HIV marker diluent, kit and preparation method thereof

Abstract

The present invention discloses an HIV marker diluent, a kit and a preparation method thereof, and relates to the field of immunoassay technology. The kit prepared by using the HIV marker diluent provided by the present invention has high sensitivity and specificity for antibody detection, and can also increase the difference in luminescence values ​​of positive and negative samples, which is beneficial to reducing detection errors and mistakes.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to an HIV marker diluent, a kit and a preparation method thereof. Background Art

[0002] The detection of antibodies against human immunodeficiency virus is currently one of the important bases for diagnosing HIV infection, and its clinical diagnostic value has been widely recognized.

[0003] Chemiluminescence is a new detection technology that has only emerged in the 1990s. In addition to the advantages of simple and rapid detection methods, it also has the advantages of high sensitivity and a wide linear detection range. Therefore, chemiluminescence has received more and more attention from clinical diagnostic laboratories and has become a hot topic in research in this field in recent years.

[0004] The use of chemiluminescence to detect HIV antibodies is a routine method for HIV etiology diagnosis. At present, in order to make the test results as accurate as possible, HIV testing uses a special strategy, that is, first use a highly sensitive method for initial screening, and then use a highly specific method to confirm the positive samples. At the same time, in order to reduce the errors and errors of the test, strict quality assurance, quality control and quality evaluation measures must be taken.

[0005] The existing detection methods have problems such as repeated detection, low efficiency, and small contrast in luminescence values ​​of positive and negative specimens.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide an HIV marker diluent, a kit and a preparation method thereof to solve the above technical problems.

[0008] The purpose of the present invention is to provide a more accurate, stable and convenient detection method for clinical immunoassay laboratories to adapt to the continuous progress of laboratory medicine.

[0009] The present invention is achieved in that:

[0010] The invention provides an HIV marker diluent, which comprises cellulose methyl ether and 2-chloroacetamide.

[0011] In one embodiment, the mass percentage content of cellulose methyl ether in the HIV marker diluent is 0.001%-0.1%, and the mass percentage content of 2-chloroacetamide in the HIV marker diluent is 0.01%-1%.

[0012] In one embodiment, the mass percentage content of cellulose methyl ether in the above HIV marker diluent is 0.005%-0.8%, and the mass percentage content of 2-chloroacetamide in the HIV marker diluent is 0.05%-0.8%.

[0013] In one embodiment, the mass percentage content of cellulose methyl ether in the above HIV marker diluent is 0.01%-0.05%, and the mass percentage content of 2-chloroacetamide in the HIV marker diluent is 0.1%-0.5%.

[0014] In one embodiment, the HIV marker diluent further comprises a stabilizer, and the stabilizer comprises bovine serum albumin and casein.

[0015] In one embodiment, the mass percentage content of albumin in the above HIV marker diluent is 1-5%, and the mass percentage content of casein in the HIV marker diluent is 0.01%-0.2%.

[0016] In one embodiment, the HIV marker diluent further comprises a buffer, an inorganic salt and a surfactant.

[0017] In one embodiment, the buffer is selected from Tris-HCl, and the inorganic salt is selected from sodium chloride.

[0018] In one embodiment, the HIV marker diluent further comprises 0.05M-0.2M Tris-HCl, 0.05%-0.2% surfactant by volume of the HIV marker diluent, and 0.8%-1% sodium chloride by mass of the HIV marker diluent.

[0019] In one embodiment, the surfactant is selected from at least one of Tween-20, Tween-80, Triton X-100 and Tween-60.

[0020] The present invention also provides an HIV labeled antigen reagent, which comprises the above HIV labeled diluent and labeled antigen.

[0021] In one embodiment, the marker on the antigen is selected from alkaline phosphatase, horseradish peroxidase or acridinium ester.

[0022] In one embodiment, the above antigen is HIV-I type antigen and / or HIV-II type antigen.

[0023] In one embodiment, the HIV-I type antigen is GP41, and the HIV-II type antigen is GP36;

[0024] In one embodiment, the antigen is a chimeric antigen of GP41 and GP36.

[0025] The present invention also provides a kit, which comprises the above HIV marker diluent or HIV marker antigen reagent.

[0026] The present invention also provides a human immunodeficiency virus antibody diagnostic kit, which comprises an HIV-labeled antigen reagent, wherein the HIV-labeled antigen reagent comprises an HIV-labeled diluent and enzyme-labeled HIV-I type antigen and / or HIV-II type antigen.

[0027] In one embodiment, the HIV marker antigen reagent comprises an HIV marker diluent and an enzyme-labeled chimeric antigen of GP41 and GP36.

[0028] The present invention has the following beneficial effects:

[0029] The present invention provides an HIV marker diluent, which contains methyl cellulose and 2-chloroacetamide. Compared with the prior art, the use of the HIV marker diluent provided by the present invention can ensure that the detection method and the kit have both high sensitivity and specificity, and can also increase the difference in luminescence values ​​of positive and negative samples, that is, the contrast of the luminescence values ​​of positive and negative samples is more obvious, which is conducive to reducing detection errors and mistakes. DETAILED DESCRIPTION

[0030] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0031] The present invention provides

[0032] A HIV marker diluent comprises cellulose methyl ether and 2-chloroacetamide.

[0033] Cellulose methyl ether acts as a thickener.

[0034] 2-Chloroacetamide is also known as chloroacetamide, 2-chloroacetamide, p-chloroacetamide, and chloroacetamide.

[0035] The inventors found that an HIV-labeled diluent added with methyl cellulose and 2-chloroacetamide has higher sensitivity and specificity when performing human immunodeficiency virus antibody detection, and the luminescence difference between positive and negative samples is more obvious.

[0036] In one embodiment, the mass percentage content of cellulose methyl ether in the above HIV marker diluent is 0.001%-0.1%, and the mass percentage content of 2-chloroacetamide in the HIV marker diluent is 0.01%-1%. Optionally, the mass percentage content of cellulose methyl ether in the HIV marker diluent is 0.001%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% or 0.8%. Optionally, the mass percentage content of 2-chloroacetamide in the HIV marker diluent is 0.01%, 0.05%, 0.07%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.

[0037] In one embodiment, the mass percentage content of cellulose methyl ether in the above HIV marker diluent is 0.005%-0.8%, and the mass percentage content of 2-chloroacetamide in the HIV marker diluent is 0.05%-0.8%.

[0038] In one embodiment, when the contents of other components in the enzyme-binding diluent are controlled to remain unchanged, when the mass percentage of cellulose methyl ether in the HIV-labeled diluent is 0.01%-0.05%, the luminescence difference between the positive and negative samples is large.

[0039] In one embodiment, when the contents of other components in the enzyme-binding diluent are controlled to remain unchanged, when the mass percentage content of 2-chloroacetamide in the HIV-labeled diluent is 0.1%-0.5%, the luminescence difference between the positive and negative samples is large.

[0040] When the mass percentage content of cellulose methyl ether in the HIV labeling diluent is 0.01%-0.05%, and the mass percentage content of 2-chloroacetamide is 0.1%-0.5%, the luminescence difference between the positive and negative samples is the largest.

[0041] In one embodiment, the HIV-labeled diluent further comprises a stabilizer, and the stabilizer comprises bovine serum albumin (BSA) and casein. The stabilizer is added to stabilize and protect the enzyme-labeled antigen, thereby preventing the activity of the enzyme-labeled antigen from being partially or completely inactivated.

[0042] In one embodiment, the mass percentage content of bovine serum albumin in the above HIV marker diluent is 1%-5%, and the mass percentage content of casein in the HIV marker diluent is 0.01%-0.2%. Optionally, the mass percentage content of bovine serum albumin in the HIV marker diluent is 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or 5%. Optionally, the mass percentage content of casein in the HIV marker diluent is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.15% or 0.2%.

[0043] In one embodiment, the mass percentage content of bovine serum albumin in the HIV marker diluent is 3%, and the mass percentage content of casein in the HIV marker diluent is 0.1%.

[0044] In one embodiment, the HIV marker diluent further comprises a buffer, an inorganic salt and a surfactant.

[0045] In one embodiment, the buffer includes but is not limited to Tris-HCl, and the inorganic salt includes but is not limited to sodium chloride.

[0046] In one embodiment, the HIV marker diluent further comprises 0.05M-0.2M Tris-HCl, 0.05%-0.2% surfactant by volume of the HIV marker diluent, and 0.8%-1% sodium chloride by mass of the HIV marker diluent.

[0047] The Tris-HCl in the HIV labeling diluent is used as a buffer, and preferably, the pH of the Tris-HCl is 7.4.

[0048] In one embodiment, the surfactant is selected from at least one of Tween-20, Tween-80, Triton X-100 and Tween-60. Optionally, the surfactant is selected from Tween-20 and Triton X-100.

[0049] Triton X-100 is polyethylene glycol octylphenyl ether, octylphenyl polyoxyethylene ether, polyethylene glycol p-isooctylphenyl ether, isooctylphenyl polyoxyethylene ether or emulsifier OP-10.

[0050] The present invention also provides an HIV labeled antigen reagent, which comprises the above HIV labeled diluent and labeled antigen.

[0051] In one embodiment, the marker on the antigen is selected from alkaline phosphatase, horseradish peroxidase or acridinium ester. In actual use, the quantitative analysis of the antibody to be tested can be achieved by combining with the corresponding enzyme-labeled detection technology.

[0052] In one embodiment, the above antigen is HIV-I type antigen and / or HIV-II type antigen.

[0053] In one embodiment, the HIV-I type antigen is GP41, and the HIV-II type antigen is GP36.

[0054] In one embodiment, the antigen is a chimeric antigen of GP41 and GP36. The chimeric antigen of GP41 and GP36 is a chimeric antigen obtained after gene recombination.

[0055] The present invention also provides a kit, which includes the above HIV marker diluent or the above HIV marker antigen reagent. It should be noted that the above kit can be a sandwich detection kit of antigen-test sample antibody-antigen, or a one-step detection kit of antigen-test sample antibody.

[0056] The above kit can also be used to detect the content of other types of viral antibodies and is not limited to the detection of human immunodeficiency virus antibodies.

[0057] The present invention also provides a human immunodeficiency virus antibody diagnostic kit, which comprises an HIV-labeled antigen reagent, wherein the HIV-labeled antigen reagent comprises an HIV-labeled diluent and enzyme-labeled HIV-I type antigen and / or HIV-II type antigen.

[0058] In one embodiment, the HIV marker antigen reagent includes HIV marker diluent and enzyme-labeled chimeric antigens of GP41 and GP36. The chimeric antigen further improves the detection sensitivity and further shortens the window period.

[0059] In one embodiment, the kit further comprises magnetic particles (Dynal), and the magnetic particles are coated with antigens. The method of coating the magnetic particles with antigens comprises:

[0060] (1) washing the magnetic particles to be coated;

[0061] (2) Activating the surface of the magnetic beads (i.e., magnetic microparticles): Adding EDC and NHS to the magnetic beads and placing them on a mixer for mixing and activation, the mass ratio of EDC to magnetic beads is 1:10, and the molar ratio of EDC to NHS is 1:2;

[0062] (3) washing;

[0063] (4) Coat the surface of the magnetic beads with antigen. The ratio of antigen to magnetic beads is: magnetic beads: antigen = 4 (ml): 1

[0064] (mg);

[0065] (5) Blocking: Block with 20% lysine blocking solution for 1 hour;

[0066] (6) Storing the magnetic beads.

[0067] The antigen selected for coating is human immunodeficiency virus (type 1+2) gene recombinant chimeric antigen (including GP41 and GP36).

[0068] When in use, the enzyme-labeled HIV-I type antigen and / or HIV-II type antigen is diluted with HIV labeling diluent.

[0069] The method of using the above-mentioned diagnostic kit for detection includes:

[0070] Add the magnetic particles coated with the chimeric antigen, negative control, positive control and the sample to be tested to the wells of the luminescent plate, and incubate at 37°C for 10-15 minutes (the positive control and the negative control are the commercially available HIV antibody national reference standard). After incubation, wash to remove the antibody that is not fully bound, then add the HIV labeled antigen reagent (including enzyme-labeled HIV-I type antigen and / or HIV-II type antigen), incubate at 37°C for 10-15 minutes to form an antigen-antibody-enzyme labeled antigen complex, wash to remove the unbound matter, then add the chemiluminescent substrate, and then place the wells of the luminescent plate in a luminescent signal measuring instrument to measure the light quantum yield.

[0071] Photons are generated under the action of the enzyme, and within a certain range, the number of photons is proportional to the content of specific antibodies, thereby achieving quantitative analysis of specific antibodies in the sample to be tested.

[0072] The present invention adopts an enzymatic chemiluminescent immunoassay method, that is, an enzyme is used to catalyze a luminescent substrate to undergo a chemical reaction and release a large amount of energy to produce an excited state intermediate. This excited state intermediate emits photons when it returns to a stable ground state. The light quantum yield is measured using a luminescent signal measuring instrument, and the light quantum yield is proportional to the amount of the substance to be tested in the sample.

[0073] Compared with several traditional immunological detection methods, the chemiluminescence method has outstanding advantages in sensitivity, detection time, linear range, reagent stability, and environmental pollution.

[0074] The above-mentioned human immunodeficiency virus antibody diagnostic kit (magnetic particle chemiluminescence method) is a highly sensitive and specific detection kit developed on a mature magnetic particle and chemiluminescence technology platform combined with the technical advantages of the enzyme-linked immunosorbent double antigen sandwich method. It can be used to detect human immunodeficiency virus (type 1+2) antibodies in human serum or plasma.

[0075] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0076] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0077] Example 1

[0078] This embodiment provides an HIV marker diluent, and its formula is shown in Table 1. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.001% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.01% 2-chloroacetamide by mass, and water.

[0079] Example 2

[0080] This embodiment provides an HIV marker diluent, and its formula is shown in Table 1. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.002% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.02% 2-chloroacetamide by mass, and water.

[0081] Example 3

[0082] This embodiment provides an HIV marker diluent, and its formula is shown in Table 2. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.003% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.02% 2-chloroacetamide by mass, and water.

[0083] Example 4

[0084] This embodiment provides an HIV marker diluent, and its formula is shown in Table 2. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.003% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.03% 2-chloroacetamide by mass, and water.

[0085] Example 5

[0086] This embodiment provides an HIV marker diluent, and its formula is shown in Table 2. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.004% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.04% 2-chloroacetamide by mass, and water.

[0087] Table 1. Formulations of HIV marker diluents in Examples 1-5.

[0088]

[0089] Example 6

[0090] This embodiment provides an HIV marker diluent, and its formula is shown in Table 1. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.005% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.05% 2-chloroacetamide by mass, and water.

[0091] Example 7

[0092] This embodiment provides an HIV marker diluent, and its formula is shown in Table 1. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.01% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.1% 2-chloroacetamide by mass, and water.

[0093] Example 8

[0094] This embodiment provides an HIV marker diluent. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.05% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.5% 2-chloroacetamide by mass, and water.

[0095] Example 9

[0096] This embodiment provides an HIV marker diluent, and its formula is shown in Table 1. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.07% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.7% 2-chloroacetamide by mass, and water.

[0097] Example 10

[0098] This embodiment provides an HIV marker diluent, and its formula is shown in Table 2. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.08% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 0.8% 2-chloroacetamide by mass, and water.

[0099] Table 2 Formulations of HIV marker diluents in Examples 6-10.

[0100]

[0101]

[0102] Embodiment 11

[0103] This embodiment provides an HIV marker diluent, and its formula is shown in Table 2. Specifically, the HIV marker diluent includes 0.1M Tris-HCl with a pH of 7.4, 3.00% BSA by mass, 0.1% casein by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 0.1% cellulose methyl ether by mass, 0.9% sodium chloride by mass, 1% 2-chloroacetamide by mass, and water.

[0104] Example 12

[0105] This embodiment provides a human immunodeficiency virus antibody diagnostic kit, which includes an HIV marker antigen reagent, wherein the HIV marker antigen reagent includes an alkaline phosphatase-labeled chimeric antigen of GP41 and GP36 and an HIV marker diluent.

[0106] It should be noted that in this embodiment, the chimeric antigens of GP41 and GP36 labeled with alkaline phosphatase are selected. In other embodiments, other types of antigens labeled with other markers can also be selected as needed. The marker type and antigen type provided in this embodiment have no direct correlation with the kit.

[0107] Comparative Example 1

[0108] This comparative example provides an HIV marker diluent. Specifically, the HIV marker diluent includes 0.05M Tris-HCl with a pH of 7.4, 0.5% BSA by mass, 4% mannitol by mass, 0.1% Tween-20 by volume, 0.1% TritonX-100 by volume, 1.5% sodium chloride by mass, 5% skim milk powder by mass, and water.

[0109] Experimental Example 1

[0110] The HIV marker diluents of Examples 1-5 described above were used as HIV marker diluents in the diagnostic kit of Example 12, and parallel comparative experiments were performed on 5 kit combinations.

[0111] During the experiment, negative control and positive control (including HIV-I type quality control and HIV-II type quality control, both of which are commercially available national standard products) were added to each combination.

[0112] Methods for testing include:

[0113] Add 30 μl of the magnetic particles coated with chimeric antigens, 50 μl of sample diluent, and 75 μl of negative control / positive control to the wells of the luminescent plate, and incubate at 37°C for 10-15 minutes (negative control / positive control are both commercially available standards). After incubation, wash to remove the antibodies that are not fully bound, and then add 100 μl of labeled antigen solution (the added labeled antigen solution is obtained by diluting the chimeric antigens of GP41 and GP36 labeled with alkaline phosphatase with HIV marker diluent. In other embodiments, other types of antigens labeled with other markers can also be selected as needed), incubate at 37°C for 10-15 minutes to form an antigen-antibody-labeled antigen complex, and then add a chemiluminescent substrate after fully washing to remove unbound substances, and then place the wells of the luminescent plate in a luminescent signal measuring instrument to measure the light quantum yield.

[0114] The test results of Examples 1-5 are shown in Table 3, and the P / N values ​​are shown in Table 4. It can be seen from Tables 3 and 4 that when the amount of 2-chloroacetamide added to the HIV marker diluent is 0.1%, the ratio of positive treatment to negative treatment is the largest, that is, the difference in luminescence values ​​of positive and negative samples is the largest at this time. Table 5 shows the test processing procedure of Experimental Example 1.

[0115] Table 3 is a data table showing quality control product testing using the HIV marker diluents of Examples 1-5 as HIV marker diluents in a diagnostic kit.

[0116]

[0117] Table 4 shows the P / N values ​​of the quality control products tested by using the HIV marker diluents of Examples 1-5 as HIV marker diluents in the diagnostic kit.

[0118]

[0119] Table 5 Detection processing procedures for Experimental Example 1.

[0120]

[0121] Experimental Example 2

[0122] In this experimental example, the HIV marker diluents of Examples 6-10 described above were used as HIV marker diluents in Example 12, and parallel comparative experiments were performed on 5 kit combinations.

[0123] During the experiment, negative control and positive control (including HIV-I type quality control and HIV-II type quality control, both of which are commercially available national standard products) were added to each combination.

[0124] Methods for testing include:

[0125] Add 30 μl of the magnetic particles coated with chimeric antigens, 50 μl of sample diluent, and 75 μl of negative control / positive control to the wells of the luminescent plate, and incubate at 37°C for 10-15 minutes (negative control / positive control are both commercially available standards). After incubation, wash to remove the antibodies that are not fully bound, then add 100 μl of labeled antigen solution (the added labeled antigen solution is obtained by diluting the chimeric antigens of GP41 and GP36 labeled with alkaline phosphatase with HIV marker diluent. In other embodiments, other types of antigens labeled with other markers can also be selected as needed), incubate at 37°C for 10-15 minutes to form an antigen-antibody-labeled antigen complex, wash thoroughly to remove unbound substances, then add chemiluminescent substrate, and then place the wells of the luminescent plate in a luminescent signal measuring instrument to measure the light quantum yield.

[0126] The test results of Examples 6-10 are shown in Table 6, and the P / N values ​​are shown in Table 7. It can be seen from Tables 6 and 7 that when the addition amount of cellulose methyl ether in the HIV marker diluent is 0.01%, the ratio of positive treatment to negative treatment is the largest, that is, the difference in luminescence values ​​of positive and negative samples is the largest at this time.

[0127] Table 6 is a data table showing quality control product testing using the HIV marker diluents of Examples 6-10 as HIV marker diluents in a diagnostic kit.

[0128]

[0129] Table 7 P / N values ​​of quality control products tested using the HIV marker diluents of Examples 6-10 as HIV marker diluents in diagnostic kits.

[0130]

[0131] Experimental Example 3

[0132] In this experimental example, the HIV marker diluents of the above-mentioned Example 11 and Comparative Example 1 were used as HIV marker diluents in Example 12, and parallel comparative experiments were performed on 5 kit combinations.

[0133] During the experiment, negative control and positive control (including HIV-I type quality control and HIV-II type quality control, both of which are commercially available national standard products) were added to each combination.

[0134] Methods for testing include:

[0135] Add 30 μl of the magnetic particles coated with chimeric antigens, 50 μl of sample diluent, and 75 μl of negative control / positive control to the wells of the luminescent plate, and incubate at 37°C for 10-15 minutes (negative control / positive control are both commercially available standards). After incubation, wash to remove the antibodies that are not fully bound, and then add 100 μl of labeled antigen solution (the added labeled antigen solution is obtained by diluting the chimeric antigens of GP41 and GP36 labeled with alkaline phosphatase with HIV labeling diluent. In other embodiments, other types of antigens labeled with other markers can also be selected as needed), and incubate at 37°C for 10-

[0136] After 15 minutes, the antigen-antibody-labeled antigen complex is formed. After sufficient washing to remove unbound substances, the chemiluminescent substrate is added, and then the luminescent plate wells are placed in a luminescent signal measuring instrument to measure the light quantum yield.

[0137] The test results of Example 11 and Comparative Example 1 are shown in Table 8, and the P / N values ​​are shown in Table 9. It can be seen from Tables 8 and 9 that the ratio of positive treatment to negative treatment is greatly improved by using the HIV marker diluent provided by the present invention. That is to say, when the kit prepared by the HIV marker diluent provided in Example 11 is used for quality control product testing, the difference in luminescence values ​​of positive and negative samples is greater than that of Comparative Example 1, and the accuracy is higher.

[0138] Table 8 is a data table showing quality control product testing of the kit prepared with the HIV marker diluent of Example 11 and Comparative Example 1.

[0139]

[0140] Table 9 P / N values ​​of quality control product testing of the kit prepared with the HIV marker diluent of Example 11 and Comparative Example 1.

[0141]

[0142] Experimental Example 4

[0143] Referring to the design of Experimental Example 1-3, the HIV antigen marker was replaced with horseradish peroxidase, and the luminescent substrate was replaced with luminol. The P / N results are shown in Table 10.

[0144] Table 10 P / N values ​​of quality control product testing of the kits prepared with the HIV marker diluents of Examples 6-10 and Comparative Example 1.

[0145]

[0146] Experimental Example 5

[0147] In this experimental example, the kit prepared by the HIV marker diluent of Example 7 was used to detect HIV clinical negative samples (see Tables 12 to 18). The average S / CO value of the sample was calculated at the same time. The detection process is shown in Table 11.

[0148] Table 11 Detection processing procedures for Experimental Example 5.

[0149]

[0150] Table 12 S / CO values ​​and test results of 20200805HIV clinical samples.

[0151]

[0152]

[0153] Table 12 is continued.

[0154]

[0155]

[0156] Table 13 S / CO values ​​and test results of 20200806HIV clinical samples.

[0157]

[0158]

[0159] Table 14 S / CO values ​​and test results of 20200811 HIV clinical samples.

[0160]

[0161]

[0162] Table 15 S / CO values ​​and test results of HIV clinical samples from 20200812 and 20200813.

[0163]

[0164]

[0165] Table 16 S / CO values ​​and test results of 20200817 HIV clinical samples.

[0166]

[0167]

[0168] Table 17 S / CO values ​​and test results of 20200818HIV clinical samples.

[0169]

[0170]

[0171]

[0172] Table 18 S / CO values ​​and test results of 20200819 HIV clinical samples.

[0173]

[0174]

[0175] In this experimental case, a total of 779 negative clinical samples were tested, all of which were negative, with a specificity of 100% and a coincidence rate of 100%.

[0176] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An HIV marker diluent, characterized in that It includes: Cellulose methyl ether and 2-chloroacetamide.

2. The HIV marker diluent according to claim 1, characterized in that The mass percentage content of the cellulose methyl ether in the HIV marker diluent is 0.001%-0.1%, and the mass percentage content of the 2-chloroacetamide in the HIV marker diluent is 0.01%-1%.

3. The HIV marker diluent according to claim 1, characterized in that The mass percentage content of the cellulose methyl ether in the HIV marker diluent is 0.005%-0.08%, and the mass percentage content of the 2-chloroacetamide in the HIV marker diluent is 0.05%-0.8%.

4. HIV marker diluent according to claim 3, characterized in that, The mass percentage content of the cellulose methyl ether in the HIV marker diluent is 0.01%-0.05%, and the mass percentage content of the 2-chloroacetamide in the HIV marker diluent is 0.1%-0.5%.

5. The HIV marker diluent according to any one of claims 1 to 4, characterized in that The HIV marker diluent also includes a stabilizer, which includes bovine serum albumin and casein.

6. The HIV marker diluent according to claim 5, characterized in that The mass percentage content of the albumin in the HIV marker diluent is 1%-5%, and the mass percentage content of the casein in the HIV marker diluent is 0.01%-0.2%.

7. The HIV marker diluent according to any one of claims 1 to 4, characterized in that The HIV marker diluent also includes a buffer, an inorganic salt and a surfactant.

8. The HIV marker diluent according to claim 7, characterized in that The buffer is selected from Tris-HCl, and the inorganic salt is selected from sodium chloride.

9. The HIV marker diluent according to claim 8, characterized in that The HIV marker diluent includes 0.05M-0.2M Tris-HCl, 0.05%-0.2% by volume of a surfactant in the HIV marker diluent, and 0.8%-1% by mass of sodium chloride in the HIV marker diluent.

10. The HIV marker diluent according to claim 7, characterized in that The surfactant is selected from at least one of Tween-20, Tween-80, Triton X-100 and Tween-60.

11. An HIV marker antigen reagent, characterized in that: The labeled antigen reagent comprises the HIV labeled diluent according to any one of claims 1 to 10 and a labeled antigen.

12. The HIV marker antigen reagent according to claim 11, characterized in that: The marker on the antigen is selected from alkaline phosphatase, horseradish peroxidase or acridinium ester.

13. The HIV marker antigen reagent according to claim 11, characterized in that: The antigen is HIV-I type antigen and / or HIV-II type antigen.

14. The HIV marker antigen reagent according to claim 13, characterized in that: The HIV-I type antigen is GP41, and the HIV-II type antigen is GP36.

15. The HIV marker antigen reagent according to claim 13, characterized in that: The antigen is a chimeric antigen of GP41 and GP36.

16. A kit, characterized in that: It comprises the HIV marker diluent according to any one of claims 1 to 10 or the HIV marker antigen reagent according to any one of claims 11 to 15.

17. A human immunodeficiency virus antibody diagnostic kit, characterized in that: It comprises the HIV-labeled antigen reagent according to any one of claims 11 to 15, wherein the HIV-labeled antigen reagent comprises an HIV-labeled diluent and enzyme-labeled HIV-Ⅰ type antigen and / or HIV-Ⅱ type antigen.

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