ELISA detection kit for detecting forest encephalitis virus envelope E protein

Abstract

The invention discloses an ELISA detection kit for detecting forest encephalitis virus envelope E protein, and relates to the technical field of kits. The kit comprises a pre-coated elisa plate, an elisa antibody, a TBEV E protein quantitative standard substance, a sample diluent, a 20-time concentrated washing solution, a chromogenic substrate A, a chromogenic substrate B, a stop buffer, a plate sealing film and a drying agent. According to the present invention, the totivirus specific correlation can be identified, and the method can be used for the preparation of the kit quality control product so as to achieve the quantitative detection, and is suitable for the E protein preparation and the identification of the specific virus protein monoclonal antibody.

Description

Technical Field

[0001] This invention relates to the field of reagent kit technology, and in particular to an ELISA detection kit for detecting the envelope E protein of tick-borne encephalitis virus. Background Technology

[0002] Tick-borne encephalitis virus (TBEV), also known as tick-borne encephalitis virus, has its envelope E protein, which is a major structural and protective antigenic protein. ELISA kits are standardized detection tools based on enzyme-linked immunosorbent assay (ELISA) technology. Through the specific binding reaction of antigens and antibodies, combined with the colorimetric signal catalyzed by the enzyme, they enable the qualitative or quantitative detection of target substances (antigens, antibodies, proteins, hormones, pathogens, etc.) in biological samples (serum, plasma, urine, cell culture medium, etc.). They are commonly used immunoassay tools in biomedicine, clinical diagnostics, scientific research, and industrial testing.

[0003] Existing kits have the following drawbacks: the whole virus has a complex structure containing multiple structural proteins, resulting in poor homogeneity, and existing kits cannot perform quantitative detection of the virus; existing methods do not provide clear identification of the antibodies in the kits, and cannot ensure applicability for E protein detection; existing kits are qualitative reagents and do not contain quantitative control materials for E antigen, thus they cannot perform quantitative detection. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for quantitative detection.

[0005] The technical solution adopted to solve the above-mentioned technical problems is: an ELISA detection kit for detecting the envelope E protein of tick-borne encephalitis virus, including a pre-coated enzyme-labeled plate, enzyme-labeled antibody, quantitative standard of TBEV E protein, sample dilution buffer, 20-fold concentrated washing buffer, chromogenic substrate A, chromogenic substrate B, stop solution, sealing film, and desiccant.

[0006] Furthermore, the pre-coated ELISA plate is coated with 2A2.

[0007] Furthermore, the enzyme-labeled antibody is 3A9-HRP.

[0008] Furthermore, the chromogenic substrate A is hydrogen peroxide.

[0009] Furthermore, the chromogenic substrate B is tetramethylbenzidine.

[0010] Furthermore, the termination solution is 2 mol / L sulfuric acid.

[0011] The beneficial effects of the present invention are as follows: The detection kit of the present invention includes a pre-coated ELISA plate, an ELISA-labeled antibody, a TBEV E protein quantitative standard, a sample diluent, a 20-fold concentrated washing buffer, a chromogenic substrate A, a chromogenic substrate B, a stop solution, a sealing film, and a desiccant. The E protein is prepared by recombinant synthesis, which can identify specific correlations with the whole virus and can be applied to the preparation of kit quality control materials to achieve quantitative detection. The invention is applicable to the preparation of E protein and the identification of specific viral protein monoclonal antibodies. Attached Figure Description

[0012] Figure 1 This is a flowchart of the detection process for the ELISA detection kit for detecting the E protein of the tick-borne encephalitis virus envelope, as described in this invention.

[0013] Figure 2 This is a graph showing the linear correlation results of the detection kit for the detection of the envelope E protein of recombinant tick-borne encephalitis virus.

[0014] Figure 3 This is a graph showing the detection specificity results of the test kit. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] The ELISA kit for detecting the envelope E protein of tick-borne encephalitis virus (TBEV) in this embodiment includes a pre-coated ELISA plate, an enzyme-labeled antibody, quantitative standards for TBEV E protein, sample diluent, 20-fold concentrated wash buffer, chromogenic substrate A, chromogenic substrate B, stop solution, sealing film, and desiccant. Specifically, the pre-coated ELISA plate is coated with 2A2, the enzyme-labeled antibody is 3A9-HRP, chromogenic substrate A is hydrogen peroxide, chromogenic substrate B is tetramethylbenzidine (TMB), and the stop solution is 2 mol / L sulfuric acid.

[0017] like Figure 1 As shown, the detection procedure for the ELISA kit for detecting the E protein of the tick-borne encephalitis virus envelope is as follows: S1. Add the test sample at an appropriate dilution to the wells of the pre-coated 2A2 antibody ELISA plate, 100 μl / well, incubate at 37°C for 60 min, discard the supernatant, wash 3 times with PBST, and pat dry. S2, add 100 μl of HRP-labeled 3A9 antibody to the washed specimen wells, incubate at 37°C for 30 min, discard the supernatant, wash 5 times with PBST, and pat dry; S3. After washing the specimen wells, add 50 μl of chromogenic substrate A (hydrogen peroxide) and 50 μl of chromogenic substrate B (TMB) to each well. Incubate at 37°C for 15 min. Then add 50 μl of stop solution (2 mol / L sulfuric acid) to each well to terminate the reaction. S4. Read the OD450nm / 630nm values ​​in the microplate reader, plot the standard curve based on the E protein standard, and calculate the E protein content in the sample to be tested.

[0018] The method for preparing the E protein of the tick-borne encephalitis virus envelope in this embodiment is as follows: The envelope E protein gene was amplified using RT-PCR with inactivated tick-borne encephalitis virus (Senzhang strain) as a template. The amplified gene was cloned into the pET28a plasmid vector and transformed into the expression host bacterium E. coli BL21(DE3). Positive clones were selected and amplified in culture. Expression was induced at 37°C with IPTG at a final concentration of 0.1 mmol / L. After induction, the bacterial cells were harvested by centrifugation, resuspended in 20 mmol / L Tris-HCl buffer, sonicated, and centrifuged again. The precipitate was dissolved thoroughly in 8 mol / L urea and purified by nickel affinity chromatography. The 100 mmol / L imidazole eluate was collected and identified as the target protein by SDS-PAGE. The evaluation of the performance of the dosage box in this embodiment is as follows: (1) Precision: The content of E protein in three samples of different concentrations was detected by this method. Ten replicates were performed for each concentration, and the test was repeated three times. The mean value (Mean), standard deviation (SD), and coefficient of variation (CV) of the antigen concentration within and between the tests were calculated to verify the precision of the method.

[0019] (2) Validation of the correlation and specificity of monoclonal antibodies: 2A2 and 3A9 monoclonal antibodies were used as primary antibodies, and goat anti-mouse-HRP was used as secondary antibodies. Western blotting was used to detect whole tick-borne encephalitis virus, recombinant tick-borne encephalitis virus E protein, human serum albumin, bovine serum albumin, serum-free culture supernatant of primary hamster kidney cells, and Japanese encephalitis virus.

[0020] Linear correlation like Figure 2As shown, reference standards for the E protein kit were prepared at seven concentrations (0-100 ng / ml) (E protein concentrations of 0, 10, 20, 40, 80, and 100 ng / ml). Using this kit for detection, a linear correlation was established between OD450nm / 630nm and E protein concentration, yielding the regression equation: y = 0.0249x + 0.0233. OD450nm / 630nm was highly correlated with E protein concentration, with a correlation coefficient R² = 0.9955. Based on this standard curve, the E protein content of the sample can be calculated by substituting the OD450nm / 630nm values ​​into the equation. The linear detection range of this kit is 0-100 ng / ml.

[0021] (4) Specificity like Figure 3 As shown, using the detection method of this kit, the raw and auxiliary materials used in the production of inactivated tick-borne encephalitis vaccine, including tick-borne encephalitis virus stock solution, live attenuated Japanese encephalitis virus stock solution, recombinant tick-borne encephalitis virus E protein, bovine serum albumin, human serum albumin, and primary hamster kidney cell culture supernatant, as well as Japanese encephalitis virus belonging to the same Flaviviridae genus, were tested. The test results showed that the kit specifically reacted with whole tick-borne encephalitis virus and recombinant E protein, but did not react with the other components mentioned above, proving that it has good detection specificity.

[0022] (5) Limit of detection: E protein was quantitatively detected by serial dilution. The cut-off value was 2.1 times the value of the negative control well A450 / 630nm. If the value was greater than the cut-off value, it was judged as positive. The limit of detection was determined.

[0023] (6) Stability: The content of protein E was repeatedly detected within the set validity period and under accelerated conditions, and the coefficient of variation (CV) between experiments was calculated to verify the stability of the method.

[0024] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. An ELISA detection kit for detecting the envelope E protein of a forest brain virus, characterized by: The kit comprises a pre-coated enzyme-labeled plate, enzyme-labeled antibody, TBEV E protein quantitative standard, sample diluent, 20-fold concentrated washing solution, color developing substrate A, color developing substrate B, termination solution, plate sealing film and desiccant.

2. The ELISA detection kit for detecting the envelope E protein of the forest encephalitis virus according to claim 1, characterized by: The pre-coated enzyme-labeled plate is 2A2 coated.

3. The ELISA detection kit for detecting the envelope E protein of the forest encephalitis virus according to claim 1, characterized by: The enzyme-labeled antibody is 3A9-HRP.

4. The ELISA detection kit for detecting the envelope E protein of the forest encephalitis virus according to claim 1, characterized by: The color developing substrate A is hydrogen peroxide.

5. The ELISA detection kit for detecting the envelope E protein of the forest encephalitis virus according to claim 1, characterized by: The color developing substrate B is tetramethyl benzidine.

6. The ELISA detection kit for detecting the envelope E protein of the forest brain fever virus according to claim 1, characterized by: The termination solution is 2 mol / L sulfuric acid.

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