Molecular analysis of the heavy chain variable region genes of human hybridoma clones specific for coagulation factor VIII
Molecular analysis of the heavy chain variable region genes of human hybridoma clones specific for coagulation factor VIII
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DOI：
10.1160/th05-06-0445
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发表时间：
2005-12-01
影响因子：
6.7
通讯作者：
Shokri, F
中科院分区：
文献类型：
--
作者：
Gharagozlou, S;Kardar, GA;Shokri, F
HemophiliaA is a X-linked hematologic disorder characterized by undetectable or low amounts of functional coagulation factor VIII (FVIII). Replacement therapy induces FVIII neutralizing antibody (Ab) (inhibitor) in a proportion of patients which makes further treatment of these patients ineffective and costly. To envisage mechanisms underlying inhibitor development, seven hybridoma clones specific for FVIII were generated from two hemophiliaA patients with high titer of inhibitor. Specificity and isotype of the monoclonal antibodies (mAbs) were determined by ELISA. Immunoglobulin (1g) variable region heavy (VH) chain gene family usage was identified by RT-PCR using V(H)1-6 specific primers. Nucleotide sequences of the V(H) gene of FVIII specific clones were determined and aligned to the most homologous germ line genes in the GenBank. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized either the V(H)1 (71%) or the V(H)3 (29%) gene family.Three V(H) domains were encoded by V 1-69 (DP-10),V 1-2 (DP-8),and V 1 -8 (DP-15) genes and two by V 1-18 (DP-14) gene, all from the VH 1 gene family. Of the V(H)3-gene family expressing clones, one belonged to V3-66 (DP-86) and the other one to V3-21 (DP-77) germline genes. The CDR3 length was found to be highly different amongst these clones ranging from I I to 22 amino acid residues. These data suggest that FVIII-specific Abs preferentially use VH gene segments derived from V(H) I gene family. Diversity of the expressed VH genes and their CDR3 length implies that different epitopes are recognized by these mAbs.
