Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning

Abstract

We have developed an efficient strategy that combines immunoglobulin (Ig) gene repertoire analysis and Ig reactivity profiling at the single cell level. Based on surface marker expression individual cells at different stages of human B cell development are isolated by fluorescence-activated cell sorting. For each cell Ig heavy and corresponding Ig light chain gene transcripts are amplified by nested RT-PCR and cloned into eukaryotic expression vectors to produce monoclonal human antibodies of the same specificity in vitro. All reactions are performed in 96-well plates and allow cloning of large numbers of Ig genes. The recombinant antibodies are tested for reactivity with diverse self- and non-self antigens and the reactivity profile can be directly linked to the complete Ig heavy and Ig light chain gene sequence information that is obtained as part of the cloning strategy. In summary, our method to clone and express human monoclonal antibodies is unbiased, highly efficient, requires only small cell numbers and the recombinant antibodies allow direct conclusions on the frequency of specific human B cells in a diverse repertoire.

Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows

“…PCR reactions were set up in 25 μL volume with 2.5 μL cDNA or PCR1 product using HotStarTaq Master Mix (Qiagen). Heavy and light chain paired retrieved form single sorted cells were cloned into human antibody expression vectors as described previously 33 .…”

Naturally Occurring Human Phosphorylcholine Antibodies Are Predominantly Products of Affinity-Matured B Cells in the Adult

“…1C). Single cells were deposited into 96-well Twintec PCR-plates (Eppendorf) containing lysis buffer as described previously (20). The purity of PC-reactive B cells was typically 67 and 100% after the first and second rounds of sorting, respectively (Fig.…”

“…Individual IgH (g or m) and IgL chain (k or l) genes rearrangements were amplified independently, using the cDNA as template, by two successive rounds of PCR (50 cycles each) as described previously (20). The PCR products were then sequenced using BigDye 3.1 terminator chemistry (Applied Biosystems) and read in an ABI 3730 instrument (Applied Biosystems).…”

“…Restriction sites for expression vector cloning were introduced by using gene-specific primers and first PCR products as template as described previously (20,21). The digested PCR products from each single cell were cloned into expression vectors containing human Igg1, Igk, or Igl constant regions (20,21).…”

The Lupus Susceptibility Locus Sle1 Facilitates the Peripheral Development and Selection of Anti-DNA B Cells through Impaired Receptor Editing

“…Ongoing studies are aimed at elucidating the molecular mechanisms through which signaling lymphocyte activation molecule family molecules might contribute to these events. Given that central and peripheral B cell tolerance checkpoints are also abrogated in human SLE, the B6.Sle1 congenic strain continues to be an informative mouse model for this human disease (34,35).…”