Affinity chromatography-enzyme linked immunity test method for ractopamine and dedicated kit
A ractopamine and enzyme-linked immunosorbent assay technology, which is applied in the field of immunoassays, can solve the problems of inability to adapt to fast, simple, inexpensive, difficult mass detection, and time-consuming instrumental analysis techniques.
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Embodiment 1
[0086] 1. Sample pretreatment:
[0087] Weigh 10g of pork products (including liver, lung, heart, kidney, muscle), add 50ml 0.1mol / L HCl for tissue homogenization, freeze and thaw twice, centrifuge to get the supernatant, adjust the pH value to 11 with 10mol / L NaOH, Then add 30ml of isobutanol, shake for 30min, let it stand for 4h, quantitatively absorb isobutanol, evaporate to dryness in a water bath, then add 1ml of phosphate solution (PBS, 0.01mol / L, pH7.4) to dissolve the eluate, and freeze it for testing ;
[0088] Pig urine samples were centrifuged to remove impurities, and the supernatant was taken for testing.
[0089] 2. Preparation of enzyme-labeled antigen: mix activated horseradish peroxidase with purified RCT, perform labeling reaction in carbonic acid buffer system, and then add sodium borohydride to reduce unbound sites;
[0090] 3. Preparation of RCT antibody
[0091] Coupling RCT with bovine serum albumin BSA to prepare antigen; immunization dose 1mg / ml BSA...
Embodiment 2
[0107] The composition of the RCT detection kit:
[0108] The kit includes: ELISA plate coated with monoclonal RCT antibody; A reagent: standard RCT reagent; B reagent: diluent; C reagent: enzyme-labeled antigen reagent; D reagent: enzyme-labeled antigen diluent; E reagent: containing Tween -20 phosphate PBS solid; F reagent: o-phenylenediamine reagent; G reagent: sodium acetate-citric acid buffer; H reagent: 30% H 2 o 2 ); I reagent: sulfuric acid solution; J reagent: glycine solution; K reagent: sepharose-4B gel coupled with monoclonal RCT antibody.
Embodiment 3
[0110] The composition of the RCT detection kit:
[0111] The kit includes: ELISA plate coated with polyclonal RCT antibody; A reagent: standard RCT reagent; B reagent: diluent; C reagent: enzyme-labeled antigen reagent; D reagent: enzyme-labeled antigen diluent; Phosphate PBS solid at temperature -20; F reagent: o-phenylenediamine reagent; G reagent: sodium acetate-citric acid buffer; H reagent: 30% H 2 o 2 ); Reagent I: sulfuric acid solution; Reagent J: glycine solution; Reagent K: sepharose-4B gel coupled with polyclonal RCT antibody.
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