The general hybridoma antibody cloning protocol and antibody sequencing protocol seem very straightforward: RNA extraction, reverse transcription to cDNA, nested PCR amplifications, cloning of PCR products, sequencing, and analysis of sequences. However, it has taken scientists a long time to optimize a variety of parameters in the protocols, especially V-gene specific primers and antibody sequence databases. Hybridoma antibody cloning and antibody sequencing protocols from public literature:
– Sequencing of antibodies.
Detailed protocol for antibody cloning and antibody sequencing.
– Antibody variable region sequencing as a method for hybridoma cell line authentication.
Variable region sequencing used for discrimination between hybridoma cell lines.
– Cloning, expression, and modification of antibody V regions.
Detailed protocol for cloning and expression of immunoglobulin variable regions using PCR with redundant primers.
– Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity.
Degenerate primers for PCR amplification of mouse VH and VL. A single highly degenerate FR1 primer sufficient for VL amplification; a series of FR1 primers for VH amplification.
– A general method allowing the design of oligonucleotide primers to amplify the variable regions from immunoglobulin cDNA.
A rational method of designing primers to amplify VH and VL for framework 1 (FR1) of immunoglobulins.
– Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.
Two sets of oligonucleotide 5′-primers hybridizing the relatively conserved motifs within the signal sequences of the 15 heavy chain and 18 kappa light chain gene families.
– Rapid cloning of any rearranged mouse immunoglobulin variable genes.
Designing of 5′ and 3′ universal primers based on regions highly conserved across all analyzed VH and VL gene families, and the joining or constant regions.
– Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR.
A set of universal primers using conserved sequences of leader (signal peptide), framework one and constant regions of the immunoglobulin heavy-chain genes.
– A general method for chimerization of monoclonal antibodies by inverse polymerase chain reaction which conserves authentic N-terminal sequences.
The method circumvents the potential problems brought by degenerate primers matching to framework region 1 and to the joining regions.
– Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.
A reliable and versatile RACE PCR method for the isolation of VH genes from human and murine lymphoma cells, especially if consensus primer PCR fails.
– Cloning and sequencing of human immunoglobulin V lambda gene segments.
14 (including 4 pseudogenes) new human variable (V) gene segments of lambda light chains from a single individual.
– Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction.
The first method established to use PCR to amplify and clone mouse immunoglobulin (Ig) variable (V) regions after selective first-strand cDNA synthesis.
We index the protocols for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.