Comparison between hybridoma and Fab/phage anti-RhD: their V gene usage and pairings

Our 11 anti-RhD's in conjunction with 37 previously published RhD antibodies, produced by hybridoma technology were analysed for germline gene usage and restriction in VH and VL pairings. The 17 VH germline genes used by the hybridoma anti-RhD IgG were derived from 4 VH families (VH1, VH2, VH3 and VH4). Eighteen kappa chains were restricted to only 5 germline genes from only 2 V kappa families (V kappa 1 and V kappa 3). However, the 13 lambda chains were not as restricted, using 10 V lambda germline genes from 4 families (V lambda 1, V lambda 2, V lambda 3 and V lambda 8). Fifty six unique Fab/phage anti-RhD were also analysed. In all cases the Fab/phage VH germline genes were derived from the VH3 family (41/41). The 29 kappa chains were restricted to 4 germline genes primarily from V kappa 1 (97%) and the 24 lambda chains used 10 V lambda germline genes from 5 families (V lambda 1, V lambda 2, V lambda 3, V lambda 4 and V lambda 7). The VH germline genes of the Fab/phage were restricted to 4 of the 17 used by the hybridoma anti-RhD IgG (DP46, DP49, DP50 and DP77). Ninety percent of the Fab/phage were restricted to 1 of the 5 V kappa germline genes used by the IgG (DPK9). However, the repertoire of the V lambda germline genes used in these two systems is different, with analysis showing greater diversity in V lambda gene usage with 8 unique germline genes used by 76% Fab/phage compared to 4 unique genes used by 46% of the IgG hybridoma anti-RhD.     

Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient.

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low-affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity.     

The V-region repertoire of Haemophilus influenzae type b polysaccharide antibodies induced by immunization of infants.

Haemophilus influenzae type b (Hib) is a significant pathogen for young children, and three Hib vaccines (named PRP-OMPC, HbOC, and PRP-T) are currently available for young children. Extensive studies of anti-Hib polysaccharide (PS) antibodies (Abs) have shown that the V regions of Abs against the Hib PS comprise a VH gene in the VH3 gene family and a VL gene from various K kappa and V lambda subgroups. To study immunogenic properties of the three vaccines in young children, we determined the VL subgroups and avidities of anti-Hib-PS Abs induced by the three clinically available conjugate vaccines. Ab avidity was measured by determining the concentration of a Hib-PS oligomer that abrogates half of the binding of immunoglobulin G anti-Hib-PS Abs to microwells. The PRP-OMPC vaccine induced lower-avidity Abs than the prelicensure HbOC vaccine (P = 0.05). When we compared anti-Hib-PS Abs expressing V kappa Ia, V kappa II, and V lambda subgroups, a greater Ab response was induced by the prelicensure HbOC vaccine than other vaccines (P < 0.05). When anti-Hib-PS Abs with the V kappa III subgroup were compared, however, both PRP-T and prelicensure HbOC vaccines induced a comparable response, which in turn was greater than those induced by the PRP-OMPC or the postlicensure HbOC vaccine (P < 0.001). The VL repertoire of Abs induced with the prelicensure HbOC or PRP-T vaccine in young children is dominated (about 80%) by anti-Hib-PS Abs using subgroup V kappa II. However, anti-Hib-PS using V kappa II VL accounts for only about 40% of the total anti-Hib-PS Abs induced with the PRP-OMPC vaccine or the postlicensure HbOC. Our data suggest that immunogenic properties of Hib vaccines in young children vary depending on the vaccine preparations as well as the vaccine types.     

Characterization of a human monoclonal autoantibody directed to cardiolipin/beta 2 glycoprotein I produced by chronic lymphocytic leukaemia B cells.

We determined the specificity and sequence of immunoglobulin molecules synthesized by monoclonal B cells from a patient with chronic lymphocytic leukaemia (CLL) who presented with a number of clinical and biological autoimmune symptoms. Heterohybrids obtained by fusion of CLL cells with the mouse X63-Ag 8.653 myeloma produced IgM lambda MoAbs directed to the cardiolipin/beta 2 glycoprotein I (beta 2GPI) complex and ssDNA. They were devoid of polyreactivity. Nucleotide sequence analysis of the variable domain of the mu chain indicated the utilization of the VH4 71.2 gene or one allotypic variant, DXP4 and JH3 segments. The lambda light chain used the single gene from the V lambda 8 subfamily, J lambda 3 and C lambda 3 genes. The VH gene displayed 11 nucleotide changes in comparison with its putative germline counterpart. However, these nucleotide changes correspond to variations observed in other published VH4 sequences, suggesting gene polymorphism rather than somatic mutation. DXP4 and JH3 were also in germline configuration. The VL gene exhibited a single replacement mutation in CDR1. These data suggest that the monoclonal CLL B cells in this patient retained VH and VL genes in germline configuration although they secreted a pathogenic anti-cardiolipin antibody associated with clinical symptoms, vasculitis and thrombosis, which may be provoked by antibodies to the phospholipid/beta 2GPI complex.     

Diversity and somatic hypermutation of the Ig VHDJH,V kappa J kappa,and V lambda J lambda gene segments in lymphoma B cells: relevance to the origin of the neoplastic B cell clone

Burkitt's lymphoma (BL) is a malignancy of B cells characterized by chromosomal translocations involving the immunoglobulin (Ig) and c-MYC gene loci. To address the putative role of antigen in the clonal expansion of these neoplastic B cells, we analyzed the VHDJH and VLJL gene segments expressed by the established cell lines derived from six endemic BL and six sporadic BL. Eight BL cell lines used genes of the VH3 family, two of the VH4, and two of the VH1. Eight VL chains were kappa (four members of the V kappa3, two of the V kappa1, and two of the V kappa2 subgroups) and four lambda (three members of the V lambda1 and one of the Vl ambda3 subgroup). The VH gene utilization was stochastic (i.e., it reflected the relative representation of the different VH gene family members in the human haploid genome). In contrast, the VL gene utilization was skewed toward the overutilization of the V kappa3 and V lambda1 gene subgroups. When compared with those of the respective germline genes, the sequences of the expressed Ig VDJ genes displayed nucleotide differences that resulted from somatic hypermutation. In three endemic and three sporadic BL cells, nucleotide changes yielding amino acid substitutions segregated within the complementarity determining region, indicating the application of a positive pressure for replacement mutations and suggesting that these neoplastic lymphocytes underwent a process of clonal selection driven by antigen, perhaps emerging from or transitioning through germinal centers.     

Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody.

Recent evidence suggests that natural autoantibodies may play an integral role in the development of the normal immune repertoire. To explore the genetic origins of these antibodies, we have isolated and sequenced the variable (V) region genes encoding both the heavy (H) and light (L) chains of a natural anti-DNA antibody, Kim11.4. The genes appear to be derived from the VH4.18 (subgroup VHIV), JH5, Hum1L1 (subgroup V lambda I) and J lambda 3 germline genes. The origin of the H chain diversity gene is more obscure, being potentially derived from one or more of several germline genes, arranged in either the forward or reverse orientations. Both the Kim11.4 VH and VL genes share significant degrees of similarity with those utilized in other autoantibodies, indicating that at least some degree of V restriction may exist in human autoreactive B cells. The pattern of nucleotide differences between the Kim11.4 VH and VL genes and their putative germline counterparts suggests that the Kim11.4 genes may have undergone somatic mutation and arisen as a result of antigen selection.     

Variable region primary structures of monoclonal anti-DNA autoantibodies from NZB/NZW F1 mice

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.     

The antibody site in Atlantic salmon; phage display and modeling of scFv with anti-hapten binding ability.

Cloning and sequencing the cDNA of around 50 VH (VDJ) and 15 VL genes in Atlantic salmon demonstrated nine VH families (above 80% identity within each family) and one dominating but relatively diverse VL family in this species. The highest variability of the VH was seen in the CDR3, but CDR2 also expressed a modest variability. The 'whole' antibody repertoire was expressed as single chain Fv (scFv) in a phage display library by combining 12 VH and two VL specific primers (FR1/microl and FR1/CL, respectively). The PCR products (VH and VL) were ligated (with a G-rich spacer) into the lambda Surf-Zap (Stratagene) vector and expressed as a surface fusion protein on the M13 phage. Anti-TNP and anti-FITC specific scFv clones were isolated by panning using hapten-coated magnetic beads and the coding DNA sequenced. The specificities of the anti-TNP and anti-FITC clones were similar to mouse monoclonal antibodies. 3D-models of the active sites (CDRs) of the anti-TNP and anti-FITC clones suggest hapten-interacting structures of the salmon antibody site similar to mammalian antibodies.     

Variable region genes of human monoclonal autoantibodies to histones H2A and H2B from a systemic lupus erythematosus patient

An antibody phage library obtained from peripheral blood lymphocytes of a systemic lupus erythematosus (SLE) patient was used to isolate four monoclonal autoantibodies against histones H2A and H2B. Analysis of the variable region sequences revealed that the anti-histone monoclonal antibodies were not clonally related; they used VH genes from three different VH gene families (VH3, VH4, and VH5) and distant members of the Vkappa group (L25, L6, A27, and O8) in conjunction with different D and J gene segments. These observations suggest that certain gene families or segments are not critical in producing anti-histone autoantibodies in SLE. Most of the utilized VH and Vkappa sequences were highly mutated and the complementarity-determining regions (CDRs) varied greatly in length. The VH region of the antibody SLEhis18 had an isoelectric point of 6.1, and 29% of the mutations were changes to acidic amino acid residues. The second CDR (CDR2) of SLEhis18 VH contained one basic and three acidic residues. Acidic residues were observed in the CDR3 regions of VH, but not VL, in all isolated clones; this is unusual, as most autoantibodies are comprised predominantly of non-acidic residues. This is the first report of a systematic sequence analysis of human anti-histone monoclonal antibodies. Our results suggest that certain V genes are not important for autoreactive specificity to histones in SLE; instead, other mechanisms such as an existence of acidic residues and somatic mutations in CDRs are required for specific binding to histones, which might play a role as a stimulatory autoantigen for the activation of autoantibody-producing B-cells and the selection of high affinity antibody.     

Identification of novel VH1/J558 immunoglobulin germline genes of C57BL/6 (Igh b) allotype

Although a rich database of Igh a allotype mouse immunoglobulin germline genes exists, current information on Igh b allotype immunoglobulin germline genes is limited. Among the immunoglobulin VH genes, single-cell amplified from six Igh b (C57BL/6 background) spleens in this study, 602 clonally independent immunoglobulin VH sequences belonging to the VH1/J558 family were identified. Whereas 335 of these sequences could be traced to have originated from 29 different VH1/J558 germline genes deposited in the NCBI Igblast database, the remaining 267 sequences appeared to have originated from 21 novel germline genes. Of the 50 VH1/J558 germline genes utilized in the peripheral repertoire of these Igh b allotype mice, the most frequently used genes included 45.21.2, V165.1, J558.6, J558.18A, and V23. Whereas the majority of the novel genes uncovered represented allelic counterparts of previously described Balb/c (Igh a allotype) genes, some appeared to represent truly novel germline genes. Collectively, the VH1/J558 germline genes exhibited high amino acid residue usage variability at the CDR1 positions, H31, H33, and H35, and the CDR2 positions, H50, H52, H53, H54, H56, and H58. The 50 VH1/J558 germline genes expressed in the peripheral Igh b repertoire also varied widely in the net charge of their CDR regions, raising the possibility that they may be differentially utilized to encode anti-nuclear autoantibodies.     

The role of somatic mutation in determining the affinity of anti-DNA antibodies

Combinatorial antibody libraries were constructed from the spleen of a patient with concomitant systemic lupus erythematosus and idiopathic thrombocytopenia. Following selection of the libraries with DNA, a panel of 15 anti-DNA Fabs was isolated. Sequence analysis of these antibodies coupled with measurements of their affinities for ss- and dsDNA were used to investigate the role of somatic mutation in affinity maturation of the anti-DNA response. Examination of the germline genes used by these Fabs supports previous studies that suggest there is no restriction of the gene usage in the anti-DNA response. However, data are presented indicating that VH3 genes and the A27 V(kappa) paired with the J(kappa)1 may be over-expressed in the anti-DNA repertoire. Analysis of the role of somatic mutation in increasing affinity for DNA indicates that affinity maturation has occurred and suggests that the CDR1 and CDR2 of the heavy chain are of importance in this process.     

Immunologic memory to phosphorylcholine. VI. Heterogeneity in light chain gene expression

BALB/c mice immunized with phosphorylcholine-conjugated keyhole limpet hemocyanin (PC-KLH) produce two types of anti-PC antibodies, designated group I and group II, which differ in their fine specificity and idiotype expression. Group II hybridomas can utilize VH genes and VL genes (in particular, V kappa 1-3) distinct from those expressed in the group I-like anti-PC myelomas. Here we have analyzed additional anti-PC hybridomas from BALB/c and (CBA/N X BALB/c)F1 male mice and also anti-PC antibodies purified from BALB/c and C57BL/6N antisera. Isoelectric focusing indicates that one of the group II hybridomas utilizes V kappa 1-3 and that related L chains are expressed in a major portion of group II serum antibodies. Other group II antibodies in antisera express different L chains, some of which are presently unidentified. However, isoelectric focusing analysis also indicates that the L chains of some group II hybridomas and serum antibodies are related to those found in the group I anti-PC myelomas and group I hybridoma and serum antibodies. In addition, one hybridoma was found to utilize lambda 2. Thus, it appears that the anti-PC antibodies with group II-like fine specificity can utilize a variety of VL genes related to, or distinct from, those expressed in group I antibodies.     

Restricted usage of VH and V kappa genes by murine monoclonal antibodies against 3-fucosyllactosamine

We are studying the structure and regulation of murine antibodies against the 3-fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/c IgM, kappa monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by V kappa 24B and J kappa 1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a JH3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity-determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7-9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.     

Molecular characterization of a human monoclonal antibody to B antigen in ABO blood type

A human anti-B antibody of clone BT97 was obtained from a healthy individual of type A of the ABO blood group without immunization. Cloning was performed by means of heterohybridoma formation of cell fusion between human peripheral lymphocytes and mouse myeloma cells. The antibody selectively reacted with B-antigen in flow cytometry using red blood cells and enzyme-linked immunosorbent assay. The VH and VL genes of BT97 were derived from the germline genes of DP-47 and 3p.81A4, respectively, with a couple of somatic mutational events. Comparative analysis with other reported anti-A, B and H antibodies revealed that the amino acid sequence of the VH region was more homologous than that of the VL region. The sequence of BT97 showed complete identity with one anti-H natural antibody reported by Marks et al., with the exception of the CDR3 region. It is not known whether the homologies include the common properties of the natural antibodies; however, a particular germline gene potentially changes to anti-ABH antibodies. We think that this method is suitable for cDNA preparation of human monoclonal antibodies to blood group antigens and for sequence analysis.     

Immunocytochemical detection of kappa and lambda light chain V region subgroups in human B-cell malignancies.

We have used a sensitive immunoperoxidase method and highly specific anti-light chain antisera to determine the light chain variable region (VL) subgroup nature of cytoplasmic (c) and cell surface (s) Ig expressed by human monoclonal plasma cells and B lymphocytes. The immunocytochemical characterization of cIg and sIg used antisera specific for the established kappa light chain V kappa subgroups (V kappa I, V kappa II, V kappa III, and V kappa IV) and the lambda light chain V lambda subgroups (V lambda I, V lambda II/V, V lambda IV, and V lambda VI). Studies were performed using cytospin preparations of bone marrow-, peripheral blood-, and lymph node-derived cells from patients with multiple myeloma, amyloidosis AL, and Waldenström''s macroglobulinemia and with low-, mid-, and high-grade B-cell malignancies. The V kappa or V lambda subgroup of the cIg or sIg also could be identified after deparaffinization and enzyme treatment of formalin-fixed, paraffin-embedded specimens. For those patients who had monoclonal serum or urinary Igs, there was complete concordance between the VL subgroup of the secreted Ig and that of the cIg or sIg. The percentage distribution of V kappa or V lambda subgroups on the sIg of cells from patients with chronic lymphocytic leukemia (CLL) and other cytomorphologic types of B-cell malignancies differed from that found for kappa- or lambda-type Bence Jones proteins obtained from patients with multiple myeloma, amyloidosis AL, and Waldenström''s macroglobulinemia. In contrast to the plasma cell and lymphocytoid plasma cell diseases, a relative predominance of certain VL subgroups, ie, V kappa IV, V lambda III, and V lambda IV, and the absence of the amyloid-associated V lambda VI subgroup were found in CLL and related diseases. The immunocytochemical techniques used make possible a rapid means to demonstrate B-cell monoclonality and provide further evidence for the selective expression of certain VL genes in human B-cell neoplasia.     

Specificity and variable region cDNA sequence of an isogeneic monoclonal antiidiotype to an anti-alpha(1----6)dextran.

We have characterized a monoclonal isogeneic antiidiotype, IdB5.7, from a BALB/c mouse immunized with the anti-alpha(1----6)dextran C57BL/6 45.21.1. It defined a hapten-inhibitable idiotope expressed on four of the 2 myeloma and 37 hybridoma anti-alpha(1----6)dextrans tested. Sequence comparison of Id+ and Id- anti-alpha(1----6)dextrans suggested that two extra amino acids at VH 100A and 100B and different residues at VH 101 abolish the expression of the idiotope in the Id- anti-alpha(1----6)dextrans. Sequence analysis of the VH of IdB5.7 showed a CDR1 longer than usual and a D segment in CDR3 formed by the fusion of two D minigenes. The IdB5.7 V kappa uses the V kappa 1 germline gene K5.1 with a few substitutions. The D-D fusion in VH CDR3 is a feature which has been reported in several other antiidiotypic antibodies.     

Variable region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b capsular polysaccharide.

A hybridoma secreting a human immunoglobulin G2 kappa monoclonal antibody (MAb) specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib) was isolated. This MAb, designated CA4, was bactericidal to Hib in vitro and protected infant rats from Hib bacteremia. Nucleotide sequence analysis of CA4 variable (V) region cDNA showed that the heavy (H)-chain V region was of subgroup III and was 96% identical to the VH germ line gene segment DP77 (V3-21). The light (L)-chain V region was of the kappa subgroup III and was 94% identical to the A27 (Humkv325) germ line gene, which is commonly used by rheumatoid factors and other autoantibodies. MAb CA4 did not have rheumatoid factor activity and did not react with histones, DNA, or chromatin. These findings identify an additional VHIII gene segment which can contribute to the anti-Hib capsular polysaccharide repertoire and demonstrate that a VL gene commonly encoding autoantibodies can be utilized for protective immunity.     

Variable region sequences and idiotypic expression of a protective human immunoglobulin M antibody to capsular polysaccharides of Neisseria meningitidis group B and Escherichia coli K1.

We determined the heavy (H)- and light (L)-chain variable (V) region nucleotide and translated amino acid sequences of the human immunoglobulin M(kappa) monoclonal antibody (MAb) 5E1, which is specific for the polysaccharide capsule of Escherichia coli K1 and Neisseria meningitidis group B (poly[alpha(2-->8)-N-acetylneuraminic acid]) and which is protective in animal models of infection. The 5E1 VH gene is a member of the VHIIIb family and is 97% homologous to the 9.1 germ line gene. The 5E1 VL gene is a member of the kappa I subgroup and is 98% homologous to the germ line gene, 15A, also known as KLO12. The VL and/or VH genes used by 5E1 are highly homologous to the V genes encoding antibodies to the Haemophilus influenzae type b polysaccharide and to antibodies reactive with self-antigens such as erythrocyte "i," DNA, and thyroid peroxidase. We also produced three murine anti-idiotype (Id) MAbs against 5E1. All three anti-Ids recognize a minor subset of antimeningococcal B polysaccharide antibodies present in serum from normal adults. Two of the anti-Ids define distinct Ids associated with antibodies having kappa I-15A V regions. These 15A-associated Ids are expressed by some heterologous human antimeningococcal B polysaccharide MAbs, and they also are independently expressed by two human MAbs that are specific for either the H. influenzae b polysaccharide or the i erythrocyte antigen and that utilize the kappa I-15A V region. Taken together, these data indicate that the 5E1 antibody uses V regions that recur in the human antibody repertoires to this polysaccharide and to structurally dissimilar polysaccharides and autoantigens. Thus, the poor immunogenicity of poly[alpha(2-->8)-N-acetylneuraminic acid] cannot be explained by the unavailability of certain critical VH and VL genes required for generation of antibody response.     

Sequence, specificity and crystallization of an oestrone-3-glucuronide antibody (3910).

We describe the specificity profile and V region sequences of a high-affinity monoclonal antibody (mAb), 3910, directed against oestrone-3-glucuronide (E3G). Inhibition studies show that the D-ring is critical for steroid specificity, while the glucuronic acid attached to the A ring is required for high binding affinity, suggesting that both 'ends' of the E3G ligand are recognized. The VH domain is encoded by a gene from the VH7183 family, while VL appears to be encoded by the Vk5.1 gene (kappa II subgroup) with a deletion of six residues from complementarity-determining region-1 (CDR1). The VH CDR3 is 10 amino acid residues in length, of which D/N contributes five residues. Comparison of VH CDR of 3910 with those of mAb against progesterone (DB3) and digoxin (26-10, 40-50), for which crystal structures have been determined, suggests that aromatic side chains are important for E3G binding and that tyrosine residues H50, H97 and H100 may interact with the ligand. The Fab fragment of 3910 has been crystallized in its native and steroid (E3G and oestriol-3-glucuronide) complexed forms. An X-ray diffraction data set to 3 A resolution has been collected for the native Fab.     

The human antibody V region repertoire to the type B capsular polysaccharide of Haemophilus influenzae.

The V region repertoire of the human antibody response to the type b capsular polysaccharide of Haemophilus influenzae (Hib-PS) is being defined at the molecular level using antibodies purified from serum of immunized adults. The VH of this response is restricted to the VHIII subgroup while the VL can be divided into two categories. The most common VL, expressed in > 90% of adults and usually constituting the majority of a subjects anti-Hib-PS antibody response, is restricted to the product of a single V kappa II gene known as A2 that probably lacks somatic mutations. The product of the A2 gene is invariably joined to one of several J kappa products by an inserted arginine at the V kappa-J kappa junction. In contrast to the restricted nature of the dominant VL clonotype, the second category of VL constitutes a heterogeneous group of at least seven different VL gene products that often contain somatic mutations and generally exhibit crossreactivity with a related polysaccharide from E. coli. Elucidation of anti-Hib-PS V regions at the molecular level will permit examination of structure-function relationships among these clinically important antibodies and should make the V region repertoire to Hib-PS a useful model for studying human V gene responses.