Porcine circovirus type 4 ELISA antibody detection kit, application and method for detecting porcine circovirus type 4 antibody
A porcine circovirus and antibody detection technology, applied in the biological field, can solve problems such as threats to the pig industry, and achieve strong specificity and sensitivity, accurate detection and/or identification
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Embodiment 1
[0086] Preparation and purification of porcine circovirus type 4 Cap protein of embodiment 1
[0087] The present embodiment provides the preparation process of porcine circovirus type 4 Cap protein, comprising the following steps:
[0088] A1. After codon optimization, the nucleotide sequence of the porcine circovirus type 4 Cap gene obtained is shown in SEQ ID NO.2, and is connected on the expression vector pET-28a, obtains the recombinant vector and double enzyme digestion identification ( figure 1 );
[0089] A2. Transform the recombinant vector into the recipient bacterium BL21(DE3) to obtain the recombinant bacterium;
[0090] A3. Pick a positive single colony and culture it in the liquid medium until the OD600 is about 0.8, add IPTG to make the final concentration 0.5mM, induce at 20°C for 12-16 hours, and centrifuge to collect the precipitate;
[0091] A4. Add 1×PBS containing protease inhibitors to resuspend the pellet, sonicate, and centrifuge to get the supernatan...
Embodiment 2
[0097] Example 2 ELISA detection of porcine circovirus type 4 antibody
[0098] The present embodiment provides a kind of ELISA detection process of porcine circovirus type 4 antibody, comprising the following steps:
[0099] B1. Antigen coating: Dilute the purified recombinant protein with the coating solution to a final concentration of 0.625 μg / mL, add it to the microtiter plate, 100 μL / well, and coat overnight at 4°C;
[0100] B2. Washing: Wash the microtiter plate 3 times with PBST, let stand for 5 minutes each time, and pat dry with absorbent paper for the last time;
[0101] B3. Blocking: add blocking solution containing 0.25% gelatin PBST, 150 μL / well, block at 37°C for 1 hour;
[0102] B4. Washing: Wash the microtiter plate 3 times with PBST, pat dry on absorbent paper, and let stand for 5 minutes each time;
[0103] B5. Primary antibody incubation: Serum samples were diluted 200 times and added to the microtiter plate, and negative and positive controls were added ...
experiment example
[0112] In order to verify the beneficial effect of the different ELISA conditions that the present invention selects, carry out following experiment:
[0113] 1. Determination of the optimal dilution concentration of antigen and primary antibody
[0114] The purified recombinant protein was diluted with coating solution, coated with 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 100 μL / well, overnight at 4 °C; washed 3 times with PBST, each Add blocking solution for 1 hour at 37°C; after washing, dilute negative and positive sera at 1:100, 1:200, 1:400, and 1:800, add 100 μL / well, and incubate at 37°C for 90 minutes; wash Afterwards, add 1:10000 diluted HRP-rabbit anti-pig IgG, 100 μL / well, incubate at 37°C for 45 minutes; wash, pat dry, add 100 μL TMB color developing solution to each well, and protect from light at 37°C for 6 minutes; add 2M sulfuric acid, 50 μL / well, terminate the reaction, and read with a microplate reader. According to Table 1, when the coating concentra...
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