Hepatitis E virus IgG antibody detection kit and preparation method and application thereof
An antibody detection and kit technology, applied in the field of hepatitis E virus IgG antibody kits, can solve the problems of high environmental cleanliness requirements and high dust, and achieve the effects of controlling the cost of reagents, improving sensitivity and improving specificity
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Embodiment 1
[0072] The preparation of embodiment 1 HEV-gene recombinant antigen coated plate
[0073] Source of HEV-gene recombinant antigen: purchased from Shanghai Biji Biotechnology Co., Ltd.
[0074] Proceed as follows:
[0075] (1) Print the barcode on the coated microporous reaction plate frame, and draw a red marking line on the raised part on the right side of the plate;
[0076] (2) After dissolving the HEV gene recombinant antigen in the coating buffer, soak the solid phase material and let it stand for 20 to 30 hours; wherein, the coating buffer is 0.05-0.1MpH8.0 (±0.2) PBS, and the coating The volume is 100 μL / well buffer solution, wherein, the buffer solution contains: 0.005-0.05% SDS; the coating working solution is added HEV gene recombinant antigen (150-400ng / mL) and 0.05%-0.5% (volume ratio) Antigen protection agent (β-mercaptoethanol) coating buffer; then add 100 μl / well into the microwell, let it stand at room temperature for 20-30 hours, and pay attention to prevent ...
Embodiment 2
[0081] The preparation of embodiment 2 europium markers
[0082] Source of mouse anti-human IgG monoclonal antibody: purchased from Beijing Xieshun Biotechnology Development Center
[0083] (1) Adjust the mouse anti-human IgG monoclonal antibody to 2.5mg / mL with ultrapure water, and dialyze overnight with a certain volume (1-5L) of dialysis solution 1 at a temperature of 20-25°C; It is 0.1mol / L, pH8.1±0.2 sodium bicarbonate buffer.
[0084] The dialysis time is 24h±2h, and the liquid is changed twice with an interval of more than 3 hours; after that, the dialysis solution 2 is used in the temperature range of 20-25°C; the dialysis buffer 2 is 0.1mol / L, pH 9.4± 0.2 CBS buffer. Dialyze for 24h±2h, change the medium twice, the time interval is more than 3 hours, and change the medium after get off work, and then dialyze overnight.
[0085] (2) At room temperature, according to the standard europium ratio of 1:3 (europium: protein), Eu-DTTA was completely dissolved in purified ...
Embodiment 3
[0087] Embodiment 3 detection application of hepatitis E IgG antibody
[0088] (1) Preparation of reagents
[0089]Mix 40mL concentrated lotion [Tris-HCl-NaCl (pH8.3±0.2) solution containing 0.2-1.0% Tween 20, diluted 40 times before use] and 960mL deionized water or distilled water in a clean lotion bottle , as a working cleaning solution.
[0090] Balance the negative and positive controls, sample diluent, analysis buffer, required number of microwell reaction strips and samples to be tested in the kit to 18-28°C), shake and mix the liquid reagents.
[0091] Prepare europium marker working solution: For each 12-well plate, take 75 μL europium marker (20×), add 1.5 mL analysis buffer [pH7.8±0.2 casein buffer, containing 0.878% NaCl, 0.5% Casein sodium salt, 0.606% Tris, 0.5% hydrochloric acid, 0.00074% EDTA-Na 2 , 0.2% TritonX-100, 3% goat serum, 0.005% cockscomb and 0.05% NaN 3 】, mix thoroughly (1:20 dilution). Prepare within 30 minutes before the second shaking reacti...
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