A human monoclonal IgA rheumatoid factor using the VkIV light chain gene

Abstract

A human hybridoma stably secreting IgA rheumatoid factor (RF) was produced by cell hybridization with peripheral blood lymphocytes of a patient with rheumatoid arthritis. The RF was of the IgA1 isotype with kappa-light chains and was useful for standardization or specificity controls in class-specific RF assays. RF activity was detected only when the IgA molecular were in a polymeric state, and could be measured by enzyme linked immunosorbent assay as well as in conventional agglutination based tests. The RF had the modified Ga fine specificity described previously for several RFs and for protein A. The immunoglobulin V genes used were isolated and sequenced. The light chain was encoded by the VkIV gene rearranged to Jk2; compared to the published VkIV germ line gene there was 90% nucleotide homology. The heavy chain gene used belonged to the VHI family and was rearranged to JH4. Comparisons with published sequences revealed 90% homology with the recently characterized VH gene expressed by RF-TS3, a rheumatoid synovia RF hybridoma.

Persistence of a rheumatoid factor (RF)-producing B cell clone with a somatically mutated Igκ chain in a patient with rheumatoid arthritis

“…The consensus sequence of eight clones shown in Fig. 2 is identical to the published RF light chain sequence [13,16]. Altogether there were three nucleotide exchanges: one from C to T (nucleotide position 63), one from T to C (nucleotide position 184) and one from A to G (nucleotide position 104).…”

“…1) [16]. Three clones of the remaining four MboII-digestible clones were nearly 100% homologous to an unrelated VHI gene.…”

“…Escherichia coli strain DH5 alpha was transformed with the annealed plasmid using the procedure of Hanahan [25]. The plasmids were screened based on restriction sites which were characteristic and present in the originally described clone [16]. One of the somatic mutations of the light chain has created a new EcoRl restriction site, and the heavy chain differs from the most related sequences by an MboII restriction site.…”

“…Correspondence: Angela Gause, Medizinische Klinik I, Universitatskliniken des Saarlandes, 66421 Homburg, Germany, Previously we were able to characterize a human heterohybridoma derived from peripheral blood lymphocytes (PBL) of a patient with RA which was producing an IgA RF [16], The V-region genes encoding the heavy and light chains of this RF were sequenced. The VH gene is related to two other RFencoding VH genes [17,18] that belong to the recently described VH7 gene family [19] which is closely related to the VH I family.…”

A somatically mutated VκIV gene encoding a human rheumatoid factor light chain

“…In positions -157, -141, 97 and 234 (underlined) the thymidine was substituted by a cytosine in one of eight sequences, in positions -18 and 26 adenosine was substituted by guanidine in one of eight sequences by mis-incorporation through the Taq polymerase. The consensus sequence is compared to the published germline sequence (gl) [14] and to the RF sequence (P61B27) [11]. Numbering is according to Klobeck [14], numbers at the ends ofthe lines designate nucleotides, numbers above the sequence designate amino acids, the asterisks designate additional amino acids in CDR I characteristic for the VKIV family.…”