Process Of Monoclonal Antibody Sequencing
The B lymphocytes of the immune system produce special proteins
, called as antibodies, in response to foreign proteins, which are called antigens. Antibodies function as markers, binding to the antigen so that the antigen molecules can be recognized and destroyed by phagocytes. Antibody binds to a part of the antigen called the epitope. Therefore, the epitope is short amino acid sequence that the antibody is able to recognize. In 1969, the first complete antibody sequence, which was determined, was the largest complete protein sequence that had ever been determined.
In the field of molecular biological research, antibody sequencing is used to know the antigen binding residues of antibody, and the nature of antigen binding residues in an antibody. Monoclonal antibodies have become an important tool in biochemistry, molecular biology, and medicine. Monoclonal antibodies (mAb or moAb) are monospecific antibodies, which are identical in structure. They are produced by one type of B lymphocytes, which are all clones of a single parent cell. Using this method, it is possible to synthesize monoclonal antibodies that specifically bind to a given substance almost any substance for detecting or purifying that substance. From the several antibody sequencing methods, protein sequencing method like Edman degradation, MS, HPLC/MS, etc. are more commonly used for deriving results in antibody sequencing. Antibody sequencing can be accomplished using several methods adopted worldwide, many of which are exorbitantly expensive. Alternatively, DNA sequencing is a relatively, cheaper technology for application in antibody sequencing.
Antibody sequencing involves several steps as follows:
Stage 1: mRNA extraction from the hybridoma cell pellet
Using standard protocols, mRNA is extracted and purified from the hybridoma cell pellet.
Stage 2: Reverse transcription
Next, mRNA transcription 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 is completed. The initial cDNA production occurs most probably with the oligo dT primer, usually, but not necessarily. Specific murine and human constant domain primers could be then used to PCR after cDNA production to determine the isotype of the antibody.
Stage 3: PCR or RACE amplification of heavy and light chains
The variable domains from the cDNA are amplified using degenerate VH and VL primers. A homopolymeric dCTP tail is added to the end of the cDNA for RACE. Following which, 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
For removing small fragments, the PCR products are gel purified, following which, these are cloned into a blunt or TA vector for sequencing.
Stage 5: Sequencing analysis
The specified number of clones to be sequenced can be ascertained by the client. As standard protocol, a minimum of 5 independent clones for each chain have to be sequenced.
Stage 6: Final Report
The entire process involving all the blots and sequence alignments of the heavy and light chains is documented and compiled into a detailed report, which is sent to the client.
by: Paul Kerr
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