A kit for detecting circulating antigens of Agkistrodon acutina using specific polyclonal antibodies
A technology of Agkistrodon acutus and Ligulata, applied in the field of biological detection, to achieve rapid diagnosis, considerable economic and social benefits, and high practical value
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Embodiment 1
[0022] Example 1 A kit for detecting circulating antigens of Agkistrodon acutina using specific polyantibody
[0023] The invention provides a kit for detecting circulating antigens of Agkistrodon acutinosa larvae using specific polyantibodies, including rabbit polyclonal antibody (rabbit anti-Aag-IgG), horseradish Oxidase-labeled polyclonal antibody against Agkistrodon acutinae (HRP-Aag-IgG), nitrocellulose membrane, 1% bovine serum albumin (BSA) in PBST solution, PBST washing solution, substrate color development solution, positive and negative control samples, and 96-well polyethylene reaction plates.
[0024] Further, the 1% bovine serum albumin in PBST solution is composed of bovine serum albumin and PBST washing solution, and the weight ratio of the bovine serum albumin and PBST washing solution is 1:99.
[0025] Further, the PBST washing solution is composed of sodium chloride, KH 2 PO 4 、Na 2 HPO4 . 2 PO 4 、Na 2 HPO 4 , KCl and water, the weight percentage of t...
Embodiment 2
[0030] The present invention also provides a preparation method of a kit for detecting circulating antigens of Agkistrodon akistrodon larvae by using specific polyclonal antibodies, including a step of preparing a soluble antigen against Agkistrodon larvae, and a step of preparing a rabbit antibody -Aag-IgG step, an enzyme-labeled antibody (HRP-Aag-IgG) step, also includes a nitrocellulose membrane, 1% bovine serum albumin (BSA) in PBST solution, PBST wash in the kit Solution, substrate chromogenic solution, positive control sample, negative control sample, and polyethylene reaction plate.
[0031] Further, in a step of preparing the soluble antigen of Agkistrodon larvae, the larvae are isolated from the abdomen of mice infected with Agkistrodon larvae eggs for 3-5 months, and the larval soluble antigen is prepared.
[0032] Further, in a step of preparing rabbit anti-Aag-IgG, mix the soluble antigen (AagA) of the larvae of Agkistrodon akistrodon and the complete Freund's adju...
Embodiment 3
[0053] A kind of operation step of the kit that adopts specific polyclonal antibody to detect the circulating antigen of Agkistrodon acutina of the present invention is as follows (principle is as follows: figure 1 shown):
[0054] a) Dilute the rabbit anti-Aag-IgG to 200 μg / ml, pipette 1 μl of antibody onto the nitrocellulose membrane, and coat at room temperature for 10 minutes.
[0055] b) Blocking: immerse the coated membrane in a PBST solution of 1% bovine serum albumin (BSA). 37°C, 15 minutes.
[0056] c) Washing: Wash the sealed membrane 3 times with washing solution, and dry it for 3 minutes each time, which is the "quick diagnosis membrane".
[0057] d) Antigen to be tested: Cut out the "quick diagnosis membrane" according to the grid, put the glossy side up, put it into the well of a 96-well polyethylene reaction plate, add 100 μl of the serum to be tested in each well, and set a blank (PBS), negative compared with positive samples. 37°C for 1h.
[0058] e) Washin...
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