CN115372606A - Method for determining antigen neutralization equivalent based on antigen detection reagent - Google Patents
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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
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
Abstract
The invention relates to a method for determining antigen neutralization equivalent based on an antigen detection reagent, which specifically comprises the following steps: s1 dilution of antigen: carrying out gradient dilution on the antigen with known purity and concentration by using a matrix to obtain a series of gradient dilution solutions with different concentrations; and (2) incubation reaction of the S2 antigen and the antibody: adding an equal amount of antibody standard substance or antibody-containing sample into each gradient diluent with different concentrations obtained in the step S1, and reacting to generate a mixed solution; determination of neutralizing equivalents of S3 antigen: adding a specific antigen detection reagent corresponding to the antigen in the step S1 to each mixed solution obtained in the step S2 for detection, and recording data to determine the antigen neutralizing equivalent. The antigen detection reagent is used for determining the antigen neutralization equivalent, and the antibody is assigned by the antigen neutralization equivalent and the antibody titer, so that the method can be used for preparing enterprise standards and even national standards.
Description
The application is a divisional application of Chinese patent application with the application number of '202111629284.7', the application date of '12 months and 28 days in 2021', the name of the invention being 'a method for determining the antigen neutralizing equivalent based on an antigen detection reagent'.
Technical Field
The invention relates to the technical field of medical detection, in particular to a method for determining antigen neutralization equivalent based on an antigen detection reagent.
Background
In Vitro Diagnostic (IVD) medical devices are products that obtain clinical diagnostic information by detecting samples (blood, body fluids, tissues, etc.) of the human body, including reagents, calibrators, control substances, devices or systems. Diagnostic reagents are one of the basic tools for detecting the presence and extent of a patient's disease, and the consequences of this are directly related to the diagnosis of the physician and the physical health and life safety of the patient. With the progress of modern medical science and technology, more accurate requirements are put forward on the results of medical examination, so that the quality requirements on in-vitro diagnosis products are higher and higher.
The preparation of standard substances (such as international standards, national standards, enterprise standards, and clinical-application calibrators) and the traceability of the quantity thereof are prerequisites for obtaining accurate measurement results. Due to the poor storage stability of the antibody, the activity gradually decreases with the increase of the storage time even in the case of cryopreservation; furthermore, the activity of the antibody is often expressed in relative dilution titers and is difficult to quantify objectively and precisely. Therefore, it is difficult to prepare a standard or reference substance for each antibody. At present, the commercial supply of related antibody standard products is lacked at home and abroad, and a plurality of insurmountable difficulties are brought to the production and the application of an antibody detection reagent.
On the other hand, one important performance parameter of antibody detection reagents is the detection limit. The detection limit, which is the lowest value at which a biological sample is processed and detected as required by the analytical method and reported to be present at a certain confidence level that can be distinguished from noise, is also used to refer to the minimum detectable concentration and the visible detection limit is also a quantitative indicator. The minimum detection limit of antibody detection reagents is also not precisely determined due to the lack of antibody standards.
Chinese patent document CN113125756A discloses a method for assigning an antibody standard and determining an antigen neutralization equivalent, which specifically comprises the following steps: determination of the antigen-neutralizing equivalent of the S1 antibody standard or sample: carrying out gradient dilution on antigens with known purity and concentration by using a matrix, respectively adding an equal amount of antibody standard substance or antibody-containing sample into each gradient dilution with different concentration, and after reaction, carrying out antibody detection on each mixed solution by using a first antibody detection reagent to determine the antigen neutralization equivalent of the antibody standard substance or sample; where the unit of measure of the antigen neutralizing equivalent of an antibody in a volume of an antibody standard or sample is expressed in terms of the mass-to-volume concentration of the antibody-neutralizable antigen, in order to make clear in particular that the mass number in the mass-to-volume unit is the amount of the corresponding neutralizing antigen, the mass unit is followed by an "NAE" such as: 1ng NAE/mL, 1. Mu.g NAE/mL, 1mg NAE/mL, etc. In the technical scheme, the antibody detection reagent is adopted for determining the antigen neutralization equivalent, but the titer of some antibody detection reagents can be changed after a period of time in the using process to influence the detection result, and the antigen is relatively stable without the problem, so that the method for determining the antigen neutralization equivalent based on the antigen detection reagent needs to be developed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for determining the antigen neutralization equivalent based on an antigen detection reagent, wherein the method uses the antigen detection reagent to determine the antigen neutralization equivalent, and can be used again to quantify an antibody after the antigen neutralization equivalent is determined, and simultaneously can solve the problems that the lowest detection limit of the antibody detection reagent on the market at present cannot be determined or the determination value is inaccurate.
In order to solve the technical problems, the invention adopts the technical scheme that: the method for determining the antigen neutralization equivalent based on the antigen detection reagent specifically comprises the following steps:
s1 dilution of antigen: carrying out gradient dilution on the antigen with known purity and concentration by using a matrix to obtain a series of gradient diluents with different concentrations;
and (2) incubating reaction of the S2 antigen and the antibody: adding an equal amount of antibody standard substance or antibody-containing sample into each gradient dilution with different concentrations obtained in the step S1, and reacting to generate a mixed solution;
determination of neutralizing equivalents of S3 antigen: and (3) adding a specific antigen detection reagent corresponding to the antigen in the step (S1) into each mixed solution obtained in the step (S2) for detection, and recording data so as to determine the antigen neutralization equivalent.
A method for determining the neutralizing equivalent of an antigen is a method for assigning an amount of an antibody in a volume, wherein the unit of measure of the neutralizing equivalent of an antibody in a volume of an antibody standard or sample is expressed in terms of the mass-volume concentration of the neutralizing antigen of the antibody, and in order to make clear in particular that the mass number in the mass-volume unit is the amount of the corresponding neutralizing antigen, the mass unit is followed by an "NAE" such as: 1ng NAE/mL, 1. Mu.g NAE/mL, 1mg NAE/mL, etc. Firstly, determining the antigen neutralization equivalent (A) of an antibody standard product or a sample; determining the titer (B) of the antibody of the sample; such that the antibody detection reagent has a minimum detection limit = a × B; because the antigen is generally stable and quantifiable, and can be neutralized by the antibody, the neutralization reaction is the reaction of an antigenic determinant and an antibody recognition end Fab, and the specific antibody detection reagent is used for determining the minimum detection limit of the antibody; the antigen adopted in the step S1 has known purity and concentration, after gradient dilution is carried out, the same amount of antibody standard substance or a sample containing the antibody is added in the step S2 for mixed reaction, the sample can be a mixed sample, an original sample can be used, and the original sample can be properly and accurately diluted, after the reaction, a specific antigen detection reagent is adopted as a detection reagent for detection, one dilution degree in all the gradient dilution degrees can generate negative reaction, the antigen amount of the dilution degree generating the negative reaction is the antigen neutralization equivalent of the antibody standard substance or the sample, and if the sample is the diluted sample, the dilution factor needs to be considered.
In a preferred embodiment of the present invention, when the antigen-neutralizing equivalent of the antibody standard or the antibody-containing sample is determined and the antigen-neutralizing equivalent thereof needs to be determined again, the above steps S1 to S3 are performed again to obtain the antigen-neutralizing equivalent of the antibody standard or the antibody-containing sample.
In a preferred embodiment of the present invention, when the antigen neutralizing equivalent of the antibody standard or the antibody-containing sample is newly determined, the antigen in step S1 is a primary antigen or a different antigen having the same epitope as the primary antigen and capable of being specifically recognized by the same antibody.
In a preferred embodiment of the present invention, in the step S3, when a negative reaction occurs in a mixture of a certain dilution in the detection result when the specific antigen detection reagent is added to each mixture for detection, the amount of antigen in the dilution corresponding to the mixture is determined to be the antigen neutralization equivalent of the antibody contained in the standard or sample.
In a preferred embodiment of the present invention, the antigens in step S1 are the same or different antigens having the same epitope and being specifically recognized by the same antibody when used at different times. After the antigen neutralization equivalent of the antibody is determined, the amount of the antibody may change after a period of time, the antibody may be re-quantified by the original antigen (re-calculating the antigen neutralization equivalent thereof), or the antigen 2 different from the original used antigen (antigen 1) may be selected to calculate the antigen neutralization equivalent of the antibody contained in the sample, and the antigen 1 and the antigen 2 have the same antigenic determinant and can be specifically recognized by the same antibody.
In a preferred embodiment of the present invention, in step S2, an equal amount of antibody standard or a sample containing a specific antibody is added to each antigen dilution with different concentrations, and the mixture is reacted at 37 ℃ for 30min to generate a mixed solution.
In a preferred embodiment of the present invention, the dilutions of the antigen in the step S1 by gradient dilution are: 1: 10. 1: 20. 1: 80. 1:160 and 1:320.
the invention further improves the method, and further comprises the step S4 of determining the antibody titer of the sample: carrying out gradient dilution on the sample containing the antibody in the step S3 by using a matrix to obtain gradient diluents with different dilutions; and performing antibody detection on the gradient dilution of each dilution by using an antibody detection reagent, thereby determining the antibody titer of the sample.
The invention further improves the method, and further comprises the step S5 of obtaining the lowest detection limit of the antibody detection reagent: and (4) multiplying the antigen neutralization equivalent of the sample determined in the step (S3) by the antibody titer of the sample determined in the step (S4) to obtain the lowest detection limit of the antibody detection reagent. Determining the titer of the antibody (B) based on the determined antigen neutralizing equivalent (A); the lowest detection limit of the antibody detection reagent = a × B can be obtained.
In a preferred embodiment of the present invention, the dilution ratios of the antibody-containing sample in step S4 by gradient dilution with the matrix are: 1:2,1:4,1:8,1:16,1:32,1:64,1:128.
in a preferred embodiment of the present invention, in the step S4, the antibody detection reagent is used to perform antibody detection on each dilution, and when a negative reaction occurs in a dilution of a certain dilution in the detection result, the dilution of a previous dilution with a higher concentration is determined as the antibody titer. By adopting the technical scheme, the antibody titer of the sample in the step S4 is detected after the same sample containing the antibody in the step S2 is subjected to gradient dilution, one dilution in all the gradient dilutions has a negative reaction, and the dilution of the previous dilution with higher concentration, in which the negative reaction occurs, is the antibody titer of the sample.
The minimum detection limit of the antibody detection reagent is obtained as described above, and is confirmed and verified through steps S6, S7. The minimum detection limit of the above antibody detection reagent can be confirmed and verified as follows:
confirmation of minimum detection limit of S6 antibody detection reagent: respectively selecting at least 3 clinical samples containing antibodies to be detected, which are representative from different sources, obtaining the lowest detection limit of an antibody detection reagent by the 3 clinical samples containing the antibodies to be detected according to the steps S1-S5, respectively diluting the samples containing the antibodies to the concentration of the lowest detection limit if the obtained lowest detection limit of the samples containing the antibodies is consistent with the lowest detection limit measured in the step S5, and carrying out repeated detection for 20 times, wherein if the positive rates are more than or equal to 90%, the lowest detection limit of the antibody detection reagent is confirmed;
when the positive rate is lower than 90% after repeated detection for many times, adjusting the antibody titer in the step S4, selecting the last dilution of the currently determined antibody titer as a new antibody titer, and performing repeated detection for many times until the positive rate is more than or equal to 90%; the lowest detection limit for confirmation at this time is recalculated based on the new antibody titer;
verification of the lowest detection limit of the S7 antibody detection reagent: selecting 3 clinical samples with time and regional characteristics (and different from the samples used in the determination of the lowest detection limit in the step S6), diluting the 3 clinical samples to the concentration of the lowest detection limit for 20 times of detection, and if the detection rate reaches 90-95% of positive detection rate, the lowest detection limit passes the verification;
or the method for verifying the lowest detection limit of the antibody detection reagent in the step S7 comprises the following steps: diluting 3 clinical samples to the concentration of the lowest detection limit, performing detection for 3 times, and if all the clinical samples are positive, passing the verification; if the 3 times of detection show negative, then 7 times of detection are carried out, and if the detection shows positive, the verification is passed; if two times of negative results appear in the 3 times of detection, the detection is carried out for 18 times, and if the two times of negative results are all positive results, the verification is passed; if all of the 3 tests are negative, the test does not pass (if the test is performed 20 times or more at a time, more time is consumed, the test of the lowest detection limit of the antibody detection reagent can be performed in this progressive manner).
Compared with the prior art, the method for determining the antigen neutralization equivalent based on the antigen detection reagent has the beneficial effects that:
(1) The antigen detection reagent is used for determining the antigen neutralization equivalent, and the antibody standard substance is assigned by the antigen neutralization equivalent and the antibody titer, so that the method can be used for preparing enterprise standard substances and even national standard substances;
(2) Determining the antigen neutralization equivalent by using an antigen detection reagent, and after the antigen neutralization equivalent is determined and a period of time passes, re-quantifying the antibody by using the original antigen (re-calculating the antigen neutralization equivalent), or selecting an antigen 2 different from the original antigen (antigen 1) to calculate the antigen neutralization equivalent of the antibody contained in the sample;
(3) Meanwhile, the minimum detection limit of the antibody detection reagent can be accurately determined, the problem that the minimum detection limit of the antibody detection reagent on the market cannot be fixed or is inaccurate in fixed value is solved, and the method is an innovative solution for accurately determining the minimum detection limit of the antibody detection reagent at present;
(4) Through the accurate determination of the lowest detection limit of the antibody detection reagent, the quality stability of the reagent can be ensured, the detection accuracy is improved, and the clinical efficiency is improved.
Detailed Description
In order to enhance the understanding of the present invention, the present invention will be described in further detail with reference to the following examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the present invention.
Example (b): the method for determining the antigen neutralization equivalent based on the antigen detection reagent specifically comprises the following steps:
s1 dilution of antigen: carrying out gradient dilution on the antigen with known purity and concentration by using a matrix to obtain a series of gradient dilution solutions with different concentrations; when the antigen neutralization equivalent of the antibody standard or the sample containing the antibody is determined and needs to be determined again, the steps S1 to S3 are carried out again to obtain the antigen neutralization equivalent of the antibody standard or the sample containing the antibody; when the antigen neutralizing equivalent of the antibody standard or the antibody-containing sample is newly determined, the antigen in step S1 is a primary antigen or a different antigen having the same antigenic determinant as the primary antigen and capable of being specifically recognized by the same antibody; wherein the antigens used at different times may be the same or different antigens having the same antigenic determinant and being specifically recognized by the same antibody; after the antigen neutralization equivalent of the antibody is determined, the antibody can be quantified again by using the original antigen (the antigen neutralization equivalent is recalculated) after a period of time, or the antigen 2 different from the original antigen (antigen 1) can be selected to calculate the antigen neutralization equivalent of the antibody contained in the sample, and the antigen 1 and the antigen 2 have the same antigenic determinant and can be specifically recognized by the same antibody; the dilution of the gradient dilution of the antigen in the step S1 is respectively as follows: 1: 10. 1: 20. 1: 80. 1:160 and 1:320, each dilution gives 50 μ L of dilution;
and (2) incubating reaction of the S2 antigen and the antibody: adding an equal amount of antibody standard substance or antibody-containing sample into each gradient dilution with different concentrations obtained in the step S1, and reacting to generate a mixed solution; in the step S2, an equal amount of antibody standard substance or a sample containing a specific antibody is respectively added into each antigen diluent with different concentrations, and a mixed solution is generated after constant temperature reaction at 37 ℃ for 30min;
determination of neutralizing equivalents of S3 antigen: adding a specific antigen detection reagent corresponding to the antigen in the step S1 into each mixed solution obtained in the step S2 for detection, and recording data so as to determine the antigen neutralization equivalent;
determination of antibody titer of S4 samples: carrying out gradient dilution on the sample containing the antibody in the step S3 by using a matrix to obtain gradient diluents with different dilutions; performing antibody detection on the gradient diluent of each dilution by using an antibody detection reagent so as to determine the antibody titer of the sample; the dilution of the sample containing the antibody in step S4 by the matrix gradient dilution is respectively as follows: 1:2,1:4,1:8,1:16,1:32,1:64,1: 128; the antibody titer of the sample in the step S4 is detected by adopting the same sample containing the antibody as that in the step S2 after gradient dilution, one dilution in all the gradient dilutions has a negative reaction, and the dilution of the previous dilution with higher concentration, in which the negative reaction occurs, is the antibody titer of the sample;
obtaining the lowest detection limit of the S5 antibody detection reagent: and (3) multiplying the antigen neutralizing equivalent of the sample determined in the step (S3) and the antibody titer of the sample determined in the step (S4) to obtain the lowest detection limit of the antibody detection reagent. Determining the titer of the antibody (B) based on the determined antigen neutralizing equivalent (A); the lowest detection limit of the antibody detection reagent = a × B can be obtained.
Confirmation of the minimum detection limit of the S6 antibody detection reagent: respectively selecting at least 3 clinical samples containing antibodies to be detected, which are representative from different sources, obtaining the lowest detection limit of an antibody detection reagent by the 3 clinical samples containing the antibodies to be detected according to the steps S1-S5, respectively diluting the samples containing the antibodies to the concentration of the lowest detection limit if the obtained lowest detection limit of the samples containing the antibodies is consistent with the lowest detection limit measured in the step S5, and carrying out repeated detection for 20 times, wherein if the positive rates are more than or equal to 90%, the lowest detection limit of the antibody detection reagent is confirmed;
when the positive rate is lower than 90% after repeated detection for many times, adjusting the antibody titer in the step S4, selecting the last dilution of the currently determined antibody titer as a new antibody titer, and performing repeated detection for many times until the positive rate is more than or equal to 90%; the lowest detection limit for confirmation at this time is recalculated based on the new antibody titer;
verification of the lowest detection limit of the S7 antibody detection reagent: selecting 3 clinical samples with time and regional characteristics (and different from the samples used in the determination of the lowest detection limit in the step S6), diluting the 3 clinical samples to the concentration of the lowest detection limit for 20 times of detection, and if the detection rate reaches 90-95% of positive detection rate, the lowest detection limit passes the verification;
or the method for verifying the lowest detection limit of the antibody detection reagent in the step S7 comprises the following steps: diluting 3 clinical samples to the concentration of the lowest detection limit, performing detection for 3 times, and if all the clinical samples are positive, passing the verification; if the 3 times of detection show negative, then 7 times of detection are carried out, and if the detection shows positive, the verification is passed; if two times of negative results appear in the 3 times of detection, the detection is carried out for 18 times, and if the two times of negative results are all positive results, the verification is passed; if all of the 3 tests are negative, the test does not pass (if the test is performed 20 times or more at a time, more time is consumed, the test of the lowest detection limit of the antibody detection reagent can be performed in this progressive manner).
Specifically, when antigen 1 is used in this example, antigen 1 is hepatitis B virus surface antigen (HBsAg), 1000. Mu.g/mL, available from absolute antibody; the detection kit used was: hepatitis B virus surface antigen, surface antibody, e antigen, e antibody, core antibody detection reagent (colloidal gold method), purchased from Guangzhou Wanfu biotechnology, inc.; negative sera used: purchased from Beijing Kogyo Hongda Biotechnology Co.
The determination of the antigen neutralizing equivalent based on the antigen detection reagent specifically comprises the following steps:
s1 dilution antigen 1: known concentrations of antigen 1 were measured with negative sera as 1:10,1:20,1:40, 1:80,1:160,1:320 steps are carried out for dilution, and each dilution obtains 50 mu L of dilution liquid;
and S2, incubation reaction of antigen 1 and antibody: adding 50 μ L of sample containing hepatitis B virus surface antibody (HBsAb) to each gradient dilution, and performing water bath at 37 deg.C for 30min;
determination of neutralizing equivalents of S3 antigen: detecting each mixed solution obtained in the step S2 with a hepatitis b virus surface antigen, a surface antibody, an e antigen, an e antibody, and a core antibody detection reagent (colloidal gold method), respectively (detecting HbsAg by using the hepatitis b virus surface antigen detection reagent in the kit), and recording the data as shown in table 1 below;
table 1 shows the data of the detection results after the incubation reaction of the antigen 1 and the antibody
| Antigen dilution gradient | 1：10 | 1：20 | 1：40 | 1：80 | 1：160 | 1：320 |
| Positive and negative | + | + | - | - | - | - |
From the recorded data, it can be determined that 1: the antigen in the mixture at 40 dilutions was the antigen neutralization equivalent of 50 μ L of the antibody standard, and the antigen neutralization equivalent 1 of this HBsAb-containing sample was calculated to be 1000 μ g/mL ÷ 40=25 μ g/mL.
Subpackaging and freezing the antibody serum; when the method is used, the antibody serum which is subpackaged and frozen is taken out, the room temperature is recovered, the antigen neutralization equivalent of the antibody is determined again by the method, namely, the antibody is assigned again, the accurate determination of the antibody value is ensured, and the clinical diagnosis error caused by the activity reduction of the antibody is avoided.
The method for determining the lowest detection limit of the antibody detection reagent specifically comprises the following steps:
determination of antibody titer of S4 samples: HBsAb-containing samples were removed with negative serum according to 1:2,1:4,1:8,1:16,1:32,1:64,1: diluting with 128 gradient to obtain gradient diluents of different dilutions, and diluting each portion to obtain 50 μ L of diluent; detecting each gradient solution by using hepatitis B virus surface antigen, surface antibody, e antigen, e antibody and core antibody detection reagent (colloidal gold method) (detecting HBsAb by using hepatitis B virus surface antibody detection reagent in the kit); the data are recorded as in table 2 below;
table 2 shows the data of the results of the detection of the sample (antibody)
| Antibody dilution gradient | 1：2 | 1：4 | 1：8 | 1：16 | 1：32 | 1：64 | 1：128 |
| Positive and negative | + | + | + | + | + | - | - |
When a certain dilution of the test result shows a negative reaction, determining the dilution of the previous higher-concentration dilution of the dilution as the antibody titer, thereby determining the antibody titer; that is, the antibody titer of this HBsAb-containing sample was 1:32.
obtaining the lowest detection limit of the S5 antibody detection reagent: multiplying the antigen neutralizing equivalent (25 μ g/mL) of the antibody in the sample obtained in the step S3 by the antibody titer (1; namely, the minimum detection limit of the hepatitis B virus surface antigen, surface antibody, e antigen, e antibody and core antibody detection reagent (colloidal gold method) is 25. Mu.g/mL/32 = 0.78. Mu.g/mL.
The minimum detection limit of the above antibody detection reagent can be confirmed and verified as follows:
confirmation of the minimum detection limit of the S6 antibody detection reagent: respectively selecting at least 3 clinical samples containing antibodies to be detected, which are representative from different sources, obtaining the lowest detection limit of the antibody detection reagent by using the 3 clinical samples containing the antibodies to be detected according to the steps S1-S5, respectively diluting the samples containing the antibodies to the concentration of the lowest detection limit if the obtained lowest detection limit of the samples containing the antibodies is consistent with the lowest detection limit measured in the step S5, and carrying out repeated detection for 20 times, wherein if the positive rates are more than or equal to 90%, the lowest detection limit of the antibody detection reagent is confirmed;
when the positive rate is lower than 90% after repeated detection for many times, adjusting the antibody titer in the step S4, selecting the last dilution of the currently determined antibody titer as a new antibody titer, and performing repeated detection for many times until the positive rate is more than or equal to 90%; the lowest detection limit for confirmation at this time is recalculated based on the new antibody titer;
verification of the lowest detection limit of the S7 antibody detection reagent: selecting 3 clinical samples with time and regional characteristics (and different from the samples used in the determination of the lowest detection limit in the step S6), diluting the 3 clinical samples to the concentration of the lowest detection limit for 20 times of detection, and if the detection rate reaches 90-95% of positive detection rate, the lowest detection limit passes the verification;
or the method for verifying the lowest detection limit of the antibody detection reagent in the step S7 comprises the following steps: diluting 3 clinical samples to the concentration of the lowest detection limit, performing detection for 3 times, and if all the clinical samples are positive, passing the verification; if the 3 times of detection show negative, then 7 times of detection are carried out, and if the detection shows positive, the verification is passed; if two times of negative results appear in the 3 times of detection, the detection is carried out for 18 times, and if the two times of negative results are all positive results, the verification is passed; if all of the 3 tests are negative, the test does not pass (if the test is performed 20 times or more at a time, more time is consumed, the test of the lowest detection limit of the antibody detection reagent can be performed in this progressive manner).
Specifically, when the Antigen neutralization equivalent of the HBsAb needs to be determined again, the Antigen 2 can be used, wherein the Antigen 2 is Hepatitis B Surface Antigen (HBsAg-subtype Ad), 2000 mu g/mL, purchased from EastCoast Bio; the detection kit comprises: hepatitis B virus surface antigen, surface antibody, e antigen, e antibody, core antibody detection reagent (colloidal gold method), purchased from Guangzhou Wanfu biotechnology, inc.; negative serum: purchased from Beijing Kongkomada biotechnology, inc.
The determination of the antigen neutralizing equivalent based on the antigen detection reagent specifically comprises the following steps:
s1 dilution antigen 2: known concentrations of antigen 2 were measured with negative sera as 1:10,1:20,1:40, 1:80,1:160,1:320 steps are carried out for dilution, and each dilution obtains 50 mu L of dilution liquid;
and S2, incubation reaction of antigen 2 and antibody: adding 50 μ L of sample containing hepatitis B virus surface antibody (HBsAb) to each gradient dilution, and performing water bath at 37 deg.C for 30min;
determination of neutralizing equivalents of S3 antigen: detecting each mixed solution obtained in the step S2 with a hepatitis b virus surface antigen, a surface antibody, an e antigen, an e antibody, and a core antibody detection reagent (colloidal gold method), respectively (detecting HBsAg-subtype Ad by using the hepatitis b virus surface antigen detection reagent in the kit), and recording the data as shown in table 3 below;
table 3 shows the data of the detection results after the incubation reaction of the antigen 2 and the antibody
| Antigen dilution gradient | 1：10 | 1：20 | 1：40 | 1：80 | 1：160 | 1：320 |
| Positive and negative | + | + | + | - | - | - |
From the recorded data, it can be determined that 1: the antigen in the mixture at 80 dilutions was the antigen neutralization equivalent of 50 μ L of the antibody standard, and the antigen neutralization equivalent 2 of this HBsAb-containing sample was calculated to be 2000 μ g/mL ÷ 80=25 μ g/mL.
Subpackaging and freezing the antibody serum; when in use, the antibody serum which is subpackaged and frozen is taken out, and the antigen neutralization equivalent of the antibody is determined again after the room temperature is recovered.
The method for determining the lowest detection limit of the antibody detection reagent specifically comprises the following steps:
determination of antibody titer of S4 samples: the HBsAb-containing samples were removed from the negative sera as follows 1:2,1:4,1:8,1:16,1:32,1:64,1: diluting with 128 gradient to obtain gradient dilution solutions with different dilution degrees, and diluting each part to obtain 50 mu L dilution solution; detecting each gradient solution by using hepatitis B virus surface antigen, surface antibody, e antigen, e antibody and core antibody detection reagent (colloidal gold method) (detecting HBsAb by using hepatitis B virus surface antibody detection reagent in the kit); the data recorded are as follows in table 4;
table 4 shows the data of the results of the detection of the sample (antibody)
| Antibody dilution gradient | 1：2 | 1：4 | 1：8 | 1：16 | 1：32 | 1：64 | 1：128 |
| Positive and negative | + | + | + | + | + | - | - |
When a certain dilution of the diluent in the detection result has a negative reaction, determining the dilution of the previous higher-concentration dilution of the diluent as the antibody titer, thereby determining the antibody titer; that is, the antibody titer of this HBsAb-containing sample was 1:32.
obtaining the lowest detection limit of the S5 antibody detection reagent: multiplying the antigen neutralizing equivalent (25 μ g/mL) of the antibody in the sample obtained in the step S3 by the antibody titer (1; namely, the minimum detection limit of the hepatitis B virus surface antigen, surface antibody, e antigen, e antibody and core antibody detection reagent (colloidal gold method) is 25. Mu.g/mL/32 = 0.78. Mu.g/mL.
The minimum detection limit of the above antibody detection reagent can be confirmed and verified as follows:
confirmation of the minimum detection limit of the S6 antibody detection reagent: respectively selecting at least 3 clinical samples containing antibodies to be detected, which are representative from different sources, obtaining the lowest detection limit of an antibody detection reagent by the 3 clinical samples containing the antibodies to be detected according to the steps S1-S5, respectively diluting the samples containing the antibodies to the concentration of the lowest detection limit if the obtained lowest detection limit of the samples containing the antibodies is consistent with the lowest detection limit measured in the step S5, and carrying out repeated detection for 20 times, wherein if the positive rates are more than or equal to 90%, the lowest detection limit of the antibody detection reagent is confirmed;
when the positive rate is lower than 90% after repeated detection for many times, adjusting the antibody titer in the step S4, selecting the last dilution of the currently determined antibody titer as a new antibody titer, and performing repeated detection for many times until the positive rate is more than or equal to 90%; the lowest detection limit for confirmation at this time is recalculated based on the new antibody titer;
verification of the lowest detection limit of the S7 antibody detection reagent: selecting 3 clinical samples with time and regional characteristics (and different from the samples used in the determination of the lowest detection limit in the step S6), diluting the 3 clinical samples to the concentration of the lowest detection limit for 20 times of detection, and if the detection rate reaches 90-95% of positive detection rate, the lowest detection limit passes the verification;
or the method for verifying the lowest detection limit of the antibody detection reagent in the step S7 comprises the following steps: diluting 3 clinical samples to the concentration of the lowest detection limit, performing detection for 3 times, and if all the clinical samples are positive, passing verification; if the 3 times of detection show negative, then 7 times of detection are carried out, and if the detection shows positive, the verification is passed; if two times of negative results appear in the 3 times of detection, the detection is carried out for 18 times, and if the two times of negative results are all positive results, the verification is passed; if all of the 3 tests are negative, the test does not pass (if the test is performed 20 times or more at a time, more time is consumed, the test of the lowest detection limit of the antibody detection reagent can be performed in this progressive manner).
It is obvious to those skilled in the art that the present invention is not limited to the above embodiments, and it is within the scope of the present invention to adopt various insubstantial modifications of the method concept and technical scheme of the present invention, or to directly apply the concept and technical scheme of the present invention to other occasions without modification.
Claims (10)
1. A method for determining the antigen neutralization equivalent based on an antigen detection reagent is characterized by comprising the following steps:
s1 dilution of antigen: carrying out gradient dilution on the antigen with known purity and concentration by using a matrix to obtain a series of gradient diluents with different concentrations;
and (2) incubating reaction of the S2 antigen and the antibody: adding an equal amount of antibody standard substance or antibody-containing sample into each gradient dilution with different concentrations obtained in the step S1, and reacting to generate a mixed solution;
determination of neutralizing equivalents of S3 antigen: adding a specific antigen detection reagent corresponding to the antigen in the step S1 to each mixed solution obtained in the step S2 for detection, and recording data to determine the antigen neutralizing equivalent.
2. The method for determining an antigen-neutralizing equivalent amount based on an antigen detection reagent according to claim 1, wherein when an antigen-neutralizing equivalent amount of an antibody standard or a sample containing an antibody is determined and an antigen-neutralizing equivalent amount thereof is to be newly determined, said steps S1 to S3 are newly performed to obtain an antigen-neutralizing equivalent amount of the antibody standard or the sample containing an antibody.
3. The method for determining an antigen-neutralizing equivalent amount based on an antigen detection reagent according to claim 2, wherein the antigen in said step S1 is the original antigen or a different antigen having the same epitope as the original antigen and being specifically recognized by the same antibody when the antigen-neutralizing equivalent amount of the antibody standard or the antibody-containing sample is newly determined.
4. The method of determining the antigen neutralizing equivalent amount by the antigen detecting reagent according to claim 1, wherein in the step S3, when the specific antigen detecting reagent is added to each mixed solution and the detection is performed, if a negative reaction occurs in a mixed solution of a certain dilution in the detection result, the amount of the antigen of the dilution corresponding to the mixed solution is determined to be the antigen neutralizing equivalent amount of the antibody contained in the standard or sample.
5. The method for determining the neutralizing equivalent to an antigen based on an antigen detecting reagent according to claim 1, wherein the antigen in step S1 is the same or different antigen having the same epitope and being specifically recognized by the same antibody when used at different times.
6. The method for determining the antigen-neutralizing equivalent amount of an antigen detection reagent according to claim 1, wherein an equal amount of the antibody standard or the specific antibody-containing sample is added to each of the antigen dilutions having different concentrations in step S2, and the mixture is formed after the reaction at 37 ℃ for 30 min.
7. The method for determining an antigen-neutralizing equivalent amount based on an antigen detection reagent according to claim 4, wherein the dilutions of the gradient dilution of the antigen in the step S1 are: 1: 10. 1: 20. 1: 80. 1:160 and 1:320.
8. the method for determining an antigen-neutralizing equivalent amount based on an antigen detection reagent according to claim 4, further comprising the step of S4 determining an antibody titer of the sample: carrying out gradient dilution on the sample containing the antibody in the step S3 by using a matrix to obtain gradient diluents with different dilutions; and performing antibody detection on the gradient dilution of each dilution by using an antibody detection reagent, thereby determining the antibody titer of the sample.
9. The method for determining an antigen-neutralizing equivalent amount based on an antigen detection reagent according to claim 8, wherein the dilution of the antibody-containing sample subjected to gradient dilution with the matrix in step S4 is: 1:2,1:4,1:8,1:16,1:32,1:64,1:128.
10. the method for determining an antigen-neutralizing equivalent amount based on an antigen detection reagent according to claim 8, wherein the antibody detection is performed for each dilution with an antibody detection reagent in step S4, and when a negative reaction occurs in a dilution of a certain dilution in the detection result, the dilution of a previous higher concentration dilution of the certain dilution is determined as the antibody titer.
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| JPH075174A (en) * | 1993-06-18 | 1995-01-10 | Nippon Steel Corp | High detectibility immunoassay |
| IT1284874B1 (en) * | 1996-08-02 | 1998-05-22 | Farmigea Spa | BIOADHESIVE COMPLEXES OF POLYCARBOPHIL AND AZOLIC ANTIFUNGAL OR ANTIPROTOZOAR DRUGS |
| US6034045A (en) * | 1997-02-25 | 2000-03-07 | Church & Dwight Co., Inc. | Liquid laundry detergent composition containing a completely or partially neutralized carboxylic acid-containing polymer |
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| JP5487393B2 (en) * | 2009-03-31 | 2014-05-07 | 静岡県 | Standardization method of antigen-specific IgG antibody titer measurement in human serum using ELISA method |
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