Novel ELISA (enzyme-linked immunosorbent assay) quantitative detection kit for IgG (immunoglobulin G) antibody of coronavirus
A coronavirus, quantitative detection technology, applied in the biological field, can solve the problems of low sensitivity and specificity
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Embodiment 1
[0053] The preparation method of an enzyme-labeled reaction plate coated with a complex antigen of the novel coronavirus N protein, S1 protein and M protein, comprising the following steps:
[0054] (1) Mix the N protein, S1 protein and M protein of the novel coronavirus according to a mass ratio of 2:2:1 to obtain a complex antigen;
[0055] (2) The complex antigen was diluted to 100 μg / ml with the coating solution, and then coated with 100 μl per well in the microwells of the ELISA plate, then incubated at 2-8°C overnight, and then 300 μl of washing solution was added to each well for washing , repeated cleaning 2 times;
[0056] (3) Add 100 μl of coating stabilization solution to the microwell, incubate at 18-25°C for 2-4 hours, and then drain;
[0057] (4) Adding 200 μl of blocking solution to the microwell, and blocking at 37° C. for 2 hours, an enzyme-labeled reaction plate coated with the complex antigen of the novel coronavirus N protein, S1 protein and M protein can ...
Embodiment 2
[0059] Coating amount and compounding combination optimization of coating antigen
[0060] (1) Take a 96-well ELISA plate; compound the N, S1 and M proteins of the novel coronavirus according to different mass ratios, and dilute the compounded antigens with coating solution to 100 μg / ml and 10 μg / ml, respectively , 100 μl per well; dry overnight in a 37°C incubator; take the overnight-dried antigen-coated plate and add 200 μl of blocking solution to block at 37°C for 1.5 hours to obtain a gradient dilution antigen-coated enzyme-labeled reaction plate;
[0061] (2) Dilute the negative standard and positive standard with a diluent of 1g:100mL by mass and volume, add 100μl to each well, incubate at 37°C for 30 minutes, and dilute the diluent ten times with distilled water, that is, add 900ml to 100ml of washing solution Distilled water, and then rinse each well 3 times with 300 μl of diluted washing solution;
[0062] (3) Dilute the enzyme-labeled secondary antibody by 100 times...
Embodiment 3
[0070] Selection of optimal titer of enzyme-labeled secondary antibody
[0071] a) Take a 96-well ELISA plate, coat it with 100ng / ml human IgG 100μl / well at 4°C overnight, wash the plate 3 times with washing solution, 350μl / well, spin dry;
[0072] b) The HRP-labeled mouse anti-human IgG monoclonal antibody was diluted in a series of dilutions, and then added to the coated wells, 100 μl / well, incubated at 37°C for 40 min, and then washed with 3 times, 350 μl / well, and dried. ;
[0073] c) Add substrate to develop color, add 100 μl of substrate solution to each well, and develop color at 37°C or room temperature for 15 minutes in the dark; then add 50 μl of stop solution to stop the reaction, and read the absorbance at 450nm wavelength on the microplate reader, that is, the A value , take the dilution of the enzyme-labeled antibody when the A value is 1.0, as the optimum titer of the enzyme-labeled secondary antibody, and the optimum titer is 1:5000.
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