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We have isolated and characterized a cDNA for IGF-binding protein-2 (IGFBP-2) and its gene from the chick embryo. Using primers from a conserved region of the mammalian IGFBP-2 sequence, a cDNA clone (1.6 kb) was isolated from an embryonic day-18 chick retina cDNA library. Although the clone was truncated at the 5' end, the complete coding sequence was obtained from 5' rapid amplification of cDNA ends and genomic sequencing. The open reading frame encoded a 311 amino acid precursor protein which contains a putative 36 residue signal peptide. The mature 275 amino acid protein had a predicted M(r) of 33,500 and exhibited 71, 68, 68 and 66% identity to rat, bovine, ovine and human IGFBP-2 cDNA respectively, with conservation of all 18 cysteines. The cDNA contained an RGD peptide but lacked a putative ATP-binding motif. A single transcript of approximately 2.3 kb was present in embryonic day-15 eye, brain, skeletal muscle, heart and intestine, but was virtually absent from embryonic day-15 liver. The chicken IGFBP-2 gene spanned approximately 38 kb, consisted of four exons, and was similarly organized to that of the rat and human. Southern blot analysis of chicken genomic DNA suggested that it is encoded by a single gene. The sequence information from the avian IGFBP-2 should be of value in examining the role of IGFBP-2 in vertebrate development.

We report and characterize a novel mutant of the hepatitis B virus surface antigen (HBsAg). The mutant was isolated from a symptomatic patient who was found to be persistently positive for both HBsAg and anti-hepatitis B surface antibody (anti-HBs) and a long-lasting anti-HBc (core) IgM. Due to the unusual immune serological profile, polymerase chain reaction and sequencing were performed and revealed a genotype D mutant HBV (LBN). Aligned with known HBsAg sequences from GenBank, the LBN variant matched to consensus subtype ayw2 and revealed five mutation positions. Interestingly, none of the mutations was found within the group-specific "a" determinant region (124-147) and, specifically, two of the five mutations, T118K and P120Q, were located only a few amino acids adjacent to the 124-147 region. Using a panel of six monoclonal antibodies and vaccine raised human neutralizing antibodies, the recombinant wild-type HBsAg and the novel variant LBN HBsAg were investigated for their immunological reactivity. Vaccine raised human anti-HBs showed less reactivity to the variant LBN HBsAg than to wild-type HBsAg in enzyme immune assays. Furthermore, our observations demonstrate the influence of adjacent residues on the group-specific "a" determinant structural configuration; this resulted in severe antigenic changes of the immunodominant region as well as in the subtype serology. The importance of the variant LBN lies in the observation that mutations close to the "a" determinant can change the immunodominant region structure and therefore alter the group specific determinant antigenicity even though no mutations are present within this region. Hence, the classical definition of the "a" determinant cluster may need to be extended to cover a broader region to include the requirement of adjacent amino acids to support its conformation. In conclusion, by understanding the HBsAg major immunodominant region structure and by using a combination of antibodies with specificity covering all key mutation locations, maximal anti-HBs-based protection, and highly sensitive diagnostic assays can be achieved.

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