Process for preparing cross-linking antibody by solid-phase immunoadsorption method
A technology of immunoadsorption and immunoadsorbent, which is applied in the field of preparation of cross-linked antibodies by solid-phase immunoadsorption method, which can solve the problems of shielding or steric hindrance, affecting the antigen-binding activity of labeled antibodies, hindering the binding of antibodies and antigens, and achieving activity small loss effect
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Embodiment 1
[0040] Taking the fluorescent probe R-phycoerythrin (macromolecular protein, molecular weight 240KD) as an example, the cross-linked antibody of R-phycoerythrin and hepatitis B virus surface antigen (HBsAg) monoclonal antibody was prepared. 1. Preparation of Solid-Phase Immunosorbent
[0041] The surface antigen of hepatitis B virus (HBsAg) is immobilized on the solid-phase carrier to make a stable immunoadsorbent-solid-phase antigen. Cyanogen bromide-activated agarose gel 4B is used as the solid phase carrier, which is a commonly used solid phase filler for affinity purification of monoclonal antibodies. When used for the preparation of solid phase antigens, a standardized operating procedure has been established. The preparation method that adopts is as follows:
[0042] ① Weigh 0.3g of freeze-dried powder, swell at 10 -3 N hydrochloric acid 30min, and with 60mL
[0043] Wash with hydrochloric acid solution, then quickly with 0.1mol / L, pH8.3 NaHCO 3 Solution wash once
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Embodiment 2
[0081] Taking the solid-phase preparation of the cross-linked antibody of R-phycoerythrin and prostate-specific antigen monoclonal antibody as an example, the operation process of the invention is described as follows again: 1. Preparation of solid-phase immunosorbent
[0082] Using cyanogen bromide-activated Sepharose 4B as a solid-phase carrier, the prostate-specific antigen (PSA) was immobilized on the solid-phase carrier to make a stable solid-phase immunosorbent. The preparation method is as follows:
[0083] ① Weigh 0.3g of freeze-dried powder, swell at 10 -3 N hydrochloric acid 30min, and with 60mL
[0084] Wash with hydrochloric acid solution, then quickly wash with 0.1mol / L, pH 8.3 NaHCO 3 solution wash one
[0085] times, to obtain 1mL activated gel;
[0086] ② Immediately add the antigen to be coupled PSA (2mg), the antigen in advance with 0.1mol / L, pH8.3
[0087]NaHCO 3 The solution was balanced, and stirred slowly at room temperature for 8 hours; ③Centri...
Embodiment 3
[0112] Using polystyrene microbeads (60-250 μm in diameter) as a solid-phase carrier, the cross-linked antibody of R-phycoerythrin and HBsAg monoclonal antibody was prepared in solid phase: 1. Preparation of solid-phase immunosorbent
[0113] ①Weigh 0.5g of polystyrene beads, at room temperature, in fresh glutaraldehyde solution (volume
[0114] Percentage is 25%) after soaking in 4h, quickly with 0.1mol / L, pH 8.3 NaHCO 3
[0115] The solution was washed once;
[0116] ② Immediately add the antigen to be coupled HBsAg (5mg), the antigen in advance with 0.1mol / L, pH
[0117] 8.3 NaHCO 3 The solution was balanced and stirred slowly for 10 hours at 1°C;
[0118] ③ Centrifuge, absorb the supernatant, add 2 mL of 0.1 mol / L glycine solution to the microbeads,
[0119] Shake the reaction for 1h to block the redundant active groups;
[0120] ④ with 50 mM Tris, 1 M NaCl, pH 8.0 and 50 mM Glycine, 1 M NaCl, pH
[0121] 3.5 Alternately wash the gel 8 times to remove non-...
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