kits.traumatologi.no

Background of Single B Cell Antibody Technology

Currently, monoclonal antibodies (mAbs) are extensively used as a critical tool for scientific research, and diagnostic & therapeutic applications, most of which on the market are obtained by hybridoma technology or display technology. To better be applied for human diagnosis and treatment, the single B cell antibody technology has been pushed out, showing huge potential and value in natural antibody production. Compared with the present approaches, single B cell antibody technology can obtain specific mAbs with natural conformation in a time-saving way. The workflow of antibody discovery by single B cell technology generally involves immunization, single B cell isolation, antibody encoded gene amplification, and target antibody expression and characterization.

B Cell Antibody Gene Amplification

After harvesting B cells, the next step is to obtain the genetic information and amplification of the specific antibody, which will facilitate the subsequent expression of the target antibody. Similar to recombinant antibody production, single B cell antibody technology also needs to recover antibody genes from source cells, determine the sequence of the desired antibody, amplify and clone the target genes.

Antibody Gene Transcript Amplification

As different types of B cells are remarkably different in the ability of antibody secretion, B cells with strong ability of antibody secretion and high antibody gene mRNA transcripts are preferred to select. Specific antibody-secreting B cells are kept in a reaction system containing cell lysates and RNase inhibitors. The sorted B cells are performed a reverse transcription-polymerase chain reaction (RT-PCR) to convert mRNA to cDNA. And full-length antibody gene transcripts are amplified by nested or semi-nested RT-PCR.

Antibody Gene Sequencing

After the amplification of the target antibody gene, the specific antibody gene transcript information is subsequently sequenced. And the sequencing results are compared using a series of databases, such as IgBLAST at the NCBI website, to evaluate gene integrity and identify the mutations of the rearranged V, D, and J gene segments.