HIV-1 p24 elisa detection kit and its use and application

Abstract

The application relates to the field of biotechnology, in particular to the field of IPCG01N33, and more particularly to an HIV-1 p24 ELISA detection kit and a use method and application thereof. The HIV-1 p24 protein in a sample is detected by adopting a double-antibody sandwich method, an enzyme-linked plate is coated with double antibodies, and the kit development is completed by using a biotin-labeled p24 antibody as a detection antibody. The plate coating does not need to be performed one day in advance, the workload of an operator is reduced, and the whole operation time is saved. The quantitative range of the kit is 6.25-200 pg / mL, and the LOD (limit of detection) is less than or equal to 3.125 pg / mL. After verification, the detection method of the kit has high sensitivity, good precision and accuracy, and can be used for detecting the content of the p24 protein in a virus sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the field of IPCG01N33, and more particularly to an HIV-1 p24 ELISA detection kit and a method of use and application thereof. BACKGROUND

[0002] P24 is the main structural protein of HIV virus particles, which is the product of structural gene gag and plays an important role in the packaging and maturation of viruses. The amino acid sequence of p24 protein is highly conserved in HIV type inspection, and the deletion of p24 will cause the virus to be unable to be normally assembled.

[0003] Lentiviral vector is a viral vector system generated by modification on the basis of HIV-1 virus, and lentivirus can efficiently transfer the target gene into the related cells. The content of p24 protein represents the particle number of lentivirus. Immune analysis method (ELISA) is the preferred method for detecting the physical titer (p24 protein) of lentivirus in the industry.

[0004] The conventional ELISA experiment operation needs to be performed one day in advance, and other test reagents are prepared, the time period is long, and the preparation work in advance is complex and the workload is large. And the ELISA detection experiment is greatly affected by the environment and the operation of the personnel, the conventional operation, the whole material preparation and detection process are complex, and the error rate is high, and often the sample and the sample are confused, the wells are repeated or the wells are skipped during the addition. The purpose of the kit is to provide an ELISA detection kit, which contains a coated plate and a prepared reagent. The kit can improve the complex operation, high error rate and other deficiencies in the existing experimental detection operation, solve the technical problems of complex preparation work in advance, facilitate the smooth development of the experiment, and obtain more reliable and accurate data. SUMMARY

[0005] In order to solve the above problems, the first aspect of the present application provides a HIV-1 p24 ELISA detection kit, comprising: a coated enzyme label plate, a detection antibody, an enzyme conjugate, a standard, a lysis solution, a detection antibody and an enzyme conjugate diluent, a sample diluent, a 20x PBST washing solution, a substrate color developing liquid A, a substrate color developing liquid B, and a termination liquid.

[0006] Preferably, the coated enzyme label plate is coated with HIV-1 p24 specific monoclonal antibody.

[0007] Preferably, the HIV-1 p24 specific monoclonal antibody is composed of two antibodies P-AB-1 and P-AB-2.

[0008] Preferably, the concentration ratio of P-AB-1 and P-AB-2 is (1-2):(1-2); further preferably, 1:1.

[0009] The applicant unexpectedly found that the HIV-1 p24 specific monoclonal antibody composed of P-AB-1 and P-AB-2 with a concentration ratio of (1-2):(1-2) applied to the coated enzyme plate can not only reduce the detection time of the kit, but also improve the precision of the kit. This may be because the traditional double antibody sandwich method for detecting HIV-1 p24 protein in the sample only uses one HIV-1 p24 specific monoclonal antibody for pre-coating, which has a long detection time and low precision. The applicant unexpectedly found that the HIV-1 p24 specific monoclonal antibody composed of P-AB-1 and P-AB-2 with a concentration ratio of (1-2):(1-2) applied to the coated enzyme plate not only improves its specificity, making it more precise, but also speeds up the reaction through the synergistic effect of the two antibodies, thereby reducing the detection time of the kit. However, it will make the background too high, affecting the presentation of specific staining color.

[0010] In some preferred embodiments, P-AB-1 and P-AB-2 can be commercially available, such as ab32352 and ab63958 from supplier abcam.

[0011] Preferably, the preparation method of the coated enzyme plate is as follows: dilute the HIV-1 p24 specific monoclonal antibody with coating buffer to obtain a mixed coating, add 100 μL of the mixed coating to each well and incubate at 4°C overnight; discard the liquid in each well, wash the microwells with washing buffer (350 μL / well) for 3 times, then add 200 μL of blocking solution to each well and incubate in a 37°C incubator for 1.5 h; discard the liquid in each well, dry on a paper towel after washing the microwells with washing buffer for 3 times; add 200 μL of preservative to each well, react at 37°C for 1 h, then discard the liquid in each well, dry on a paper towel and dry, and the coated enzyme plate is obtained.

[0012] In some preferred embodiments, the coating buffer can be commercially available, such as ELISA coating buffer (1x) produced by Shanghai Ziliano Biological Technology Co., Ltd.

[0013] Preferably, the concentration ratio of the coating buffer and the antibody is 1:(2000-4000).

[0014] Preferably, the preparation method of the washing buffer is as follows: dilute an appropriate amount of 20x PBST washing solution with deionized water at a ratio of 1:20, and the washing buffer is obtained.

[0015] In some preferred embodiments, the 20x PBST washing solution can be commercially available, such as PBST washing buffer produced by Beijing Zhongjian Baotai Biological Technology Co., Ltd.

[0016] In some preferred embodiments, the blocking solution can be commercially available, such as the blocking buffer produced by the supplier, Yingchuang Biotech.

[0017] In some preferred embodiments, the preservative can be commercially available, such as the sucrose solution (10%) produced by the supplier, Shanghai Enzyme-Linked Biotech Co., Ltd.

[0018] Preferably, the detection antibody is a biotin-labeled p24 antibody.

[0019] In some preferred embodiments, the detection antibody can be commercially available, such as ab68617 from the supplier, abcam.

[0020] Preferably, the enzyme conjugate is horseradish peroxidase (HRP) labeled streptavidin (avidin-HRP).

[0021] In some preferred embodiments, the enzyme conjugate can be commercially available, such as 016-030-084 produced by the supplier, Jackson ImmunoResearch, with a product concentration of 1 mg / mL.

[0022] Preferably, the standard is human serum containing HIV-1 p24 antigen.

[0023] The standard is a commonly available human serum containing HIV-1 p24 antigen on the market, and the specific manufacturer is not limited.

[0024] Preferably, the lysis solution is one or more of 0.1% SDS and 0.1 vol% Triton; further preferably, 0.1 vol% Triton.

[0025] Applicants have unexpectedly found that selecting Triton as the lysis solution can dissolve the lipid components in the biological membrane, so that the antigen can bind to the antibody. In particular, when the concentration of Triton is 0.1%, not only can the lysis effect be maintained, but the background can also be reduced and the specific color can be improved.

[0026] Preferably, the preparation method of 0.1 vol% Triton is as follows: mix 50 μL of Ttiton-100 with 50 mL of PBS, filter with a 0.22 μm filter membrane, and store at 4°C.

[0027] In some preferred embodiments, the Ttiton-100 can be commercially available, such as 9002-93-1 produced by the supplier, Sigma.

[0028] In some preferred embodiments, the PBS can be commercially available, such as PBS produced by the supplier, Nanjing Sunbeiqia Biotech Co., Ltd.

[0029] Preferably, the detection antibody and enzyme conjugate diluent is one or more of 20vol% CS Buffer and 3vol% BSA-PBS; further preferably, 20vol% CS Buffer.

[0030] In order to reduce the background and improve the sensitivity of the kit, the blocking solution and diluent are selected. The applicant unexpectedly found that after coating the enzyme-linked plate with P-AB-1 and P-AB-2 antibodies, using 0.5wt% Casein-Na for blocking, and using 20vol% CS Buffer for the diluent of biotin-labeled p24 antibody and avidin-HRP, the sensitivity can reach 3.125pg / mL while the background is low and the signal-to-noise ratio is better.

[0031] Preferably, the preparation method of the 20vol% CS Buffer is as follows: 20mL of calf serum and 0.1mL of Proclin-300 are mixed, 1×PBS is added to make up to 100mL.

[0032] In some preferred embodiments, the calf serum can be commercially available, such as 151022 produced by Kangyuan Biotech Co., Ltd.

[0033] In some preferred embodiments, the Proclin-300 can be commercially available, such as 48914-U produced by Sigma.

[0034] Preferably, the preparation method of the 1×PBS is as follows: an appropriate amount of 20×PBS washing solution is diluted with deionized water at a ratio of 1:20 to obtain the 1×PBS.

[0035] In some preferred embodiments, the 20×PBS washing solution can be commercially available, such as PBS Phosphate Buffered Saline (20×) produced by Shanghai Yueteng Biotech Co., Ltd.

[0036] In some preferred embodiments, the sample diluent can be commercially available, such as Gradient Diluent produced by Anke Bioproducts.

[0037] Preferably, the substrate color developing solution A and the substrate color developing solution B are both purchased from the company of Kinbond.

[0038] Preferably, the termination solution is a 2mol / L aqueous sulfuric acid solution.

[0039] The second aspect of the present application provides a use method of the HIV-1 p24 ELISA detection kit as described above, the steps are as follows:

[0040] S1, add 10 μL lysis solution to each well of the coated enzyme-labeled plate, then add 90 μL standard sample diluted with sample diluent to the corresponding well, seal the plate with sealing film, and then place it in a 37°C constant temperature incubator for incubation at a shaking speed of 200-300 rpm for 1 h;

[0041] S2, discard the liquid in each well, wash the microwells with washing solution (350 μL / well) for 3 times, and then pat dry on a paper towel;

[0042] S3, add 100 μL detection antibody diluted with 20 vol% CS Buffer to each well, seal the plate with sealing film, and then place it in a 37°C constant temperature incubator for incubation at a shaking speed of 200-300 rpm for 1 h;

[0043] S4, discard the liquid in each well, wash the microwells with washing solution (350 μL / well) for 3 times, and then pat dry on a paper towel;

[0044] S5, add 100 μL enzyme conjugate diluted with 20 vol% CS Buffer to each well, seal the plate with sealing film, and then place it in a 37°C constant temperature incubator for incubation at a shaking speed of 200-300 rpm for 1 h;

[0045] S6, discard the liquid in each well, wash the microwells with washing solution (350 μL / well) for 3 times, and then pat dry on a paper towel;

[0046] S7, mix substrate color developing solution A and substrate color developing solution B to prepare substrate color developing solution, add 100 μL substrate color developing solution to each well, seal the plate with sealing film, and then develop color at 25°C for 10 min;

[0047] S8, add 50 μL termination solution to each well, and then read the value with an enzyme-labeled instrument.

[0048] Preferably, the parameters of the enzyme-labeled instrument are as follows: select the main wavelength of the enzyme-labeled instrument as 450 nm, the reference wavelength as 630 nm, and measure the absorbance (OD value) of each well.

[0049] The dilution step in step S1 is as follows:

[0050]

[0051]

[0052] Preferably, the concentration of the standard sample diluted with the sample diluent in step S1 is 6.25-200 pg / mL.

[0053] Preferably, the concentration ratio of the detection antibody to 20 vol% CS Buffer in step S3 is 1:2000.

[0054] Preferably, the concentration ratio of the enzyme conjugate to 20vol% CS Buffer in step S5 is 1:10000.

[0055] The applicant surprisingly found that the use of a coating buffer with a concentration ratio of 1:2000 and P-AB-1 / P-AB-2 mixed antibodies, in combination with a detection antibody with a concentration ratio of 1:2000 and 20vol% CS Buffer, and an enzyme conjugate with a concentration ratio of 1:10000 and 20vol% CS Buffer, not only further reduces the background, but also increases the absorbance value after the kit test, making the standard curve gradient obvious.

[0056] Preferably, the weight ratio of the substrate color developing liquid A and the substrate color developing liquid B is (1-2):(1-2); further preferably, 1:1.

[0057] The third aspect of the application provides the use of the HIV-1 p24 ELISA detection kit as described above.

[0058] Advantages:

[0059] 1. The use of HIV-1 p24 specific monoclonal antibodies composed of P-AB-1 and P-AB-2 with a concentration ratio of (1-2):(1-2) in coating the enzyme-labeled plate not only reduces the detection time of the kit, but also improves the precision of the kit.

[0060] 2. The use of Triton with a concentration of 0.1% as a lysis solution not only maintains good lysis effect, but also reduces the background and improves the presentation of specific color.

[0061] 3. The use of a coating buffer with a concentration ratio of 1:2000 and P-AB-1 / P-AB-2 mixed antibodies, in combination with a detection antibody with a concentration ratio of 1:2000 and 20vol% CS Buffer, and an enzyme conjugate with a concentration ratio of 1:10000 and 20vol% CS Buffer, not only further reduces the background, but also increases the absorbance value after the kit test, making the standard curve gradient obvious.

[0062] 4. After coating the enzyme-linked plate with P-AB-1 and P-AB-2 antibodies, using 0.5wt% Casein-Na for blocking, and using 20vol% CS Buffer for the dilution of biotin-labeled p24 antibody and avidin-HRP, the sensitivity can reach 3.125pg / mL under the condition of low background and better signal-to-noise ratio.

[0063] 5、The kit of the present application removes the operation steps of plate coating and other test reagent preparation performed one day in advance, and has prepared reagents in the kit, which can be directly used for experiments, reducing the workload of operators and saving the entire operation time, which can be completed in only 3 hours. If used for continuous detection, the detection results are more comparable.

[0064] 6、The kit of the present application has completed methodological verification, and has fast detection, strong specificity and reliable performance. DETAILED DESCRIPTION

[0065] EMBODIMENT

[0066] EMBODIMENT 1

[0067] Embodiment 1 provides an HIV-1 p24 ELISA detection kit, comprising: a coated enzyme-labeled plate, a detection antibody, an enzyme conjugate, a standard, a lysis solution, a detection antibody and enzyme conjugate diluent, a sample diluent, a 20x PBST washing solution, a substrate developing solution A, a substrate developing solution B, and a termination solution.

[0068] The coated enzyme-labeled plate is coated with HIV-1 p24 specific monoclonal antibodies.

[0069] The HIV-1 p24 specific monoclonal antibodies are composed of two antibodies P-AB-1 and P-AB-2.

[0070] The concentration ratio of P-AB-1 and P-AB-2 is 1:1.

[0071] P-AB-1 and P-AB-2 are purchased from suppliers abcam company ab32352 and ab63958.

[0072] The preparation method of the coated enzyme-labeled plate is as follows: after diluting the HIV-1 p24 specific monoclonal antibodies with coating buffer to obtain a mixed coating, 100 μL of the mixed coating is added to each well and incubated at 4°C overnight; the liquid in each well is poured out, and the microwells are washed with washing solution (350 μL / well) for 3 times, then 200 μL of blocking solution is added to each well, and the 37°C incubator is placed for 1.5 h; the liquid in each well is poured out, and the microwells are washed with washing solution for 3 times, then the microwells are patted dry on a paper towel; 200 μL of preservative is added to each well, and the 37°C reaction is performed for 1 h, then the liquid in each well is poured out, patted dry on a paper towel and dried, and the coated enzyme-labeled plate is obtained.

[0073] The coating buffer is purchased from ELISA coating buffer (1x) produced by Shanghai Ziliano Biological Technology Co., Ltd.

[0074] The concentration ratio of the coating buffer and the HIV-1 p24 specific monoclonal antibodies is 1:2000.

[0075] The preparation method of the cleaning solution is: diluting the appropriate amount of 20xPBST washing solution with deionized water at a ratio of 1:20 to obtain the cleaning solution.

[0076] The 20xPBST washing solution is purchased from the PBST washing buffer produced by the supplier Beijing Zhijian Baitai Biotechnology Co., Ltd.

[0077] The blocking solution is purchased from the blocking buffer produced by the supplier Yingchuang Biotechnology Co., Ltd.

[0078] The preservative is sucrose solution (10%) produced by the supplier Shanghai Zilian Biotechnology Co., Ltd.

[0079] The detection antibody is a biotin-labeled p24 antibody.

[0080] The detection antibody is ab68617 from the supplier Abeam Co., Ltd.

[0081] The enzyme conjugate is horseradish peroxidase (HRP) labeled streptavidin (avidin-HRP).

[0082] The enzyme conjugate is 016-030-084 produced by the supplier JacksonlmmunoResearch Co., Ltd., and the product concentration is 1 mg / mL.

[0083] The standard sample is human serum containing HIV-1 p24 antigen.

[0084] The standard sample is the British NIBSC 90 / 636 HIV-1 p24 antigen standard material from the supplier Shenzhen Debuer Biotechnology Co., Ltd.

[0085] The lysis solution is 0.1 vol% Triton.

[0086] The preparation method of the 0.1 vol% Triton is: mixing 50 μL of Ttiton-100 with 50 mL of PBS, filtering with a 0.22 μm filter membrane, and then storing at 4°C.

[0087] The Ttiton-100 is 9002-93-1 produced by the supplier Sigma Co., Ltd.

[0088] The PBS is produced by the supplier Nanjing Senbeiga Biotechnology Co., Ltd.

[0089] The detection antibody and enzyme conjugate diluent are 20 wt% CSBuffer.

[0090] The preparation method of the 20wt% CS Buffer is as follows: 20 mL of calf serum and 0.1 mL of Proclin-300 are mixed, 1x PBS is added to make up to 100 mL.

[0091] The calf serum is purchased from a supplier, Kangyuan Biotech Co., Ltd., with a product number of 151022.

[0092] The Proclin-300 is purchased from a supplier, Sigma-Aldrich, with a product number of 48914-U.

[0093] The preparation method of the 1x PBS is as follows: a proper amount of 20x PBS washing solution is diluted with deionized water at a ratio of 1:20 to obtain the 1x PBS.

[0094] The 20x PBS washing solution is purchased from a supplier, Shanghai Yueteng Biotech Co., Ltd., with a product number of PBS Phosphate Buffered Saline (20x).

[0095] The sample diluent is purchased from a supplier, Anke Bioproducts, with a product number of Gradient Diluent.

[0096] The substrate color developing solution A and the substrate color developing solution B are both purchased from a supplier, Kinbond.

[0097] The termination solution is a 2 mol / L aqueous sulfuric acid solution.

[0098] The second aspect of the present application provides a use method of the HIV-1 p24 ELISA detection kit as described above, and the steps are as follows:

[0099] S1, 10 μL of lysis solution is added to each well of the coated enzyme-labeled plate, and 90 μL of standard sample diluted with the sample diluent is added to the corresponding well, and then the plate is sealed with a sealing film and placed in a 37°C constant temperature shaking incubator at a shaking speed of 250 rpm for 1 h;

[0100] S2, the liquid in each well is poured out, the microwells are washed with the washing solution (350 μL / well) for 3 times, and then dried on a paper towel;

[0101] S3, 100 μL of detection antibody diluted with 20 vol% CS Buffer is added to each well, the plate is sealed with a sealing film and placed in a 37°C constant temperature shaking incubator at a shaking speed of 250 rpm for 1 h;

[0102] S4, the liquid in each well is poured out, the microwells are washed with the washing solution (350 μL / well) for 3 times, and then dried on a paper towel;

[0103] S5, 100 μL of enzyme conjugate diluted with 20wt% CS Buffer is added to each well, the plate is sealed with a sealing film and placed in a 37°C constant temperature shaking incubator at a shaking speed of 250 rpm for 1 h;

[0104] S6, after washing the micro-well 3 times with washing solution (350 μL / well), the liquid in each well was poured out and dried on a paper towel;

[0105] S7, substrate color developing solution A and substrate color developing solution B were mixed to prepare substrate color developing solution, 100 μL of substrate color developing solution was added to each well, and after sealing with sealing film, color developing was performed at 25℃ for 10 min;

[0106] S8, 50 μL of termination solution was added to each well, and the value was read by an enzyme label instrument.

[0107] The parameters of the enzyme label instrument were as follows: the main wavelength of the enzyme label instrument was 450 nm, the reference wavelength was 630 nm, and the absorbance (OD value) of each well was determined.

[0108] The dilution step in step S1 was as follows:

[0109]

[0110]

[0111] The concentration of the standard sample diluted by the sample diluent in step S1 was 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.125 pg / mL, and NC (0 pg / mL).

[0112] The concentration ratio of the detection antibody to 20 wt% CS Buffer in step S3 was 1:2000.

[0113] The concentration ratio of the enzyme conjugate to 20 wt% CS Buffer in step S5 was 1:10000.

[0114] The weight ratio of substrate color developing solution A to substrate color developing solution B in step S7 was 1:1.

[0115] The third aspect of the present application provides that the HIV-1 p24 ELISA detection kit is applied to detect HIV-1 p24 protein in a sample.

[0116] Example 2

[0117] Example 2 provides an HIV-1 p24 ELISA detection kit, and the specific implementation manner is the same as that in Example 1, and the difference is that the coated enzyme label plate is coated by HIV-1 p24 specific monoclonal antibody.

[0118] The HIV-1 p24 specific monoclonal antibody is P-AB-1.

[0119] Example 3

[0120] Example 3 provides an HIV-1 p24 ELISA detection kit, the specific embodiment is same as Example 1, the difference is that the lysis solution is 0.1% SDS.

[0121] Example 4

[0122] Example 4 provides an HIV-1 p24 ELISA detection kit, the specific embodiment is same as Example 1, the difference is that the detection antibody and enzyme conjugate diluent is 3% BSA-PBS.

[0123] Example 5

[0124] Example 5 provides an HIV-1 p24 ELISA detection kit, the specific embodiment is same as Example 1, the difference is that the concentration ratio of coating buffer and antibody is 1:4000; the concentration ratio of detection antibody and 20vol% CS Buffer in step S3 is 1:2000; the concentration ratio of enzyme conjugate and 20vol% CS Buffer in step S5 is 1:4000.

[0125] Performance test method

[0126] 1. Coating enzyme plate precision test

[0127] The coating enzyme plate prepared in Example 1 is added with standard samples of the same concentration (150 pg / mL) to evaluate the precision of the coating plate, and the OD value of each well is tested, and the test results are shown in Table 1.

[0128] Table 1 OD value of 96 wells in coating enzyme plate

[0129]

[0130] The average OD value of 96 wells is calculated as 2.053, and the standard deviation is 0.0532. The CV (precision, %) is calculated by the formula CV (precision, %) = SD / average value*100%, and the CV is 2.59%. The qualified judgment standard is CV (%) ≤20%. The coating plate precision is qualified.

[0131] 2. OD value determination

[0132] The OD1 and OD2 of two parallel holes of the HIV-1 p24 ELISA detection kit prepared in Examples 1-5 are determined respectively, and the concentration of the diluted standard sample is 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.125 pg / mL, and NC. The test results are shown in Table 2.

[0133] Table 2 OD value

[0134]

[0135] The data were processed to calculate the sensitivity and precision. The sensitivity was judged by calculating the Cutoff value of sensitivity and the average value of 3.125 pg / mL OD. The detection results are shown in Tables 3 and 4. The Cutoff value = 0 pg / mL OD average value + 3*SD, and the Cutoff value is less than the average value of 3.125 pg / mL OD, indicating that the sensitivity (LOD) can reach below 3.125 pg / mL, otherwise the sensitivity is not enough.

[0136] Table 3 Precision analysis results

[0137]

[0138]

[0139] Table 4 Sensitivity detection

[0140] Example 0 pg / mL LOD mean 0 pg / mL LOD mean Cutoff 3.125 pg / mL LOD mean LOD 1 0.048 0.0014 0.0522 0.1165 ≤ 3.125 pg / mL 2 0.7985 0.0021 0.8049 0.797 > 3.125 pg / mL 3 0.646 0.0014 0.6502 0.621 > 3.125 pg / mL 4 0.2055 0.0148 0.2500 0.279 ≤ 3.125 pg / mL 5 0.3275 0.0233 0.3975 0.3355 > 3.125 pg / mL

[0141] 3. Accelerated stability experiment

[0142] The components of the kit in Example 1 were placed in a 37°C constant temperature box for 6 days, and compared with 5°C for testing. The results are shown in Table 5.

[0143] Table 5 Stability test results

[0144]

[0145]

[0146] The results show that the kit was subjected to an accelerated stability experiment, and the signal decreased by 5.18% after 6 days at 37°C, with a decrease of less than 20%, indicating that the stability of the kit is good.

[0147] 4. Methodology verification

[0148] The HIV-1 p24 ELISA detection kit prepared in Example 1 was subjected to 6 times of methodology verification to investigate its linearity, precision and accuracy. The concentration of the standard curve was 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.125 pg / mL, and NC. The concentration of the quality control was set to 150 pg / mL (QC1), 75 pg / mL (QC2) and 10 pg / mL (QC3). (The quality control is human serum containing HIV-1 p24 protein, and the manufacturer is not specifically limited) The verification results are shown in Table 6.

[0149] Table 6 Methodology verification experiment

[0150]

[0151]

[0152] The correlation parameters of the standard curve of 6 experiments are shown in Table 7, the sensitivity detection is shown in Table 8, and the R of the fitting curve of 6 times is greater than 0.990 2 All the sensitivities (LOD) of the kits can reach 3.125 pg / mL.

[0153] Table 7 Correlation parameters of the standard curve

[0154]

[0155]

[0156]

[0157] Note: The standard curve is obtained by four-parameter fitting of the theoretical concentration of the standard and the corresponding OD value.

[0158] Table 8 Sensitivity detection

[0159]

[0160] The quantitative range of the kit is determined by the recovery rate of the standard, and the results are shown in Table 9.

[0161] Recovery rate (%) = Recalculated concentration / Theoretical concentration * 100%. The recovery rates of the standards in the range of 6.25-200 pg / mL of 6 experiments are all between 80-120%, and the quantitative range of the kit is determined to be 6.25-200 pg / mL.

[0162] Table 9 Determination of the quantitative range

[0163]

[0164]

[0165] The intra-assay and inter-assay precisions of the standards (200, 100, 50, 25, 12.5, 6.25 pg / mL) and quality controls (150, 75, 10 pg / mL) of 6 experiments are calculated respectively, and the results are shown in Tables 10, 11 and 12. CV (%) = SD / Mean * 100%. The intra-assay and inter-assay experimental precisions of 6 experiments are all less than 10%, and all do not exceed 20%, which meet the requirements of method validation.

[0166] Table 10 Intra-assay precision of the standard

[0167]

[0168] Table 11 Intra-assay precision of quality control samples

[0169]

[0170] Table 12 Inter-assay precision of quality control samples

[0171]

[0172] The accuracy analysis results of 3 quality control samples in 6 different analysis batches are shown in Table 13. The average accuracy of the 6 batches of quality control samples is between 86.04% and 107.64%, which meets the requirements of the bioanalysis method validation guidelines, and the accuracy verification is qualified.

[0173] Table 13 Accuracy analysis results

[0174]

[0175] Note: The qualification criteria is: accuracy (%) = average of measured value / theoretical value * 100%; the accuracy (%) is between 80-120%;

[0176] The quantitative range of the kit is 6.25-200 pg / mL, and the LOD is ≤3.125 pg / mL; after 6 verifications, the intra-assay, inter-assay and whole plate CV (%) of the kit are all less than 10%, and the precision is qualified; the accuracy of 6 batches of 3 quality control samples is between 86.04% and 107.64%, which is within the range of 80-120%, and the accuracy is qualified. The detection method of the kit has high sensitivity, good precision and accuracy, and can be used for detecting the content of p24 protein in virus samples.

Claims

1. An HIV-1 p24 ELISA detection kit, characterized in that, include: The following components are included: coated microplate, detection antibody, enzyme conjugate, standard, lysis buffer, detection antibody and enzyme conjugate diluent, sample diluent, 20×PBST washing buffer, substrate chromogenic solution A, substrate chromogenic solution B, and stop solution. The coated ELISA plate was coated with an HIV-1p24-specific monoclonal antibody. The detection antibody is a biotin-labeled p24 antibody; The enzyme conjugate is horseradish peroxidase HRP-labeled streptavidin; The standard is human serum containing HIV-1p24 antigen; The antibody and enzyme conjugate dilution solution was 20 vol% CS Buffer. The HIV-1p24-specific monoclonal antibody is composed of two antibodies, P-AB-1 and P-AB-2, with a concentration ratio of (1-2):(1-2). The supplier of P-AB-1 and P-AB-2 is Abcam, and the corresponding part numbers are ab32352 and ab63958, respectively. The preparation method of the coated ELISA plate is as follows: HIV-1p24 specific monoclonal antibody is diluted with coating buffer to obtain a mixed coating. 100 μL of the mixed coating is added to each well and incubated at 4°C overnight. The liquid in each well is discarded, and the microwells are washed three times with washing buffer. 200 μL of blocking buffer is added to each well and incubated at 37°C for 1.5 h. The liquid in each well is discarded, and the microwells are washed three times with washing buffer and then patted dry on a tissue. 200 μL of preservative is added to each well, and the reaction is carried out at 37°C for 1 h. The liquid in each well is then discarded, and the microwells are patted dry on a tissue and allowed to air dry. The sealing solution is 0.5 wt% Casein-Na; The lysis buffer was 0.1 vol% Triton; The method of using the HIV-1 p24 ELISA detection kit is characterized by the following steps: S1. Add 10 μL of lysis buffer to each well of the coated microplate, then add 90 μL of standard diluted with sample diluent to the corresponding well. Seal the plate with sealing film and place it in a 37°C constant temperature shaking incubator at a shaking speed of 200-300 rpm for 1 hour. S2. Pour out the liquid in each hole, clean the micropores 3 times with cleaning solution, and then pat dry on a tissue. S3. Add 100 μL of detection antibody diluted with 20 vol% CS Buffer to each well, seal the plate with sealing film, and place it in a 37°C constant temperature shaking incubator at a shaking speed of 200-300 rpm for 1 h. S4. Pour out the liquid in each hole, clean the micropores 3 times with cleaning solution, and then pat dry on a tissue. S5. Add 100 μL of enzyme conjugate diluted with 20 vol% CS Buffer to each well, seal the plate with sealing film, and place it in a 37°C constant temperature shaking incubator at a shaking speed of 200-300 rpm for 1 h. S6. Pour out the liquid in each hole, clean the micropores 3 times with cleaning solution, and then pat dry on a tissue. S7. Mix substrate color development solution A and substrate color development solution B to prepare substrate color development solution. Add 100 μL of substrate color development solution to each well, seal the plate with sealing film, and develop the color at 25℃ for 10 min. S8. Add 50 μL of stop solution to each well and read the value using a microplate reader; The concentration ratio of the coating buffer to the antibody is 1:2000; the concentration ratio of the detection antibody to 20 vol% CS Buffer in step S3 is 1:2000; and the concentration ratio of the enzyme conjugate to 20 vol% CS Buffer in step S5 is 1:10000.

2. The HIV-1 p24 ELISA detection kit according to claim 1, characterized in that, The cleaning solution is prepared by diluting an appropriate amount of 20×PBST washing solution with deionized water at a ratio of 1:

20.

3. The HIV-1 p24 ELISA detection kit according to claim 2, characterized in that, The concentration of the diluted standard in step S1 is 6.25-200 pg / mL.

4. A non-diagnostic application of the HIV-1 p24 ELISA test kit according to claim 1 or 2.

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