CN113311159A

CN113311159A - Test strip for rapidly distinguishing recent and long-term HIV infection states through serum HIV-1 antibody detection and preparation method thereof

Info

Publication number: CN113311159A
Application number: CN202110404802.9A
Authority: CN
Inventors: 唐时幸; 万政伟; 王海鹰; 赵建辉; 王克寅; 孟婷婷; 刘兴旺
Original assignee: Beijing Xinchuang Bioengineering Co ltd; Southern Medical University
Current assignee: Beijing Xinchuang Bioengineering Co ltd; Southern Medical University
Priority date: 2021-04-15
Filing date: 2021-04-15
Publication date: 2021-08-27

Abstract

A test paper strip for rapidly distinguishing recent and long-term HIV infection states by serum HIV-1 antibody detection and a preparation method thereof. The test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the nitrocellulose membrane and the absorbent paper are arranged on the bottom plate, two ends of the nitrocellulose membrane are respectively lapped with the sample pad and the absorbent paper, and a colloidal gold-labeled mouse anti-human IgG antibody is sprayed on one end, close to the nitrocellulose membrane, of the sample pad to form a gold-labeled layer; the nitrocellulose membrane is provided with a first detection line for mixed coating of GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33, a second detection line for single coating of HIV recombinant antigen and a quality control line of goat anti-mouse IgG antibody. The test strip can judge the recent infection or long-term infection state of HIV-1, greatly reduces the false judgment or false judgment probability of a single GP41 recombinant antigen coating, and has convenient use and simple judgment.

Description

Test strip for rapidly distinguishing recent and long-term HIV infection states through serum HIV-1 antibody detection and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a test strip for rapidly distinguishing recent infection states and long-term infection states of HIV through serum HIV-1 antibody detection and a preparation method thereof.

Background

Human Immunodeficiency Virus (HIV) is a Virus that infects cells of the Human immune system, causing various diseases by destroying the immune cells of the Human body, resulting in the immune system becoming defenseless. Monitoring methods such as AIDS (AIDS)/HIV (HIV) case report, sentinel monitoring, behavior monitoring and the like provide a great deal of basis for finding out HIV/AIDS epidemic situation, mastering HIV/AIDS epidemic trend and change conditions of high-risk behaviors and evaluating the effect of intervention measures for a long time. However, the monitoring method is difficult to judge whether the HIV infected person is newly infected or infected for a long time, direct and accurate newly infected data cannot be obtained, the real change trend of epidemic situations and the effect of intervention measures cannot be sensitively reflected, and the incidence rate estimated by a few queue researches and statistical models cannot be far adapted to the requirements of HIV epidemic situation analysis and prevention and treatment effect evaluation.

With the wide popularization of antiviral treatment, the survival time of more and more HIV infectors and AIDS patients is gradually prolonged, so that the accumulated number of long-term infectors is more and more, and the HIV infection rate cannot sensitively and directly reflect the change trend of HIV epidemics. Therefore, the effect of AIDS prevention and treatment measures is evaluated, so that the accurate estimation of the new infection rate of AIDS becomes a new key point of the AIDS prevention and treatment work.

In recent years, with the development of laboratory techniques, it has become possible to serologically monitor new infections with HIV-1. Four more mainstream laboratory new infection detection methods have appeared in succession from 1998 to the world: the hypo-sensitive enzyme-linked assay, the AxSYM affinity index assay, the BED-CEIA assay and the LAg-Avidity EIA assay.

The four detection methods can be classified into two major categories according to the detection principle. The hypo-sensitive enzyme-linked method and the BED-CEIA method distinguish newly infected persons from those infected with long-term HIV based on the characteristic that the number of HIV antibodies in newly infected persons is small. AxSYM affinity index method and LAg-Avidity EIA distinguish newly infected persons from those with long-term infection based on the weak affinity of HIV antibodies in newly infected persons.

The low-sensitivity enzyme-linked method has subtype dependence on the detection result of the new infection due to the coating of the subtype B antigen, and is not suitable for the detection of the new infection in areas with multiple subtypes. The AxSYM affinity index method requires double-well detection of a sample, the operation method is complicated, a special detection instrument is required to be configured, and the misjudgment is shown to be high by research make internal disorder or usurp. The BED-CEIA method and the AxSYM affinity index method appear almost at the same time, some researchers use the two methods for detecting new infection at the same time, and the results show that misjudgment conditions of the BED-CEIA method are obviously reduced, but the operation is complex. At present, BED-CEIA is still the most used new infection detection method worldwide, the detection problem is increasingly highlighted along with the wide use, and multiple researches make internal disorder or usurp show that the detection result is influenced by CD4 counting and antiviral treatment, and finally the accuracy of the estimation of the new infection rate is influenced. The limited antigen affinity enzyme-linked method has low misjudgment rate and high specificity, and China realizes independent research and development and production. However, the limited antigen affinity enzyme-linked assay is still a laboratory detection method, has high requirements on experimental technicians, equipment and laboratory conditions, and is difficult to realize large-scale rapid detection of people.

The method is widely applied at present for detecting human immunodeficiency virus (HIV-1/HIV-2) antibodies in human serum, plasma and whole blood in vitro by collecting blood samples of patients and preparing a test strip colloidal gold method by an immunochromatography technology. However, these methods mainly determine whether or not infection is caused by detecting the presence or absence of HIV antibodies in blood of a suspicious individual, and cannot distinguish newly infected individuals from long-term infected individuals.

Therefore, it is necessary to provide a test strip for rapidly distinguishing the recent infection state and the long-term infection state of HIV by serum HIV-1 antibody detection and a preparation method thereof to overcome the defects of the prior art.

Disclosure of Invention

The invention aims to avoid the defects of the prior art and provides a test strip for rapidly distinguishing the recent infection state and the long-term infection state of HIV through serum HIV-1 antibody detection and a preparation method thereof.

The object of the invention is achieved by the following technical measures.

The test strip comprises a bottom plate, a sample pad arranged on the bottom plate, a nitrocellulose membrane and absorbent paper, wherein two ends of the nitrocellulose membrane are respectively lapped with the sample pad and the absorbent paper, and a colloidal gold-labeled mouse anti-human IgG antibody is sprayed at one end, close to the nitrocellulose membrane, of the sample pad to form a gold-labeled layer;

The nitrocellulose membrane is provided with a first detection line for mixed coating of GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33, a second detection line for single coating of HIV recombinant antigen (New England (Xiamen) science and technology GmbH, product number: XM1008HIV-A) and a quality control line for goat anti-mouse IgG antibody.

Preferably, in the test strip for rapidly distinguishing the recent infection state from the long-term infection state of the HIV, the coating concentration of the HIV recombinant antigen in the second detection line is 2.5-3.5 mg/ml.

Preferably, the test strip for rapidly distinguishing the recent infection state from the long-term infection state of the HIV has the coating concentration of GP41 recombinant antigen BE23 in the first detection line of 0.1-0.3mg/ml and the coating concentration of GP41 recombinant antigen BE33 in the first detection line of 0.3-0.6 mg/ml.

Preferably, the test strip for rapidly distinguishing the recent infection state from the long-term infection state of the HIV has the structure that GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33 are mixed according to the volume ratio of 1: 3, mixing and coating.

Preferably, the test strip for rapidly distinguishing the recent infection state from the long-term infection state of the HIV has the coating concentration of the goat anti-mouse IgG of the quality control line of 0.5-1.0 mg/ml.

Preferably, the test strip for rapidly distinguishing the recent infection state from the long infection state of HIV is characterized in that the concentration of the colloidal gold labeled mouse anti-human IgG antibody is 10-20 mug/ml.

Preferably, the test strip for rapidly distinguishing the recent infection state from the long-term infection state of the HIV is characterized in that the test strip refers to a colorimetric card, refers to a first detection line when strips appear in both a quality control line and a second detection line, shows the result of recent infection when the brightness of the strip in the first detection line is less than L3, and shows the result of long-term infection when the strip in the first detection line is greater than or equal to L3.

The invention also provides application of the first detection line of the mixed coating GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33 in the aspect of the test strip for rapidly distinguishing the recent infection state from the long-term infection state of HIV.

The invention also provides a preparation method of the test paper for rapidly distinguishing the recent infection state from the long-term infection state of the HIV, which is carried out by the following steps,

1) preparing a sample pad;

2) combining mouse anti-human IgG and colloidal gold particles to form a colloidal gold labeled mouse anti-human IgG antibody, uniformly coating the colloidal gold labeled antibody on a gold labeled pad, and freeze-drying the colloidal gold labeled pad at the temperature of 45 ℃ for later use;

3) preparing a first detection line, a second detection line and a quality control line, and then carrying out whole-body humidity measurement at 25 ℃ and 10 DEG C_～Airing or drying for 18-22h under the condition of 30 percent;

the specific process for preparing the first detection line is as follows: diluting GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33 recombinant antigens to 0.1-0.3mg/ml and 0.3-0.6mg/ml respectively by using buffer solution, and after mixing, scribing the mixed solution on a nitrocellulose membrane by using a membrane spraying machine to form a first detection line;

The specific process for preparing the second detection line is as follows: diluting the HIV-1 recombinant antigen to 2.5-3.5mg/ml by using a coating buffer solution, and scribing a nitrocellulose membrane by using a membrane spraying machine to form a second detection line;

the specific process for preparing the quality control line is as follows: diluting goat anti-mouse antibody to 0.5-1.0mg/ml, and scribing on the nitrocellulose membrane by using a film spraying machine to form a quality control line;

4) and sequentially overlapping the sample pad, the nitrocellulose membrane and the absorbent paper on a bottom plate to obtain the complete test strip.

Preferably, the sample dilution is 10mm PBS, pH 7.2 + -0.2.

The test strip for rapidly distinguishing the recent infection state and the long-term infection state of the HIV through serum HIV-1 antibody detection has the following advantages:

1) by judging the recent infection or long-term infection state of HIV-1 through the mixed coated GP41 recombinant antigen, the misjudgment or the missed judgment of the single GP41 recombinant antigen coating can be greatly reduced.

2) The invention has convenient use, simple operation, no need of high-end experimental equipment and complex experimental steps, lower professional requirements on operators, and reduced result misjudgment of the operators caused by misoperation and improved operation repeatability. In addition, the method has fewer operation steps, and reduces the frequency of contacting HIV positive samples by operators, thereby reducing the infection risk of the operators.

3) Compared with the current laboratory method on the market, the method has the advantages that the detection time is shortened to be within 30 minutes, the detection time is greatly shortened, and the detection efficiency is improved.

4) The method has simple interpretation result, can be judged by comparing the color of the reference line with the standard colorimetric card by an operator according to the operation flow without professional interpretation, is suitable for primary health workers, has good popularization and can be used for large-scale primary screening of HIV-1 late stage infection.

Drawings

The invention is further illustrated by means of the attached drawings, the content of which is not in any way limiting.

FIG. 1 is a schematic diagram of a test strip for rapidly differentiating recent and long-term HIV infection status by serum HIV-1 antibody detection according to the present invention.

FIG. 2 shows the determination of new infection and previous infection results in the example of the present invention.

FIG. 3 is a flow chart of sample detection according to an embodiment of the present invention.

In fig. 1, the method includes:

1. a sample pad; 2. a gold mark layer; 3. a nitrocellulose membrane; 4. absorbent paper; 5. a first detection line; 6. a second detection line; 7. a quality control line; 8. a base plate.

Detailed Description

The invention is further illustrated by the following examples.

Example 1.

A test strip for rapidly distinguishing the recent infection state and the long-term infection state of HIV through serum HIV-1 antibody detection is shown in figure 1 and comprises a bottom plate 8, a sample pad 1 arranged on the bottom plate 8, a nitrocellulose membrane 3 and absorbent paper 4. The two ends of the nitrocellulose membrane 3 are respectively lapped with the sample pad 1 and the absorbent paper 4, and one end of the sample pad 1, which is close to the nitrocellulose membrane 3, is sprayed with the colloidal gold-labeled mouse anti-human IgG antibody to form a gold label layer 2.

The nitrocellulose membrane 3 is provided with a second detection line 6 for singly coating an HIV recombinant antigen, a first detection line 5 for mixed coating of GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33 and a quality control line 7 for goat anti-mouse IgG antibody.

The test strip for rapidly distinguishing the recent infection state and the long-term infection state of the HIV has the coating concentration of GP41 recombinant antigen BE23 in a first detection line of 0.1-0.3mg/ml and the coating concentration of GP41 recombinant antigen BE33 in the first detection line of 0.3-0.6 mg/ml. Preferably, the GP41 recombinant antigen BE23 and the GP41 recombinant antigen BE33 are mixed in a volume ratio of 1: 3, mixing and coating.

The gene sequence of GP41 recombinant antigen BE23 is as follows:

the gene sequence of GP41 recombinant antigen BE33 is as follows:

the HIV recombinant antigens individually coated on the second test line were purchased from british innovation (mansion) science ltd, cat #: XM1008 HIV-A. The coating concentration of the HIV recombinant antigen in the second detection line is 2.5-3.5 mg/ml.

The coating concentration of the goat anti-mouse IgG of the quality control line 7 is 0.5-1.0 mg/ml. The concentration of the colloidal gold-labeled mouse anti-human IgG antibody was 10-20. mu.g/ml.

The test paper strip is prepared by the following steps,

1) preparing a sample pad;

It should be noted that, the sequence of preparing the first detection line, the second detection line and the quality control line can be flexibly adjusted. Only three lines are finally prepared, and the preparation sequence of the three lines can be flexibly adjusted.

The test strip for rapidly distinguishing the recent infection state and the long-term infection state of the HIV through serum HIV-1 antibody detection judges the recent infection state or the long-term infection state of the HIV-1 through the mixed coated GP41 recombinant antigen, and can greatly reduce the misjudgment or the missed judgment of the single GP41 recombinant antigen coating.

The test strip for rapidly distinguishing the recent infection state and the long-term infection state of the HIV refers to a colorimetric card, refers to the first detection line 5 when strips appear on the quality control line 7 and the second detection line 6, displays the result of recent infection when the brightness of the strips on the first detection line 5 is less than L3, and displays the result of long-term infection when the brightness of the strips on the first detection line 5 is greater than or equal to L3.

The test strip is convenient to use, simple to operate, low in professional requirement on operators and capable of reducing result misjudgment of the operators caused by misoperation and improving repeatability of operation, and does not need high-end experimental equipment and complex experimental steps. In addition, the method has fewer operation steps, and reduces the frequency of contacting HIV positive samples by operators, thereby reducing the infection risk of the operators. Compared with the current laboratory method on the market, the method has the advantages that the detection time is shortened to be within 30 minutes, the detection time is greatly shortened, and the detection efficiency is improved. The method has simple interpretation result, can be judged by comparing the color of the reference line with the standard colorimetric card by an operator according to the operation flow without professional interpretation, is suitable for primary health workers, has good popularization and can be used for large-scale primary screening of HIV-1 late stage infection.

Example 2.

The test paper strip for rapidly distinguishing the recent infection state and the long-term infection state of HIV through serum HIV-1 antibody detection has the same structure as that of the example 1, and the preparation process of the test paper strip of the embodiment is as follows:

1) preparing a sample pad, wherein the sample pad 1 is purchased from scientific and technological corporation of encyclopedia of English;

3) preparing a second detection line, a first detection line and a quality control line, and then carrying out whole-body humidity measurement at 25 ℃ and 10 DEG C_～At 30% of the total weight of the compositionAiring or drying for 18-22 h;

the specific process for preparing the second detection line is as follows: diluting the HIV-1 recombinant antigen to a working concentration of 2.5-3.5mg/ml by using a coating buffer solution, and scribing a nitrocellulose membrane by using a membrane spraying machine to form a second detection line;

the specific process for preparing the first detection line is as follows: diluting GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33 recombinant antigens to working concentration (BE 23: 0.1-0.3mg/ml, BE 33: 0.3-0.6mg/ml) by buffer solution, and scribing a nitrocellulose membrane by a membrane spraying machine to form a first detection line;

the specific process for preparing the quality control line is as follows: diluting the goat anti-mouse antibody to a working concentration, and scribing the nitrocellulose membrane by using a film spraying machine to form a quality control line;

In this embodiment, the first detection line mixed coating BE23 and BE33 are mixed according to a volume ratio of 1: 2 and mixing. For comparison effect, the comparison test strip 1 with the GP41 protein BE23 coated on the first detection line independently is prepared according to the method, and the comparison test strip 2 with the GP41 protein BE33 coated on the first detection line independently is prepared according to the method.

The prepared test strip, the test strip 1 and the test strip 2 are adopted to detect a sample.

Sample loading: 10ul of serum is sucked by a pipette and dripped on a sample pad, 2-3 drops (about 100-150ul) of sample diluent are dripped, the sample diluent is 10mm PBS, the pH value is 7.2 +/-0.2, and the color brightness of the test paper detection line is read after 20-30 minutes.

And (4) judging a result: and comparing the detection line strips with a standard colorimetric card, wherein the range is L0-L10. Firstly, looking at a quality control line, and describing that the operation is correct if the positive result is positive; looking next at the second reference line, a color intensity greater than L0 indicates that the sample is HIV-1 positive, and a color intensity of L0 indicates that the sample is HIV negative. When the sample is judged to be HIV-1 positive, the color intensity is larger than L0 to indicate that the sample is HIV-1 long-term infection, and the color intensity is L0 to indicate that the sample is HIV recently infected. The test strip result determination mode is shown in figure 3.

Interpretation of the results: in the experiment, a recent infection sample N1-N5, a long-term infection sample P1-P5 and strong-yang, medium-yang and weak-yang reference substances are selected for detection. And the color of the quality control line and the second detection line of all the samples is normal. The results of the first detection line are shown in the table I, and it can BE seen that a plurality of false long periods and false short-term results occur when BE23 and BE33 are coated independently, and BE230.3mg/ml and BE330.45mg/ml are coated in a mixed mode according to the volume ratio of 1:2, so that the false short-term results can BE reduced, the accuracy is improved, and therefore, the test paper strip in the mixed coating mode is high in accuracy.

Table one: BE23 and BE33 are coated separately, and BE23 and BE33 are coated with a first detection line detection reference product result in a mixed mode.

Note: L0-L10 are the contrast of the color of the strip reaction to the brightness of a standard color chart. The larger the number, the greater the band brightness, L0 representing no band, and L10 representing the band color brightness brightest. P is long-term infection serum with definite HIV infection time exceeding 250 days; n is recently infected serum with a definite HIV infection time of less than 90 days (infection time was obtained from epidemiological follow-up data).

Example 3.

The test results of example 2 demonstrate that the coated GP41 mixed protein weakly reacts with HIV recently infected serum antibodies to result in weak color development, and this example provides a different test strip.

1) The GP41 fusion protein in the first detection line of the test example is coated by BE23 and BE33 in a mixed way. Wherein BE230.3mg/ml and BE330.45mg/ml are mixed according to the ratio of 1:1,1:2,1:3,3:1,2: 1.

2) The method for spraying gold particles and assembling the test strip in this test example is the same as that in test example 1.

3) The test paper of this test example was prepared in the same manner as in test example 1.

4) The loading method of this test example was the same as that of test example 1.

5) The method for determining the test results in this test example was the same as in test example 1.

6) We use the detection result of the laboratory differential HIV near-term and far-term infection kit which is already marketed as a reference standard to search the mixed coating amount of GP41 protein in the first detection line. We obtained 98 HIV serum samples from the Chinese CDC and tested this batch simultaneously using our prepared test strip and the LAg-Avidity EIA test kit to determine the optimal mixed coating of GP41 protein on the first test line of the test strip. The results of the different coating test are shown in Table 2.

7) Interpretation of the results: when the color reading of the quality control line is normal, the reading of the second detection line is normal, and the reading of the first detection line is less than L3, the detected sample is considered to be newly infected; when the quality control line and the second detection line are normal, the first detection line is greater than or equal to L3, the infection is considered as long-term infection, and conversely, when the first detection line is less than L3, the infection is considered as recent infection. The comparison of the detection results of the mixed package of the GP41 proteins in different proportions in the first detection line and the detection results of the LAg-Avidity EIA method shows that the detection effect is optimal when the volume ratio of the GP41 proteins BE23(0.3mg/ml) to BE33(0.45mg/ml) is 1:3, and the sensitivity, the specificity and the consistency are respectively 86.96%, 88.46% and 87.75%. When the volume ratio of GP41 protein BE23(0.3mg/ml) to BE33(0.45mg/ml) is 1:3, the color of the first detection line is L3 which is convenient for ordinary people to distinguish, so that visual and visual judgment can BE realized, and the result can BE judged by a detector only through simple vision in the actual use process.

Table two: the results of the mixed detection of the BE23 and the BE33 proteins in different ratios are compared with the detection result of the LAg-Avidity EIA method.

Example 4.

The experimental example is further incorporated into a new sample for detection, and the test paper strip is verified to distinguish the recent infection and long-term infection capacity of HIV-1.

1) The GP41 fusion protein in the first detection line of the test example is coated by BE23 and BE33 in a mixed way. Wherein BE230.3mg/ml and BE330.45mg/ml are mixed in a ratio of 1: 3.

2) The method for spraying gold particles and assembling the test strip in the test example is the same as that in test example 2.

3) The test paper of this test example was prepared in the same manner as in test example 2.

4) This experiment was carried out in the same manner as in example 2.

5) The loading method of this test example is the same as that of test example 2.

6) The method for judging the detection result of the test example is the same as the test example 2, and the specific interpretation process is shown in the attached figure 3.

7) In the test example, the test strip is further verified by adopting an imported LAg-Avidity EIA method kit to expand samples. We obtained 65 HIV-infected IDU human serum samples from the CDC of Guangzhou city and tested this batch simultaneously using our prepared test strip and the imported LAg-Avidity EIA test kit to test the detectability of our test strips. The test results are as in table three.

8) Interpretation of the results of this test example: compared with the detection result of an imported LAg-Avidity EIA kit, the detection result of the test strip has better consistency, and the sensitivity, the specificity and the coincidence rate are respectively 84.6%, 93.3% and 82.00%.

TABLE III comparison of test paper with the consistency of imported EIA kit

Example 5.

Compared with the fourth experimental example, the experimental example further incorporates a data sample with epidemiology follow-up, and verifies the capability of the test strip to distinguish recent infection and long-term infection of HIV-1.

2) The method for spraying gold particles and assembling the test strip in this test example is the same as that in test example 4.

3) The test paper of this test example was prepared in the same manner as in test example 4.

4) This experiment was carried out in the same manner as in example 4.

5) The loading method of this test example is the same as that of test example 4.

6) The method for judging the test results in this test example was the same as in test example 4.

7) We obtained 36 serum samples from the CDC of Guangzhou city with detailed follow-up information on infection time, and tested the samples simultaneously using the test paper prepared by us and the LAg-identification EIA kit, thereby testing the detection ability of the test paper. The test results are shown in tables 4 and 5.

8) Interpretation of the results: compared with the detection result of an imported LAg-Avidity EIA kit, the detection result of the test strip has better consistency, and the sensitivity, the specificity and the coincidence rate are respectively 92.31%, 91.30% and 91.70%.

TABLE IV, test paper and import kit for detecting 9 parts of serial serums

TABLE V, test paper and import kit for detecting consistency comparison of 9 serial serums

The test result confirms that the test strip can judge the recent infection or long-term infection state of the HIV-1, the judgment result is accurate, and the misjudgment or the misjudgment missing probability is small.

The test strip is convenient to use, simple to operate, low in professional requirement on operators, low in result misjudgment of the operators due to misoperation and high in repeatability of operation, and does not need high-end experimental equipment and complex experimental steps. In addition, the method has fewer operation steps, and reduces the frequency of contacting HIV positive samples by operators, thereby reducing the infection risk of the operators.

Compared with the current laboratory method on the market, the test strip has the advantages that the test time is shortened to be within 30 minutes, the test time is greatly shortened, and the test efficiency is improved.

The test strip disclosed by the invention is simple in result interpretation, can be judged only by comparing the reference line color with a standard color comparison card according to the operation flow by an operator, does not need professional interpretation, is suitable for primary health workers, is good in popularization, and can be used for large-scale primary screening of HIV-1 late-stage infection.

The technical features of the above-mentioned test examples can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above-mentioned test examples are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

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Claims

1. A test strip for rapidly distinguishing recent and long-term HIV infection states comprises a bottom plate, a sample pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the nitrocellulose membrane and the absorbent paper are arranged on the bottom plate, two ends of the nitrocellulose membrane are respectively lapped with the sample pad and the absorbent paper, and a colloidal gold-labeled mouse anti-human IgG antibody is sprayed on one end, close to the nitrocellulose membrane, of the sample pad to form a gold-labeled layer; the method is characterized in that:

2. The test strip of claim 1 for rapidly differentiating between recent and long-term HIV infection states, wherein: the coating concentration of the HIV recombinant antigen in the second detection line is 2.5-3.5 mg/ml.

3. The test strip of claim 2 for rapidly differentiating between recent and long-term HIV infection states, wherein: the coating concentration of the GP41 recombinant antigen BE23 in the first detection line is 0.1-0.3mg/ml, and the coating concentration of the GP41 recombinant antigen BE33 in the first detection line is 0.3-0.6 mg/ml.

4. The test strip of claim 3 for rapidly differentiating between recent and long-term HIV infection states, wherein: the GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33 are mixed according to the volume ratio of 1: 3, mixing and coating.

5. The test strip according to any one of claims 1 to 4 for rapidly differentiating between recent and long-term HIV infection status, characterized in that: the coating concentration of the goat anti-mouse IgG of the quality control line is 0.5-1.0 mg/ml.

6. The test strip of claim 5 for rapidly differentiating between recent and long-term HIV infection states, wherein: the concentration of the colloidal gold-labeled mouse anti-human IgG antibody is 10-20 mu g/ml.

7. The test strip of claim 6 for rapidly differentiating between recent and long-term HIV infection states, wherein: and referring to a color comparison card, referring to the first detection line when the strips appear on the quality control line and the second detection line, displaying the result of recent infection when the brightness of the strips on the first detection line is less than L3, and displaying the result of long-term infection when the brightness of the strips on the first detection line is greater than or equal to L3.

8. Use of a first test line mixedly coated with GP41 recombinant antigen BE23 and GP41 recombinant antigen BE33 in a test strip according to any one of claims 1 to 7 for rapidly differentiating between recent and long-term infection states of HIV.

9. A method of making a test strip according to any one of claims 1 to 7 for rapidly differentiating between recent and long-term HIV infection, comprising: the method is carried out by the following steps,

1) preparing a sample pad;

3) preparing a first detection line, a second detection line and a quality control line, and then airing or drying the whole under the conditions of 25 ℃ and 10-30% of humidity for 18-22 h;