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- W2510162592 abstract "A technique is described that allows single hybridoma cell colonies to be assayed for the productive rear- rangement of a single immunoglobulin variable region (V) gene segment by utilizing expression of V mRNA for analysis. Hybridomas growing in microwell tissue culture plates are lysed in situ, cellular RNA is directly transferred to nitrocellu- lose by filtration, and specific immunoglobulin mRNA is de- tected by hybridization of the filter with a DNA probe. The method is simple and sensitive. A single species of mRNA can be detected in a lysate of 1000 cells; 5000 hybridoma colonies can be easily screened per day. The technique has been suc- cessfully used to isolate cell lines from nonimmune mice ex- pressing a particular heavy chain variable region (VH) gene segment. Strain A mice produce a relatively simple polyclonal immune response when immunized with p-azophenylarsonate (Ars)- protein conjugates. The majority of the antibodies made in either the VCHRI or VCRI gene segment. We felt it necessary to avoid potential bias due to both the effects of antigen and serological screening methodologies. Serological screening techniques using poly or monoclonal anti-idiotypic antibody reagents have frequently been used in investigations of this sort (10-12). The presence of idiotype on an antibody can often be positively correlated with the expression in that molecule of a single VH or VL gene segment (13). The nature of the determinant(s) recognized by these serological re- agents are often ill-defined, however. Determinants ana-" @default.
- W2510162592 created "2016-09-16" @default.
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- W2510162592 date "2016-01-01" @default.
- W2510162592 modified "2023-09-26" @default.
- W2510162592 title "Isolation of hybridomas expressing region gene segment by using a sci mRNA sequences in whole cell lysa (monoclonal antibodies/filter hybridization/bacterial lipopolysacchar" @default.
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