WO2022012673A1 - 抗体标准品赋值和抗体检测试剂最低检出限确定的方法 - Google Patents

抗体标准品赋值和抗体检测试剂最低检出限确定的方法 Download PDF

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WO2022012673A1
WO2022012673A1 PCT/CN2021/106796 CN2021106796W WO2022012673A1 WO 2022012673 A1 WO2022012673 A1 WO 2022012673A1 CN 2021106796 W CN2021106796 W CN 2021106796W WO 2022012673 A1 WO2022012673 A1 WO 2022012673A1
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antibody
detection limit
detection reagent
dilution
minimum
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PCT/CN2021/106796
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English (en)
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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Priority to US17/762,088 priority Critical patent/US11630113B2/en
Priority to EP21843122.9A priority patent/EP4187249A4/en
Publication of WO2022012673A1 publication Critical patent/WO2022012673A1/zh

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the invention relates to the technical field of medical detection, in particular to a method for assigning antibody standard products and determining the minimum detection limit of antibody detection reagents.
  • IVD In Vitro Diagnosis
  • diagnostic reagents are one of the basic tools to detect whether a patient is sick or not.
  • the accuracy of the results directly affects the doctor's diagnosis and the patient's health and life safety.
  • the detection limit refers to the lowest value that can be distinguished from noise when the biological sample is processed and detected according to the requirements of the analytical method and reported with a certain confidence level. It is also used to refer to the minimum detectable concentration, and it can be seen that the 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.
  • the activity of the antibody is unstable, the antigen corresponding to the antibody is generally stable and can be accurately quantified.
  • the neutralization reaction is the reaction between the antigenic determinant and the Fab at the recognition end of the antibody
  • the activity of the antibody can be quantified according to how much antigen the antibody needs to neutralize its activity, that is, assigning a value to the antibody.
  • NAE Neutralized Antigen Equivalent
  • the amount of antibody is quantified by the size of the antigen neutralizing equivalent. The larger the antigen neutralizing equivalent of the antibody, the more antibody is in a unit volume of matrix. In this way, we can quantify antibodies, assign values to antibody reference materials, and determine the minimum detection limit of antibody detection reagents.
  • the technical problem to be solved by the present invention is to provide a method for the assignment of antibody standard products and the determination of the minimum detection limit of antibody detection reagents. It can also be used as an assignment method for the activity of antibody standards.
  • the technical solution adopted in the present invention is: the method for assigning the antibody standard product and determining the minimum detection limit of the antibody detection reagent, which specifically includes the following steps:
  • Determination of antigen neutralization equivalent of S1 antibody standard or sample Dilute the antigen of known purity and concentration with matrix to obtain multiple gradient dilutions of different concentrations; add to each gradient dilution of different concentrations respectively The same amount of antibody standards or samples containing antibodies, after the reaction, use the primary antibody detection reagent to detect the antibody in each mixture, so as to determine the antigen neutralization equivalent of the antibody standard or sample; the step S1 antibody standard Or the method for determining the antigen neutralization equivalent of the sample is the method for assigning the amount of antibody in a certain volume.
  • the unit of measurement of the antigen neutralization equivalent of the antibody standard in a certain volume or the antibody in the sample is expressed by the mass-volume concentration of the antigen in which the antibody can neutralize. Therefore, add "NAE" after the mass unit, such as: 1ng NAE/mL, 1 ⁇ g NAE/mL, 1mg NAE/mL, etc.
  • Matrix which refers to everything in the book except the analyte. In terms of serum cholesterol (Chol) measurement, it refers to all components in serum other than Chol. Since antigens or antibodies generally exist in serum, the matrix of the present invention specifically refers to serum components (or dilution buffer components) other than "antigen” in "antigen of known purity and concentration”; and “antibody standards” or the serum components (or dilution buffer components) other than the “antibody standard” and “antibody” in the “sample containing antibodies”.
  • a further improvement of the present invention is that it also includes the following step S2 for determining the antibody titer of the sample: performing gradient dilution of the antibody-containing sample in step S1 with a matrix to obtain gradient dilutions of different dilutions; using a second antibody detection reagent Antibody detection is performed on serial dilutions of each dilution to determine the antibody titer of the sample;
  • the method of assigning the antibody standard product and determining the minimum detection limit of the antibody detection reagent firstly determines the antigen neutralization equivalent (A) of the antibody standard product or sample; and then determines the antibody titer (B) of the sample; thus the antibody
  • the minimum detection limit of detection reagents A*B; because the antigen is usually stable and quantifiable, it can neutralize the antibody, and the neutralization reaction is the reaction between the antigenic determinant and the Fab at the recognition end of the antibody, and then use the specificity Antibody detection reagent is used to determine the minimum detection limit of its antibody; wherein the antigen used in step S1 is of known purity and concentration, and after gradient dilution, an equal amount of antibody standard or sample containing antibody is added, and the sample can be mixed.
  • the sample can be diluted with the original sample, which can be properly and accurately diluted, and then tested after the reaction.
  • the gradient dilutions there will be a positive reaction at one dilution, and the amount of the antigen at the higher dilution before the positive reaction is Antigen neutralization equivalent of antibody standard or sample, if the sample is a diluted sample, the dilution factor shall be taken into consideration; the antibody titer of the sample in step S2 adopts the same antibody-containing sample as in step S1 through gradient dilution.
  • the first antibody detection reagent in step S1 and the second antibody detection reagent in step S2 may be different kinds of detection reagents, and the first antibody detection reagent used in step S1 may be the second antibody detection reagent used in step S2 High-level, more accurate, and lower detection limit detection reagents.
  • a further improvement of the present invention is that it also includes the confirmation of the minimum detection limit of the antibody detection reagent in step S4: obtaining the minimum detection limit of the second antibody detection reagent for at least three samples containing antibodies according to steps S1 to S3, if the obtained The minimum detection limit of the antibody-containing sample is consistent with the minimum detection limit obtained in step S3, then the antibody-containing sample is diluted to the concentration of the minimum detection limit, and repeated detection is carried out multiple times, if the positive rate is ⁇ 90% , the minimum detection limit of the secondary antibody detection reagent is confirmed. Select at least 3 representative clinical samples containing the antibody to be tested from different sources for confirmation.
  • a further improvement of the present invention is that it also includes the verification of the minimum detection limit of the antibody detection reagent in step S5: at least three samples containing antibodies are diluted to the concentration of the minimum detection limit, and repeated detection is carried out multiple times. % positive detection rate, the minimum detection limit is verified. Select at least 3 clinical samples with temporal and regional characteristics (and are different from the samples used for the minimum detection limit confirmation in step S4), preferably 20 repeated detections, so that the verification can be further confirmed through steps S1 ⁇ S3. Whether the minimum detection limit of the antibody detection reagent is accurate.
  • the dilution degrees of the gradient dilution of the antigen in the step S1 are respectively: 1:2, 1:4, 1:8, 1; 16, 1:32, 1:64, 1: 128 and 1:256.
  • step S1 an equal amount of antibody standard or a sample containing specific antibodies is added to each gradient dilution solution respectively, reacted at 37°C for 30 min, and then each mixed solution is treated with the first step.
  • an antibody detection reagent is used for detection, when a certain dilution of the mixture has a positive reaction in the test result, the antigenic amount of the mixture with the previous higher concentration of the mixture is determined as the antigen neutralization equivalent of the antibody standard or sample.
  • the gradient dilutions of the antibody-containing samples in step S2 are: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1, respectively. : 128; in the step S2, the second 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 detection result, the previous dilution of the dilution is determined.
  • the dilution of the higher concentration dilution is the antibody titer of the sample.
  • the antigen neutralization equivalent of the sample determined in the step S1 and the antibody titer of the sample determined in the step S2 are multiplied and calculated to obtain the second antibody detection.
  • the minimum detection limit of the reagent is the minimum detection limit of the reagent.
  • the antibody titer in step S2 is adjusted, and the previous dilution of the currently determined antibody titer is selected as For the new antibody titer, repeat the test several times until the positive rate is ⁇ 90%; then the second minimum detection limit confirmed at this time is recalculated according to the new antibody titer.
  • the present invention also includes the verification of the minimum detection limit of the antibody detection reagent in step S5: dilute at least three samples containing antibodies to the concentration of the minimum detection limit, and perform three tests first, if all are positive, then Pass the verification; if one of the three tests is negative, then do another seven tests, if all are positive, then pass the verification; if two of the three tests are negative, then do another 18 tests, and if they are all positive, pass the verification; If all three tests are negative, the verification fails.
  • this progressive method can be used to verify the minimum detection limit of the second antibody detection reagent.
  • step S4 at least three representative clinical samples containing the antibody to be tested from different sources are respectively selected to confirm the minimum detection limit of the second antibody detection reagent; in the step S5, During the verification of the minimum detection limit of the second antibody detection reagent, at least 3 clinical samples with time and region characteristics are selected and are different from the samples used in the confirmation of the minimum detection limit in step S4.
  • the first antibody detection reagent in step S1 is a higher-level antibody detection reagent or the same antibody detection reagent as the second antibody detection reagent in step S2, and the antibody used in step S1
  • the detection reagent can be more accurate and have lower detection limit than the antibody detection reagent used in step S2.
  • the method for assigning antibody standard products and determining the minimum detection limit of antibody detection reagents of the present invention has the beneficial effects as follows: the antigenic properties are stable and can be accurately quantified, while the antibody properties are unstable. Assigning value to antibody standards can be used for the preparation of enterprise standards and even national standards; and it can accurately determine the minimum detection limit of antibody detection reagents, which solves the problem that the minimum detection limit of antibody detection reagents on the market cannot be determined or the determined value is inconsistent.
  • the problem of accuracy is an innovative solution to accurately determine the minimum detection limit of antibody detection reagents. By accurately determining the minimum detection limit of antibody detection reagents, it can ensure the stability of reagent quality, improve detection accuracy, and improve clinical efficiency.
  • Embodiment 1 the assignment method of antibody standard substance, specifically comprises the following steps:
  • Determination of Antigen Neutralization Equivalent of S1 Antibody Standard Dilute the antigen of known purity and concentration with matrix to obtain a series of gradient dilutions of different concentrations; add an equal amount to each dilution of different concentrations respectively Antibody standard, after the reaction, use the first antibody detection reagent to detect the antibody in each mixture, so as to determine the antigen neutralization equivalent of the antibody standard, and assign the value to the antibody standard; among them, the amount of antibody in the antibody standard Values are expressed in mass-volume concentration units of their antigen-neutralizing equivalents.
  • This embodiment is to prepare the antibody standard substance (standard substance) of novel coronavirus (COVID-19), and its assignment method is:
  • Determination of the antigen neutralization equivalent of the antibody standard use recombinant novel coronavirus antigen (S protein), the antigen concentration is 400 ⁇ g/mL, and the matrix (negative serum) is used for gradient dilution, and the dilution gradient is 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256 to obtain 50 ⁇ L of each dilution; add an equal volume of 50 ⁇ L of antibody serum to each serial dilution, respectively, and react at 37°C for 30 min Then use antibody detection reagent to test the antibody of the mixture.
  • S protein novel coronavirus antigen
  • the matrix negative serum
  • the amount of antigen in the mixture of 1:32 dilution is determined to be the antigen neutralization equivalent of 50 ⁇ L antibody standard.
  • the minimum detection limit of the antibody detection reagent can also be obtained through steps S2 and S3, and the minimum detection limit of the antibody detection reagent can be obtained through steps S4 and S5.
  • the detection limit was confirmed and verified (see Example 2 below for details).
  • Embodiment 2 The method for determining the minimum detection limit of an antibody detection reagent, which specifically includes the following steps:
  • the sample containing the antibody was diluted with the matrix, and the dilution of the gradient was: 1:2, 1:4, 1:8, 1:16, 1:32, 1: 64 and 1:128, to obtain gradient dilutions of different dilutions; use the secondary antibody detection reagent to perform antibody detection on the dilutions of each dilution. Determine the dilution of the previous higher concentration dilution of the dilution as the antibody titer, thereby determining the antibody titer;
  • step S2 When the positive rate is lower than 90% after repeated detection, adjust the antibody titer in step S2, select the previous dilution of the determined antibody titer as the new antibody titer, and perform multiple repeated detections. Until the positive rate is ⁇ 90%; then the minimum detection limit of confirmation at this time is recalculated according to the new antibody titer;
  • Validation of the minimum detection limit of the S5 antibody detection reagent select 3 clinical samples with temporal and regional characteristics (and are different from the samples used for the minimum detection limit determination in step S4), and dilute the 3 clinical samples to the minimum The concentration of the detection limit is tested 20 times. If the positive detection rate reaches 90% to 95%, the minimum detection limit will pass the verification;
  • the method for verifying the minimum detection limit of the antibody detection reagent in step S5 is: dilute the three clinical samples to the concentration of the minimum detection limit, do three tests first, and pass the verification if they are all positive; If it is negative once, then do 7 more tests, if all are positive, it will pass the verification; if two of the 3 tests are negative, then do 18 more tests, if all are positive, then pass the verification; if all 3 tests are negative, Then the verification fails (when the detection is performed 20 times or more at one time, it will take more time, then this progressive method can be used to verify the minimum detection limit of the second antibody detection reagent).
  • the antigen used respiratory syncytial virus RSV F protein 1000 ⁇ g/mL, purchased from absolute antibody;
  • the detection kit used the respiratory syncytial virus IgM antibody detection kit (colloidal gold method), purchased from Beijing Innotek Biotechnology Co., Ltd.;
  • Negative serum purchased from Beijing Jingke Hongda Biotechnology Co., Ltd.;
  • S1-1 dilution antigen the antigen is serially diluted with matrix-negative serum, the dilution of RSV F protein of respiratory syncytial virus is 1:2, 1:4, 1:8, 1:16, 1:32, 1 : 64, 1:128 and 1:256, 50 ⁇ L samples were obtained from each dilution, and multiple gradient dilutions of different concentrations were obtained;
  • Detection after S1-2 antigen/antibody incubation Add 50 ⁇ L of RSV IgM-containing samples to each gradient dilution solution, and after 30 min in a water bath at 37°C, each mixed solution was detected with a respiratory syncytial virus IgM antibody detection kit (colloid). Gold method) to detect, record data such as table 1;
  • Table 1 is the detection result data after antigen/antibody incubation
  • S2-2 antibody detection use the respiratory syncytial virus IgM antibody detection kit (colloidal gold method) to detect the antibody of each dilution of the dilution, and the recorded data are shown in Table 2;
  • Table 2 is the sample (antibody) detection result data
  • S2-3 Determine the antibody titer of the sample: when a certain dilution of the dilution in the test results has a negative reaction, then determine the dilution of the dilution with the previous higher concentration as the antibody titer, so that Determine the antibody titer of the sample, that is, the antibody titer is 1:32;
  • Confirmation of the minimum detection limit of S4 antibody detection reagent select at least 3 clinical samples containing the antibody to be tested representative of different sources, and obtain the minimum detection limit of the antibody from the 3 clinical samples containing the antibody to be tested according to steps S1 to S3 Detection limit, if the obtained minimum detection limit of the antibody-containing sample is consistent with the minimum detection limit measured in step S3, then the antibody-containing sample is diluted to the concentration of the minimum detection limit, and repeated detection is carried out 20 times , if the positive rate is ⁇ 90%, the minimum detection limit of the secondary antibody detection reagent is confirmed;
  • step S2 When the positive rate is lower than 90% after repeated detection for many times, adjust the antibody titer in step S2, and select the dilution of the previous higher concentration of the determined antibody titer as the new antibody titer. Repeat the test several times until the positive rate is ⁇ 90%; then the minimum detection limit for confirmation at this time is recalculated based on the new antibody titer;
  • Validation of the minimum detection limit of the S5 antibody detection reagent select 3 clinical samples with temporal and regional characteristics (and are different from the samples used for the minimum detection limit confirmation in step S4), and dilute the 3 clinical samples to the minimum The concentration of the detection limit is tested for multiple times. If the positive detection rate reaches 90% to 95%, the minimum detection limit will pass the verification;
  • the verification method of the minimum detection limit of the S5 antibody detection reagent is: dilute 3 clinical samples to the concentration of the minimum detection limit, do 3 tests first, and pass the verification if they are all positive; Negative, then do 7 more tests, if they are all positive, then pass the verification; if two of the 3 tests are negative, then do 18 more tests, if they are all positive, then pass the verification; if all 3 tests are negative, then If the verification fails (if the detection is performed 20 times or more at one time, it will take more time, then this progressive method can be used to verify the minimum detection limit of the secondary antibody detection reagent).

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Abstract

一种抗体标准品赋值和抗体检测试剂最低检出限确定的方法，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；S2样品的抗体滴度的确定：将步骤S1中含有抗体的样品用基质进行梯度稀释，用第二抗体检测试剂对每个稀释度的梯度稀释液进行抗体检测确定抗体滴度；S3抗体检测试剂最低检出限的获得：将确定的样品的抗原中和当量与确定的样品的抗体滴度进行相乘计算，获得第二抗体检测试剂的最低检出限。

Description

抗体标准品赋值和抗体检测试剂最低检出限确定的方法 技术领域
本发明涉及医学检测技术领域，尤其涉及一种抗体标准品的赋值以及抗体检测试剂最低检出限的确定的方法。
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。
因此，有必要开发一种能够准确定量抗体的方法。虽然抗体的活性不稳定，但是和抗体对应的抗原通常比较稳定，而且可以准确定量。利用抗原可以和抗体发生中和反应(中和反应是抗原决定簇和抗体识别端Fab的反应)，可根据抗体需要多少抗原来中和其活性来定量抗体活性，也就是给抗体赋值。我们把单位体积基质(如血清)中的抗体需要多少相应的抗原来中和称作抗体的“抗原中和当量(Neutralized Antigen Equivalent，NAE)”。用抗原中和当量的大小来定量抗体的多少，抗体的抗原中和当量越大表示在单位体积基质中的抗体越多。借此，我们就可以定量抗体，给抗体标准物质赋值，确定抗体检测试剂的最低检出限。
发明内容
本发明要解决的技术问题是，提供一种抗体标准品赋值和抗体检测试剂最低检出限确定的方法，此方法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，也可以作为抗体标准品活性的赋值方法。
为了解决上述技术问题，本发明采用的技术方案为：该抗体标准品赋值和抗体检测试剂最低检出限确定的方法，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，获得多个不同浓度的梯度稀释液；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，从而确定抗体标准品或样品的抗原中和当量；所述步骤S1抗体标准品或样品的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。
基质，指标本中除分析物以外的一切成分。以血清胆固醇(Chol)测定而言，就是指Chol以外血清中的一切成分。由于抗原或抗体一般存在于血清中，所以本发明的基质具体指“已知纯度和浓度的抗原”中除了“抗原”之外的血清成分(或稀释用缓冲液成分)；以及“抗体标准品或含有抗体的样品”中除了“抗体标准品”与“抗体”之外的血清成分(或稀释用缓冲液成分)。
本发明进一步改进在于，还包括以下步骤S2样品的抗体滴度的确定：将所述步骤S1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；用第二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；
S3抗体检测试剂最低检出限的获得：将所述步骤S1中确定的样品的抗原中和当量与所述步骤S2中确定的样品的抗体滴度进行相乘计算，获得第二抗体检测试剂的最低检出限。
采用上述技术方案，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，进行梯度稀释后加入等量的抗体标准品或含有抗体的样品，样品可以是混合样品，可以用原样品，可适当准确稀释，反应后进行检测，在所有梯度稀释度中会有一个稀释度出现阳性反应，而出现阳性反应的前一个较高浓度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，需考虑稀释因素；其中步骤S2中的样品的抗体滴度采用与步骤S1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。其中步骤S1中的第一抗体检测试剂与所述步骤S2中的第二抗体检测试剂可以是不同种检测试剂，步骤S1所用的第一抗体检测试剂可以是步骤S2中所用的第二抗体检测试剂的高一级的、更加精准、检出限更低的检测试剂。
本发明进一步改进在于，还包括步骤S4抗体检测试剂的最低检出限的确认：将至少三个含有抗体的样品，按照步骤S1～S3获得第二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S3中获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，进行多次重复检测，若阳性率均≥90％，则第二抗体检测试剂的最低检出限通过确认。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进行确认，首先确定抗原中和当量(A)；确定抗体的滴度(B)；从而抗体检测试剂最低检出限＝A*B；再将含抗体的样品稀释至最低检出限的浓度，确认通过步骤S1～S3获得的最低检出限是否准确。
本发明进一步改进在于，还包括步骤S5抗体检测试剂的最低检出限的验证：将至少三个含有抗体的样品稀释至最低检出限的浓度，进行多次重复检测，若达到90％～95％阳性检出率，则最低检出限通过验证。选择具有时间和区域特征性的至少3个临床样品(且与步骤S4中最低检出限确认所使用的样品不同)，优选进行20次重复检测，这样通过验证可以进一步确认通过步骤S1～S3获得的抗体检测试剂的最低检测出限是否准确。
作为本发明的优选技术方案，所述步骤S1中的抗原的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和1：256。
作为本发明的优选技术方案，所述步骤S1中向每个梯度稀释液中分别加入等量的抗体标准品或含有特异抗体的样品，经37℃反应30min，后对每个混合液分别用第一抗体检测试剂进行检测，当检测结果中某个稀释度的混合液出现阳性反应，则确定该混合液的前一个较高浓度的混合液的抗原量为抗体标准品或样品的抗原中和当量。
作为本发明的优选技术方案，所述步骤S2中的含有抗体的样品的梯度稀释度分别为：1：2、1：4、1：8、1：16、1：32、1：64和1：128；所述步骤S2中用第二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样品的抗体滴度。
作为本发明的优选技术方案，所述步骤S3中将所述步骤S1中确定的样品的抗原中和当量与所述步骤S2中确定的样品的抗体滴度进行相乘计算，获得第二抗体检测试剂的最低检出限。
作为本发明的优选技术方案，所述步骤S4中当多次重复检测后，阳性率低于90％，则调整步骤S2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％；则此时的确认的第二最低检出限是根据新的抗体滴度重新进行计算获得的。
作为本发明的优选技术方案，还包括步骤S5抗体检测试剂的最低检出限的验证：将至少三个含有抗体的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第二抗体检测试剂的最低检出限的验证。
作为本发明的优选技术方案，所述步骤S4中分别选取不同来源具有代表性的至少三个临床含待测抗体的样品进行第二抗体检测试剂最低检出限的确认；所述步骤S5中进行第二抗体检测试剂的最低检出限的验证时选取具有时间和区域 特征性的至少3个临床样品且与步骤S4中最低检出限确认时所使用的样品不同。
作为本发明的优选技术方案，所述步骤S1中的第一抗体检测试剂为所述步骤S2中的第二抗体检测试剂的高一级抗体检测试剂或同一种抗体检测试剂，步骤S1所用的抗体检测试剂可以比步骤S2中所用的抗体检测试剂更加精准和检出限更低。
与现有技术相比，本发明的抗体标准品赋值和抗体检测试剂最低检出限确定的方法具有的有益效果为：抗原性质稳定，可以精准定量，而抗体性质不稳定，此方法利用抗原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；而且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，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1：抗体标准品的赋值方法，具体包括以下步骤：
S1抗体标准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，获得一系列不同浓度的梯度稀释液；向每个不同浓度的稀释液中分别加入等量的抗体标准品，反应后，用第一抗体检测试剂对每个混合液进行抗体检测，从而确定抗体标准品的抗原中和当量，以此对抗体标准品赋值；其中，抗体标准品中抗体的量值用其抗原中和当量的质量-体积浓度单位表示。
该实施例为制备新型冠状病毒(COVID-19)的抗体标准品(标准物质)，其赋值方法为：
收集恢复期新型冠状病毒患者的高滴度抗体血清，混合均匀，作为抗体标准品；
抗体标准品的抗原中和当量的确定：用重组新型冠状病毒抗原(S蛋白)，抗原浓度为400μg/mL，用基质(阴性血清)进行梯度稀释，稀释梯度为1：2、1：4、1：8、1：16、1：32、1：64、1：128和1：256，得到每份稀释液50μL；向每个梯度稀释液中分别加入等体积50μL抗体血清，37℃反应30min后以抗 体检测试剂对混合液进行抗体检测，1：64稀释度的混合液出现阳性反应，则确定1:32稀释度的混合液中的抗原量为50μL抗体标准品的抗原中和当量，经计算得到抗体标准品的抗体浓度为400μg/mL÷32＝12.5μgNAE/mL，即新型冠状病毒(COVID-19)的抗体标准品赋值为12.5μgNAE/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标准品的抗原中和当量，即重新对抗体标准品赋值，保证抗体标准品的量值精准确定，避免抗体标准品因活性下降而造成临床诊断误差。
利用上述抗体标准品的赋值方法，即样品的抗原中和当量的确定方法，还可以通过步骤S2，S3来获得抗体检测试剂的最低检出限，通过步骤S4，S5来对抗体检测试剂的最低检出限进行确认和验证(详见以下实施例2的内容)。
实施例2：抗体检测试剂最低检出限的确定方法，具体包括以下步骤：
S1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，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和1：256，获得多个不同浓度的梯度稀释液；向每个梯度稀释液中分别加入等量的含有特异抗体的样品，经37℃反应30min，后对每个混合液分别用特异抗体检测试剂(第一抗体检测试剂)进行检测，当检测结果中某个稀释度的混合液出现阳性反应，则确定该混合液的前一个较高浓度的混合液的抗原量为该样品的抗原中和当量；
S2样品的抗体滴度的确定：将含有抗体的样品用基质进行梯度稀释，梯度稀释的稀释度分别为：1：2、1：4、1：8、1：16、1：32、1：64和1：128，获得不同稀释度的梯度稀释液；采用第二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，从而确定抗体滴度；
S3抗体检测试剂最低检出限的获得：将所述步骤S1中获得的样品中抗体的抗原中和当量与所述步骤S2中确定的样品的抗体滴度进行相乘计算，获得第二抗体检测试剂的最低检出限。
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：
S4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～S3 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S3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％，则第二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S2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；
S5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S4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S5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第二抗体检测试剂的最低检出限的验证)。
采用上述抗体检测试剂最低检出限的确定方法进行具体检测应用，确定呼吸道合胞病毒IgM抗体检测试剂盒(胶体金法)的最低检出限时，具体包括以下步骤：
其中试验材料：
抗原使用呼吸道合胞病毒RSV F protein，1000μg/mL，购自absolute antibody；
检测试剂盒使用呼吸道合胞病毒IgM抗体检测试剂盒(胶体金法)，购自北京英诺特生物技术有限公司；
阴性血清：购自北京经科宏达生物技术有限公司；
S1样品的抗原中和当量的确定：
S1-1稀释抗原：将抗原用基质-阴性血清进行梯度稀释，呼吸道合胞病毒RSV F protein的稀释度分别为1：2、1：4、1：8、1：16、1：32、1：64、1：128和 1：256，每份稀释得到50μL样品，获得多个不同浓度的梯度稀释液；
S1-2抗原/抗体孵育后检测：向每个梯度稀释液中分别加入50μL含RSV IgM的样品，37℃作水浴30min后，每个混合液分别用呼吸道合胞病毒IgM抗体检测试剂盒(胶体金法)进行检测，记录数据如表1；
表1为抗原/抗体孵育后检测结果数据
抗原稀释梯度 1：2 1：4 1：8 1：16 1：32 1：64 1：128 1：256
阴阳性 - - - - - - + +
S1-3确定样品的抗原中和当量：当检测结果中某个稀释度的混合液出现阳性反应，则确定该混合液的前一个较高浓度的混合液的抗原量为样品中抗体的抗原中和当量；
即此含RSV IgM的样品的抗原中和当量为1000μg/mL÷64＝15.625μgNAE/mL；
S2样品的抗体滴度的确定：
S2-1稀释样品：将含RSV IgM的样品用阴性血清进行梯度稀释，样品的稀释度分别为1：2，1：4，1：8，1：16，1：32，1：64，1：128，获得不同稀释度的梯度稀释液；
S2-2抗体检测：用呼吸道合胞病毒IgM抗体检测试剂盒(胶体金法)对每个稀释度的稀释液进行抗体检测，记录数据如表2；
表2为样品(抗体)检测结果数据
Figure PCTCN2021106796-appb-000001
S2-3确定样品的抗体滴度：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的浓度的稀释液的稀释度为抗体滴度，从而确定样品的抗体滴度，即抗体滴度为1：32；
S3抗体检测试剂最低检出限的获得：将所述步骤S1中确定的样品的抗原中和当量与所述步骤S2中确定的样品的抗体滴度进行相乘计算，获得第二抗体检测试剂的最低检出限；
即呼吸道合胞病毒IgM抗体检测试剂盒(胶体金法)的最低检出限为15.625μgNAE/mL×(1:32)＝0.488μgNAE/mL；
将上述应用实施例的数据经过步骤S4和步骤S5的确认与验证；
S4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～S3获得抗体的最低检出限，若获得的含有抗体的样品的最低检出限和步骤S3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％，则第二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S2中的抗体滴度，选取现确定的抗体滴度的前一个较高浓度的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；
S5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S4中最低检出限确认所使用的样品不同)，将3个临床样品稀释至最低检出限的浓度进行检测多次检测，若达到90％～95％阳性检出率，则最低检出限通过验证；
或者S5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第二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。

Claims (12)

  1. 一种抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，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，获得一系列不同浓度的梯度稀释液；向每个不同浓度的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，从而确定抗体标准品或样品的抗原中和当量。
  2. 根据权利要求1所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，还包括以下步骤：
    S2样品的抗体滴度的确定：将所述步骤S1中含有抗体的样品用基质进行梯度稀释，获得一系列不同稀释度的梯度稀释液；用第二抗体检测试剂对每个稀释度的稀释液进行抗体检测，从而确定样品的抗体的滴度；
    S3抗体检测试剂最低检出限的获得：将所述步骤S1中确定的样品的抗原中和当量与所述步骤S2中确定的样品的抗体滴度进行相乘计算，获得第二抗体检测试剂的最低检出限。
  3. 根据权利要求2所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，还包括步骤S4抗体检测试剂的最低检出限的确认：将至少三个含有抗体的样品，按照步骤S1～S3获得第二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S3中获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，进行多次重复检测，若阳性率均≥90％，则第二抗体检测试剂的最低检出限通过确认。
  4. 根据权利要求3所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，还包括步骤S5抗体检测试剂的最低检出限的验证：将至少三个含有抗体的样品稀释至最低检出限的浓度，进行多次重复检测，若达到90％～95％阳性检出率，则第二抗体检测试剂的最低检出限通过验证。
  5. 根据权利要求1所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，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：1：2、1：4、1：8、1：16、1：32、1：64、1：128和1：256。
  6. 根据权利要求5所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方 法，其特征在于，所述步骤S1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抗体的样品，经37℃反应30min，然后对每个混合液分别用第一抗体检测试剂进行检测，当检测结果中某个稀释度的混合液出现阳性反应，则确定该混合液前一个较高浓度的混合液的抗原量为抗体标准品或样品的抗原中和当量。
  7. 根据权利要求3所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，所述步骤S2中的将含有抗体的样品进行稀释的稀释度分别为：1：2、1：4、1：8、1：16、1：32、1：64和1：128；所述步骤S2中用第二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。
  8. 根据权利要求7所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，所述步骤S3中将所述步骤S1中获得的样品的抗原中和当量与所述步骤S2中确定的样品的抗体滴度进行相乘计算，获得第二抗体检测试剂的最低检出限。
  9. 根据权利要求7所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，所述步骤S4中当多次重复检测后，阳性率低于90％，则调整步骤S2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。
  10. 根据权利要求3所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，还包括步骤S5抗体检测试剂的最低检出限的验证：将至少三个含有抗体的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。
  11. 根据权利要求4或10所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，所述步骤S4中分别选取不同来源具有代表性的至少三个含待测抗体的临床样品进行第二抗体检测试剂最低检出限的确认；所述步骤 S5中进行第二抗体检测试剂的最低检出限的验证时选取具有时间和区域特征性的至少3个临床样品且与步骤S4中第二抗体检测试剂最低检出限确认时所使用的样品不同。
  12. 根据权利要求2所述的抗体标准品赋值和抗体检测试剂最低检出限确定的方法，其特征在于，所述步骤S1中的第一抗体检测试剂为所述步骤S2中的第二抗体检测试剂的高一级抗体检测试剂或同一种抗体检测试剂。
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