METHOD FOR PRODUCING AND OBTAINING VARIABLE DOMAINS OF ANTI-DIGOXIN MONOCLONAL ANTIBODY FAB FRAGMENT USING THE MOLECULAR BIOLOGY CLONING TECHNIQUE

摘要

Method for producing and obtaining variable domains of anti-digoxin monoclonal antibody Fab fragment using the molecular biology cloning technique, and more precisely the present patent of invention relates to a method for producing and obtaining clones of the anti-digoxin antibody Fab fragment using phage display technology and the characterization of the binding thereof to the antigen; the present innovation pertains to the development of the product for therapeutic use having specific potency and a more precise dose for detoxification of patients undergoing treatment with digoxin.

主权项

1. Method for producing and obtaining variable domains of anti-digoxin antibody Fab fragments using the molecular biology cloning technique, characterized by the fact that the method for producing and obtaining clones of anti-digoxin antibody Fab fragment using cloning technology in molecular biology by phage display and the characterization of its antigen binding, comprises the following steps:
Step 1)—Obtaining and characterizing bovine albumin conjugated with digoxin (Dig-BSA)
Phase 1.1—Obtaining Dig-BSA conjugates;Phase 1.2—Characterizing Dig-BSA conjugates; Step 2)—Preparing the anti-digoxin monoclonal antibody;
Phase 2.1—Purifying the monoclonal anti-digoxin antibody; Step 3)—Building the Fab library in pComb3X phagemid from anti-digoxin hybridomas
Phase 3.1—Obtaining cDNA from anti-digoxin hybridomas;Phase 3.2—Amplifying genes of LC and of Fd portion of HC;Phase 3.3—Obtaining competent cells and vector;Phase 3.4—Cloning the LC repertoire of genes in vector pComb3X;Phase 3.5—Cloning the HC repertoire of genes in the LC library; Step 4)—Phage display and enrichment of Fab library;
Phase 4.1—Preparing helper phage;Phase 4.2—Generating anti-digoxin Fab phage library (phage display); Step 5)—Expressing soluble Fab fragments;
Phase 5.1—Quantifying soluble Fab fragments by sandwich enzyme-linked immunosorbent assay (ELISA); Step 6)—Characterizing crude extracts containing Fab fragments of clones obtained by phage display
Phase 6.1—Soluble Fab fragment antigen binding assay by ELISA test;Phase 6.2—Polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE);Phase 6.3—Western blotting assay;Phase 6.4—Affinity analysis using BIAcore T100.