B4 Allotype Rabbit Monoclonal Antibody Cloning With Selective Primers
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The production of rabbit monoclonal antibodies is hindered by the challenge of efficiently amplifying the desired b4 allotype antibodies while minimizing the production of endogenous antibodies from the fusion partner, particularly in hybridomas with b5 allotype, due to sequence variations and similarities among rabbit immunoglobulin genes.
Innovation Solution
A method involving specific nucleic acid primers, particularly a four-base motif reverse primer proximal to the stop codon, is used to selectively amplify b4 kappa light chain sequences, allowing for the production of recombinant antibodies by fusing B-lymphocytes with rabbit myeloma cells, isolating cDNA, amplifying with designated primers, cloning into expression vectors, and expressing the antibodies in mammalian host cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional amplification methods are used to produce rabbit monoclonal antibodies, then antibody production occurs, but endogenous antibodies from the fusion partner (b5 allotype) contaminate the desired b4 allotype antibodies
Solution Approach 1:
The patent applies parameter changes by designing primers with specific sequence parameters that match only the b4 allotype kappa light chain. The reverse primer contains a four-base motif (GGTG) at positions 23-26 that is unique to b4 allotype, while the forward primer targets positions 1-10 of the kappa light chain. This specific parameter matching allows selective amplification of b4 allotype sequences while excluding b5 allotype sequences from the fusion partner.
Solution Approach 2:
The patent applies local quality by focusing the amplification specificity on particular regions of the kappa light chain gene. The forward primer binds to the 5' end region (positions 1-10) and the reverse primer binds to the 3' end region (positions 23-26), with the critical discriminatory four-base motif located at positions 23-26. This localized specificity ensures that only b4 allotype sequences are amplified from the hybridoma, while b5 allotype sequences from the fusion partner are excluded.
2Productivity
If rabbit B-lymphocytes are fused with rabbit myeloma cells to produce hybridomas, then monoclonal antibody production is enabled, but the fusion partner's endogenous antibodies (b5 allotype) are co-produced
Solution Approach 1:
The patent applies the taking out principle by extracting and selectively amplifying only the desired b4 allotype antibody sequences from the hybridoma population. Using the specifically designed primer pair, the method extracts the b4 allotype variable region and kappa light chain sequences through PCR amplification, separating them from the contaminating b5 allotype sequences produced by the fusion partner. This extracted material is then cloned and sequenced to obtain pure b4 allotype monoclonal antibody sequences.
Solution Approach 2:
The patent applies the intermediary principle by using a molecular intermediary (the specifically designed primer pair) to mediate between the hybridoma population and the desired b4 allotype sequences. The primers act as intermediaries that selectively bind to and amplify only b4 allotype sequences, bridging the gap between the mixed hybridoma population and the pure b4 allotype product, while excluding b5 allotype sequences from the fusion partner.
3Adaptability or versatility
If standard PCR primers are used for amplification, then general antibody sequences can be amplified, but consistent amplification of specific allotype sequences cannot be achieved due to sequence similarities
Solution Approach 1:
The patent applies parameter changes by modifying the primer sequence parameters to achieve both versatility and precision. The forward primer (positions 1-10) and reverse primer (positions 23-26 with four-base motif GGTG) are designed with specific sequence parameters that are conserved across b4 allotype variants but differ from b5 allotype. This parameter optimization allows consistent amplification of b4 allotype sequences while maintaining the ability to adapt to different b4 variants.
Data Source
AI summary
A method for cloning a full length coding sequence for a light chain of a rabbit monoclonal antibody is provided. In some embodiments, this method may involve: fusing a B cell from a rabbit having a B4 allotype with a 240E cell or a derivative thereof to produce a hybridoma, wherein the B cell and the hybridoma produce a monoclonal antibody; making cDNA from the hybridoma; amplifying from the cDNA a full length coding sequence for the light chain of a monoclonal antibody produced by the hybridoma using: a forward primer that hybridizes to a site in SEQ ID NO:10, and a reverse primer having a 3′ end of sequence CTARCAGTCX (SEQ ID NO:11), wherein R is A or G and X is A, AC, ACC or ACCC; and cloning the amplified sequence into an expression vector to produce a first plasmid.