Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells

Rohatgi, Soma; Ganju, Parul; Sehgal, Devinder

doi:10.1016/j.jim.2008.09.017

Cited by 43 publications

(39 citation statements)

References 43 publications

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“…There are several primers set proposed for the selective amplification of murine IGHV and IGHJ subfamilies, however most do not cover the whole variable region, from the first to the last codon. [23][24][25][26][27][28][29] Despite the fact that the primers set used in this study lacked strict subfamily selectivity, such primer restriction was proven not to be essential. Nevertheless, the design of an extended and more selective set of primers would improve the overall recovery outcome.…”

Section: Discussionmentioning

confidence: 99%

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

Valdés-Alemán

Téllez-Sosa

Ovilla-Muñoz

et al. 2013

mAbs

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Section: Discussionmentioning

confidence: 99%

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

Valdés-Alemán

Téllez-Sosa

Ovilla-Muñoz

et al. 2013

mAbs

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“…We opted for a nested RT-PCR-based strategy for amplifying the expressed Ig H and L chain genes. Isotype-specific C H and C L antisense primers, along with V H and V L sense primers, were designed in our laboratory (26). The V H and V L external primers bind in the leader or in framework region (FR) 1, whereas the V H and V L internal primers bind in FR1.…”

Section: Amplification Of the Expressed Ig H And L Chain Genes By Rt-pcrmentioning

confidence: 99%

Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Rohatgi

Dutta²,

Tahir³

et al. 2009

The Journal of Immunology

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Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA 3-286 ) from strain R36A. Abs to PspA 3-286 were encoded by diverse V H and V families/genes. The H chain CDR3 and L chain CDR3 lengths were 3-13 (7.8 ؎ 0.5) and 8 -9 (8.7 ؎ 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D H gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D H gene-derived amino acids did not prevent antiPspA 3-286 mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA 3-286 polyclonal Ab response in PspA 3-286 -and R36A-immunized mice. Compared with PspA 3-286 -immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D H genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D H -less Abs.

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“…The major problem in rapidly obtaining heavy and light chain variable genes from a hybridoma is the occurrence of aberrant mRNAs that can be transcribed but that have no function. 25 If the aberrant gene is used in the construction of the chimeric or humanized antibody, it may produce immunoglobulin that has low affinity for the antigen and is completely non-functional. Several strategies may decrease the amplification of aberrant variable genes, but they have limitations primarily because the aberrant genes may be highly homologous to the functional ones.…”

Section: Discussionmentioning

confidence: 99%

Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes

et al. 2010

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Murine monoclonal antibodies (mAbs) are widely used but have limitations if administered in humans. The use of chimeric or humanized mAbs can reduce immunogenicity. The first step in producing such mAbs is to clone murine variable genes from a hybridoma, but it is possible to amplify both functional and aberrant variable genes, as they coexist in the hybridoma. During the development of a murine-human chimeric antibody, we have cloned from a hybridoma the functional heavy chain variable region (V H ) and light chain variable region (V L ) genes of a mAb that blocks the binding of anthrax lethal factor to protective antigen. In this study, we report the detection of two aberrant transcripts from a hybridoma produced using myeloma cell line OUR-1, the development of a method to distinguish between the functional and abundant aberrant V L transcripts, and the origins of these aberrant genes. The aberrant V L gene is derived from OUR-1 cells, while the aberrant V H gene might derive from antibody repertoires in B cells or from gene rearrangement in the hybridoma cells. The aberrant V H and V L genes in this study may facilitate discrimination between the functional and aberrant variable genes from hybridoma cells.

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Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells

Cited by 43 publications

References 43 publications

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes

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