方法为：将3个临床样品稀释至最低检出限的浓度，先做3次检测，若均阳性则通过验证；若3次检测中出现一次阴性，则再做7次检测，若均阳性则通过验证；若3次检测中出现两次阴性，则再做18次检测，若均阳性则通过验证；若3次检测中均为阴性，则验证不通过(当一次性采用20次及20次以上进行检测时，会耗费更多的时间，则可以采用该递进方式进行抗体检测试剂的最低检出限的验证)。
具体地，当需要重新确定该HBsAb的抗原中和当量时，还可以使用抗原2进行，抗原2为Hepatitis B Surface Antigen(HBsAg-subtype Ad)，2000μg/mL，购自EastCoast Bio；检测试剂盒：乙型肝炎病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体检测试剂(胶体金法)，购自广州万孚生物技术股份有限公司；阴性血清：购自北京经科宏达生物技术有限公司。
基于抗原检测试剂的抗原中和当量的确定，具体包括以下步骤：
S1稀释抗原2：用阴性血清将已知浓度的抗原2按照1：10，1：20，1：40，1：80，1：160，1：320的梯度进行稀释，每份稀释得到50μL稀释液；
S2抗原2、抗体孵育反应：向每个梯度稀释液分别加入50μL含乙型肝炎病毒表面抗体(HBsAb)的样品，37℃作水浴30min；
S3抗原中和当量的确定：向所述步骤S2获得的每个混合液分别用乙型肝炎病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体检测试剂(胶体金法)进行检 测(检测时是利用试剂盒中的乙型肝炎病毒表面抗原检测试剂检测HBsAg-subtype Ad)，记录数据如下表3；
表3 为抗原2、抗体孵育反应后检测结果数据
抗原稀释梯度 1：10 1：20 1：40 1：80 1：160 1：320
阴阳性 + + + - - -
从记录数据中可以确定，1：80稀释度的混合液中的抗原为50μL抗体标准品的抗原中和当量，经计算得到此含HBsAb的样品的抗原中和当量2为2000μg/mL÷80＝25μg/mL。
将抗体血清分装、冻存；使用时，将分装冻存的抗体血清取出，恢复室温后，重新确定抗体的抗原中和当量。
抗体检测试剂最低检出限的确定方法，具体包括以下步骤：
S4样品的抗体滴度的确定：用阴性血清将含HBsAb的样品按照1：2，1：4，1：8，1：16，1：32，1：64，1：128的梯度进行稀释，获得不同稀释度的梯度稀释液，每份稀释得到50μL稀释液；每个梯度液分别用乙型肝炎病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体检测试剂(胶体金法)进行检测(检测时是利用试剂盒中的乙型肝炎病毒表面抗体检测试剂检测HBsAb)；记录数据如下表4；
表4 为样品(抗体)检测结果数据
抗体稀释梯度 1：2 1：4 1：8 1：16 1：32 1：64 1：128
阴阳性 + + + + + - -
当检测结果中某个稀释度的稀释液出现了阴性反应，则确定该稀释液的前一个较高浓度的稀释液的稀释度为抗体滴度，从而确定抗体滴度；即此含HBsAb的样品的抗体滴度为1：32。
S5抗体检测试剂最低检出限的获得：将所述步骤S3中获得的样品中抗体的抗原中和当量(25μg/mL)与所述步骤S4中确定的样品的抗体滴度(1：32)进行相乘计算，获得抗体检测试剂的最低检出限；即乙型肝炎病毒表面抗原、表面抗体、e抗原、e抗体、核心抗体检测试剂(胶体金法)的最低检出限为25μg/mL÷32＝0.78μg/mL。
对以上抗体检测试剂的最低检出限可以进行如下确认与验证：
S6抗体检测试剂的最低检出限的确认：分别选择不同来源具有代表性的至 少3个临床含待测抗体的样品，将3个临床含待测抗体的样品按照步骤S1～S5获得抗体检测试剂的最低检出限，若获得的含有抗体的样品的最低检出限和步骤S5中测得的最低检出限一致，则将含有抗体的样品分别稀释至最低检出限的浓度，进行20次重复检测，若阳性率均≥90％，则抗体检测试剂的最低检出限通过确认；
当多次重复检测后，阳性率低于90％，则调整步骤S4中的抗体滴度，选取现确定的抗体滴度的上一稀释度为新的抗体滴度，再进行多次重复检测，直至阳性率≥90％；则此时的确认的最低检出限是根据新的抗体滴度重新进行计算获得的；
S7抗体检测试剂的最低检出限的验证：选择具有时间和区域特征性的3个临床样品(且与步骤S6中最低检出限确定所使用的样品不同)，将3个临床样品稀释至最低检出限的浓度进行20次检测，若达到90％～95％阳性检出率，则最低检出限通过验证；
或者步骤S7抗体检测试剂的最低检出限的验证的方法为：将3个临床样品稀释至最低检出限的浓度，先做3次检测，若均阳性则通过验证；若3次检测中出现一次阴性，则再做7次检测，若均阳性则通过验证；若3次检测中出现两次阴性，则再做18次检测，若均阳性则通过验证；若3次检测中均为阴性，则验证不通过(当一次性采用20次及20次以上进行检测时，会耗费更多的时间，则可以采用该递进方式进行抗体检测试剂的最低检出限的验证)。
对于本领域的普通技术人员而言，具体实施例只是对本发明进行了示例性描述，显然本发明具体实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种非实质性的改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围之内。

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  1. 一种基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，具体包括以下步骤：
    S1稀释抗原：将已知纯度和浓度的抗原用基质进行梯度稀释，获得一系列不同浓度的梯度稀释液；
    S2抗原、抗体孵育反应：向所述步骤S1获得的每个不同浓度的梯度稀释液中分别加入等量的抗体标准品或含有抗体的样品，反应生成混合液；
    S3抗原中和当量的确定：向所述步骤S2获得的每个混合液中加入与所述步骤S1中抗原相对应的特异性抗原检测试剂进行检测，记录数据，从而确定抗原中和当量。
  2. 根据权利要求1的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，当确定过抗原中和当量的抗体标准品或含有抗体的样品，需重新确定其抗原中和当量时，则重新进行所述步骤S1～S3获得该抗体标准品或含有抗体的样品的抗原中和当量。
  3. 根据权利要求2的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，当重新确定该抗体标准品或含有抗体的样品的抗原中和当量时，所述步骤S1中的抗原为原抗原或与原抗原具有相同的抗原决定簇能被同一种抗体特异性识别的不同抗原。
  4. 根据权利要求1的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，所述步骤S3中对每个混合液中加入特异性抗原检测试剂进行检测时，当检测结果中某个稀释度的混合液出现阴性反应，则确定该混合液对应的稀释度的抗原量为该标准品或样品所含抗体的抗原中和当量。
  5. 根据权利要求1的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，所述步骤S1中的抗原在不同次数使用时为具有相同的抗原决定簇，能被同一种抗体特异性识别的相同的或不同的抗原。
  6. 根据权利要求1的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，所述步骤S2中向每个不同浓度的抗原稀释液中分别加入等量的抗体标准品或含有特异性抗体的样品，经37℃恒温反应30min后，生成混合液。
  7. 根据权利要求4的基于抗原检测试剂的抗原中和当量的确定方法，其特征在 于，所述步骤S1中抗原的梯度稀释的稀释度分别为：1：10、1：20、1：80、1：160和1：320。
  8. 根据权利要求4的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，还包括步骤S4样品的抗体滴度的确定：将所述步骤S3中含有抗体的样品用基质进行梯度稀释，获得不同稀释度的梯度稀释液；采用抗体检测试剂对每个稀释度的梯度稀释液进行抗体检测，从而确定样品的抗体滴度。
  9. 根据权利要求8的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，还包括步骤S5抗体检测试剂最低检出限的获得：将所述步骤S3中确定的样品的抗原中和当量与所述步骤S4中确定的样品的抗体滴度进行相乘计算，获得抗体检测试剂的最低检出限。
  10. 根据权利要求8的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，所述步骤S4中含有抗体的样品用基质进行梯度稀释的稀释度分别为：1：2，1：4，1：8，1：16，1：32，1：64，1：128。
  11. 根据权利要求8的基于抗原检测试剂的抗原中和当量的确定方法，其特征在于，所述步骤S4中用抗体检测试剂对每个稀释度的稀释液进行抗体检测，当检测结果中某个稀释度的稀释液出现了阴性反应，则确定该稀释液的前一个较高浓度的稀释液的稀释度为抗体滴度。
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