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Immuno-informaticsTypes of AntibodyAntibody : ResourcesTechniques and ProtocolsImportant Links |
Antibody Sequencing
To know the antigen binding residues of antibody and the nature of anitgen binding residues in antibody, one have to sequence antibody. The protein sequencing methods (like Edman degradation, MS, HPLC/MS) can be applied to antibody sequencing.Although this methods have been adopoted by some companies (Proteome factory). But the cost of this methods is very high and the research is going on in this area to fish out some cost effective methods. At the same time DNA sequencing is a cheaper technology comparitively, that also can be applied to antibody sequencing. Antibody sequencing technologyStage 1: mRNA extraction from the hybridoma cell pelletmRNA will be extracted and purified from the hybridoma cell pellet using standard protocols. Stage 2: Reverse transcription mRNA will be transcribed into cDNA using either an oligo dT anti-sense primer or a gene-specific (murine or human IgG1 CH and kappa CL) anti-sense primer. The initial cDNA production will most probably be with the oligo dT primer. Specific murine and human constant domain primers could be used to PCR after cDNA production to determine the isotype of the antibody. Stage 3: PCR or RACE amplification of heavy and light chains Degenerate VH and VL primers are used to amplify the variable domains from the cDNA. For RACE, a homopolymeric dCTP tail is added to the end of the cDNA. The heavy and light chains can now be amplified with an oligo dG sense primer and a gene specific (CH/CL) anti-sense primer. PCR products will include the sequence of the signal peptide, variable domains and constant domains up to the anti-sense primer. Stage 4: Cloning into a standard sequencing vector The PCR products will be gel purified to remove small fragments, and cloned into a blunt or TA vector for sequencing. Stage 5: Sequencing analysis The specified number of clones to be sequenced can be determined by the client. As standard, a minimum of 5 independent clones for each chain have to be sequenced. Stage 6: Final Report A report is put together to detail the work performed which encompasses all the blots and sequence alignments of the heavy and light chains and is e-mailed to the client. |