Fd Fragment Research Articles

Comparison of two forms of catalytic antibody displayed on yeast-cell surface

Two forms of the Fab fragment of the catalytic antibody 6D9 were individually displayed on yeast-cell surface in fusion to the C-terminal half of α-agglutinin: one was 6D9 Fab1, in which the light chain of the Fab (Lc) fragment is displayed on cell surface and the heavy chain of the Fab (Fd) fragment is secreted and linked to the Lc fragment with a disulfide bond; the other was 6D9 Fab2, in which the Fd fragment is displayed on cell surface and the Lc fragment is secreted and linked to the Fd fragment with a disulfide bond. Analysis by flow cytometry indicated that some 6D9 Fab2 fragments were unable to construct an appropriate conformation, and that most of the 6D9 Fab1 fragments displayed on yeast-cell surface exhibited higher binding affinity, stability, and catalytic activity. Conformation of the surface-displayed hetero-dimeric Fab fragment mainly depended on the intermolecular disulfide bond between the Lc and Fd fragments. The conformation of 6D9 Fab1 was more stable than that of Fab2. In the reducing environment of solution containing 25 nM DTT, the function of 6D9 Fab2 was almost completely lost. The successful display of 6D9 Fab1 on yeast-cell surface provides a novel approach to the engineering of catalytic antibodies.

Comparison of two forms of catalytic antibody displayed on yeast-cell surface

Two forms of the Fab fragment of the catalytic antibody 6D9 were individually displayed on yeast-cell surface in fusion to the C-terminal half of α-agglutinin: one was 6D9 Fab1, in which the light chain of the Fab (Lc) fragment is displayed on cell surface and the heavy chain of the Fab (Fd) fragment is secreted and linked to the Lc fragment with a disulfide bond; the other was 6D9 Fab2, in which the Fd fragment is displayed on cell surface and the Lc fragment is secreted and linked to the Fd fragment with a disulfide bond. Analysis by flow cytometry indicated that some 6D9 Fab2 fragments were unable to construct an appropriate conformation, and that most of the 6D9 Fab1 fragments displayed on yeast-cell surface exhibited higher binding affinity, stability, and catalytic activity. Conformation of the surface-displayed hetero-dimeric Fab fragment mainly depended on the intermolecular disulfide bond between the Lc and Fd fragments. The conformation of 6D9 Fab1 was more stable than that of Fab2. In the reducing environment of solution containing 25 nM DTT, the function of 6D9 Fab2 was almost completely lost. The successful display of 6D9 Fab1 on yeast-cell surface provides a novel approach to the engineering of catalytic antibodies.

Alteration of amino acid residues at the L-chain N-terminus and in complementarity-determining region 3 increases affinity of a recombinant F(ab) for the human N blood group antigen.

To examine the fine specificity of glycopeptide-specific antibodies, this study focused on the human MN blood group system. F(ab) phage display methods were previously used to construct an F(ab) family in which the H-chain Fd fragment was held constant whereas the L chains were "shuffled." This yielded two related F(ab), N92 and NNA7, with low and high affinity for N, respectively. Although their L-chain sequences are very similar, sharing 92 percent amino acid identity, there are intriguing differences at the N-terminus and in complementarity-determining region 3 (CDR3) at positions 89, 91, 92, and 96. Site-directed mutagenesis, ELISA, and hemagglutination were used to examine the contributions of these variations to antibody affinity. Studies with the N92-S91G and NNA7-G91S mutants demonstrated that the Gly at position 91 was critically important for ensuring high affinity. Indeed, the affinity of N92-S91G was almost as high as N92TM, in which all four CDR3 residues were changed to match NNA7. N-terminal L-chain differences were surprisingly important in determining affinity. For example, when the N-terminus of N92 was changed to match that of NNA7, affinity increased approximately 30-fold. Specific residues at the L-chain N-terminus and in CDR3 significantly affected F(ab) affinity for N. Future structural studies of these F(ab), alone and complexed with this glycopeptide antigen, will provide further insights into these phenomena.

The frequent mutation Gly/Asp in CDR1H may determine a cross-reactive idiotope in anti-I cold agglutinins.

Variable domains (VH) of all known anti i/I cold agglutinin (CA) heavy chains are codified by the VH4-21 gene. While anti-i CAs are the expression of gene rearrangement without mutations represented by amino acid changes, anti-1 CAs present, among others, a frequent somatic mutation of Gly by Asp at position 31. The hydropathy profile calculated for the CDR1H (position 30 to position 35), as well as some adjacent positions of the heavy chain belonging to anti-i and anti-I antibodies, showed the conformational changes accompanying the replacement of Gly by Asp. A MoAb (LP91), which had been obtained in BALB/c mice immunized with a Fabmu fragment from a monoclonal IgMkappaIIIb anti-I CA (protein KAU), proved capable of inhibiting human adult erythrocyte cryoagglutination by anti-I CAs but not that of fetal erythrocytes by anti-i CAs. Western blot analysis disclosed that such MoAbs recognized a sequential epitope located in the Fd fragment of all anti-I CAs employed in this study. With the purpose of checking whether Asp(31) was involved in the epitope recognized by the MoAb, two peptides, D and G, were synthesized which mimicked the CDR1H structure of anti-I and anti-i, respectively; the MoAb only reacted with peptide D by ELISA. Subsequent experimental results indicate that the Gly/Asp mutation could be associated with the diverse specificity presented by these autoantibodies, a change determining a characteristic epitope/idiotope, recognized by LP91 in the CDR1H.

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface.

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.

Construction of dimeric F(ab) useful in blood group serology.

When expressed in Escherichia coli, recombinant F(ab) contain a heavy-chain Fd fragment and a complete light-chain fragment. Because these F(ab) are monovalent, their avidity is significantly lower than that of a corresponding bivalent IgG antibody. In addition, when monovalent F(ab) are used in hemagglutination assays, antiglobulin reagents are required. Therefore, it would be useful to develop a system that expresses recombinant bivalent F(ab) in E. coli. Three modified vectors were constructed. Each contained cDNA sequences encoding a peptide linked to the C terminus of a heavy-chain CH1 region: an IgG1 hinge region (Hinge), a leucine zipper (Zip), or a peptide containing the Hinge and Zip sequences in tandem (HingeZip). The vectors were used to express two cloned F(ab) recognizing human antigens M and N: NNA7 (anti-N) and 425/2B (anti-M). The recombinant proteins were expressed in E. coli and were purified and evaluated by ELISA and hemagglutination. By gel filtration chromatography, 35, 90, and 70 percent of the purified F(ab) expressing the Hinge, Zip, and HingeZip tails, respectively, were dimers. By ELISA, the avidity of F(ab) containing the Zip or HingeZip tails was six to eight times higher than that of the corresponding monovalent F(ab). In addition, the dimeric F(ab) directly agglutinated RBCs in concentrations similar to those of corresponding bivalent IgG antibodies. An introduction of dimer-inducing peptides allowed the isolation of bacterially produced, bivalent F(ab). This approach could be useful for obtaining inexpensive, serologic reagents that may replace or complement conventional MoAbs produced by mammalian tissue culture methods.

Complete sequencing of anti-vancomycin fab fragment by liquid chromatography–electrospray ion trap mass spectrometry with a combination of database searching and manual interpretation of the MS/MS spectra

Sequencing of anti-vancomycin monoclonal antibody (mAb) Fab region (48,000 Da) was carried out using liquid chromatography–electrospray ionization ion trap mass spectrometry (LC/ESI-MS). Comprehensive strategies were employed to ensure complete sequence coverage. The sequence information was obtained from the spectra of collision-induced dissociation (CID) (MS/MS) of the protonated proteolytic peptides resulting from multiple enzymatic digestions of reduced/ S-carboxymethylated (RCM) light chain and Fd fragment. Database searching of the spectra against the published immunoglobulin G (IgG) sequences allowed the identification of all the peptides in constant domains as well as partial sequences in variable domains. The rest of the sequences were deduced by manual interpretation of the peptide tandem mass spectrometry (MS/MS) spectra. The analysis showed that the N-terminus of the heavy chain was modified by the conversion of a glutamine residue to pyroglutamic acid.

Sequencing of anti-thyroxine monoclonal antibody fab fragment by ion trap mass spectrometry.

A comprehensive mass spectrometric strategy is described for the sequencing of anti-thyroxine monoclonal antibody Fab region (48 000 Da). After reduction and S-carboxymethylation of the Fab, the modified light chain and Fd fragment were separated and subjected to multiple proteolytic digestions. The resulting digests were characterized by on-line microbore liquid chromatography/electrospray ionization ion trap mass spectrometry. Database search against published immunoglobulins (IgGs) allowed identification of all the peptides in constant domains. The homologous framework residues in the IgGs were utilized as 'sequence maps' for the sequence determination of variable domains. S-Carboxymethylation with an isotopic-enriched moiety greatly facilitated the recognition and data elucidation of cysteinyl peptides through the unique isotopic distribution patterns specific to the modified peptides. Methylation of peptide mixtures provided additional information for the interpretation of MS/MS spectra, allowing easy differentiation of Asp/Asn and Gln/Glu pairs. This study clearly demonstrates the power of mass spectrometry for the sequencing of antibodies without knowing the corresponding DNA sequences.

Sequencing of anti‐thyroxine monoclonal antibody Fab fragment by ion trap mass spectrometry

A comprehensive mass spectrometric strategy is described for the sequencing of anti-thyroxine monoclonal antibody Fab region (48 000 Da). After reduction and S-carboxymethylation of the Fab, the modified light chain and Fd fragment were separated and subjected to multiple proteolytic digestions. The resulting digests were characterized by on-line microbore liquid chromatography/electrospray ionization ion trap mass spectrometry. Database search against published immunoglobulins (IgGs) allowed identification of all the peptides in constant domains. The homologous framework residues in the IgGs were utilized as ‘sequence maps’ for the sequence determination of variable domains. S-Carboxymethylation with an isotopic-enriched moiety greatly facilitated the recognition and data elucidation of cysteinyl peptides through the unique isotopic distribution patterns specific to the modified peptides. Methylation of peptide mixtures provided additional information for the interpretation of MS/MS spectra, allowing easy differentiation of Asp/Asn and Gln/Glu pairs. This study clearly demonstrates the power of mass spectrometry for the sequencing of antibodies without knowing the corresponding DNA sequences. Copyright © 2000 John Wiley & Sons, Ltd.

Tritrichomonas foetus extracellular cysteine proteinase cleavage of bovine IgG2 allotypes.

Bovine trichomoniasis is a sexually transmitted disease associated with reproductive failure. Systemic immunization results in protective IgG antibodies in uterine and vaginal secretions. Because bovine IgG2 is a better opsonin than IgG1, it is potentially important in defense. Yet, Tritrichomonas foetus extracellular cysteine proteinase (TFECP) cleaves bovine IgG2, evading protective IgG2 responses. Variations in resistance of the 2 IgG2 allotypes to digestion may explain inherited differences in protection. To address this hypothesis, TFECP was incubated with both IgG2 allotypes at different concentrations and times. The digestion products were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained, and quantitated by image analysis. IgG2a was digested faster by TFECP than IgG2b. Differences in the sizes and numbers of digestion products were observed, but the presence of bands the size of Fc and Fd fragments indicated that both allotypes were cleaved at the hinge. Cysteine in the digestion mixture reduced the antibody molecules and increased the rate of digestion, but IgG2a was still more susceptible to cleavage than IgG2b in the absence of cysteine. Thus, not only reduced H chains can be cleaved by cysteine proteinase secreted by T. foetus but also intact functional antibody molecules. Because parasites may evade protective antibody responses by cleaving IgG2, animals with the more resistant IgG2b allotype may be better protected by immunization than animals with the more readily digested IgG2a allotype.

Light Chain of Natural Antibody Plays a Dominant Role in Protein Antigen Binding

Examinations of the contribution and the specificity of heavy (H) and light (L) chains of natural antibodies to antigen binding may help us to better understand antigen recognition and the development of naive B cells. We previously generated natural Fab antibody fragments reactive to preS1 of HBV using a naive, non-immunized Fab antibody library derived from peripheral B cells of a normal healthy volunteer. We now constructed expression vectors for the Fd (VH + CH1), L chain, and scFv fragments using the sequences encoding parental Fabs as a source of natural antibody genes. The recombinant antibody fragments were expressed as inclusion bodies in E. coli BL21 (DE) cells. When denatured and then refolded, the antibody fragments retained their binding properties. Recombinant L chains and scFvs exhibited three- to 40-fold higher affinities (in the order of 107 M−1) over the parental Fabs, whereas the affinities of Fds (in the order of 105 M−1) were much lower compared to the parental Fabs. The results obtained from sandwich ELISA revealed that the L chains bound the virus more efficiently than Fds. Additional experiments were performed to evaluate the specificity of the recombinant fragments for surface proteins of HBV. Fds and L chains were reactive towards HBsAg and the preS2 peptide as well as preS1 and showed patterns of epitope recognition quite different from those of parental Fabs. The data presented here demonstrate that the prominence of the L chain in determining protein binding activity is a property of natural antibodies and is quite unlike the antibodies induced by immunization, and that the specificity of Fab is not determined by the individual antibody chain but by the correct pairing of H and L chain.

Inhibition of cytoplasmic antigen, glucose-6-phosphate dehydrogenase, by VH-CH1, an intracellular Fd fragment antibody derived from a semisynthetic Fd fragment phage display library

A library of Fd fragment antibody binding proteins was created by random mutation of 15 nucleotides within the CDRIII region of the immunoglobulin heavy chain gene and displayed as Fd coat protein fusion constructs of M13 phage. The library was screened for those VH binding sites that bound glucose-6-phosphate dehydrogenase (G6PD). One isolate (DH27bp) inhibited G6PD activity by 85 %. The DH27 bp gene was re-engineered, placed in a eukaryotic expression vector having an isopropyl-β-δ-thiogalactopyranoside (IPTG) inducible promoter, and transfected and then expressed in Chinese hamster V79 cells. G6PD activity was completely inhibited. Removal of IPTG reverted the cell to full G6PD activity. The intracellular dynamics of the G6PD/DH27bp complex showed that when the proteasomes of cells expressing DH27 bp were inhibited (N-acetyl-Leu-Leu-norleucinal or lactacystin) G6PD activity increased. Metabolic labelling of newly synthesized IPTG-induced proteins during/absence of proteasomal inhibitors showed that both G6PD and DH27bp are signaled for degradation when the intracellular complex is formed. Furthermore, semi-quantitative RT/PCR demonstrated that G6PD mRNA is upregulated over the time course of G6PD inactivation by DH27 bpFd binding protein. These effects were not observed in those cells expressing a non-mutated Fd (UMHC) or in IPTG-treated non-transduced V79 cells.Our results demonstrate that an Fd-based intracellular binding protein can find and disable the function of a specific intracellular target and once the Fd expression is repressed the activity of intracellular targeted protein can revert to normal.

A molecular approach for isolating high-affinity Fab fragments that are useful in blood group serology.

Multiple mouse hybridoma antibodies recognize the antigens of the MNS blood group system. The Fab fragments of several of these antibodies were expressed on bacteriophage and as soluble proteins. The parental N92 anti-N IgG monoclonal antibody (parental N92 MoAb), but not its monovalent, soluble Fab fragment (N92 Fab fragment), agglutinated antigen-positive red cells by an antiglobulin method. Light-chain shuffling was used to isolate mutant N92 Fab fragments with higher affinity that would function by agglutination. Light-chain cDNA libraries, constructed from mice immunized with N-type glycophorin A, were inserted into a recombinant pComb3H vector containing the N92 Fd fragment. The N92 Fd fragment:light-chain libraries were panned on N-type glycophorin A or NN red cells, and antigen-binding clones were isolated. Purified parental N92 MoAb and the Fab fragments were evaluated by enzyme-linked immunosorbent assay and agglutination. The novel NNA7, C1, and G11 Fab fragments all bound to N-type glycophorin A with higher affinity than did the N92 Fab fragment. The affinity of the library-derived clones was equivalent to that of the parental N92 MoAb. Although their fine specificity differed slightly from the parental N92 MoAb, the clones functioned equivalently by agglutination using an antiglobulin method. Light-chain shuffling allowed the isolation of bacterially produced, high-affinity, soluble, monovalent recombinant anti-N Fab fragments that functioned well by agglutination. This approach is useful in obtaining inexpensive serologic reagents that may replace conventional MoAbs produced by tissue culture methods.

Paradoxical behavior of asymmetric IgG antibodies.

Changes in the quantity and quality of antibodies occur in the course of an immune response. This review describes the physicochemical and biological properties of asymmetric antibodies as well as their functions, beneficial or harmful to the host, according to the nature of the antigen and the particular situation in which they act. Asymmetric antibodies have two paratopes, one of high affinity, with K0 similar to that of symmetric antibodies, and the other one with an affinity for the antigen 100 times lower. Functional univalence is due to steric hindrance present in one of the paratopes by the carbohydrate moiety attached to the Fd fragment of the Fab region, so these antibodies are unable to form large antibody-antigen complexes and cannot trigger reactions, such as complement fixation, phagocytic activity and antigen clearance. When asymmetric IgG antibodies are specific for self-antigens, they prove beneficial for the host by exerting regulatory functions. In allergic manifestations, in autoimmune diseases and especially during pregnancy, despite the fact that the antigens responsible for the process are foreign to the host, they also perform beneficial activity. During pregnancy, the placenta secretes molecules or factors that regulate the synthesis of these antibodies, thus favoring fetal protection.

Demonstration of thyroid stimulating activity within H chain fragments of TSAb-IgG by protease digestion and reduction.

Whether or not the distribution of biologically active fragments in TSAb-IgG molecules parallels antigen-binding activity in other anti-thyroidal antibodies was examined. Both the thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) were examined by measuring the reduction in TSAb-IgG followed by gel-filtration on Sephadex G-100. Two forms of IgG [IgG(kappa) and IgG(lambda)] were separated from TSAb-IgG by column chromatography using Protein L-Sepharose (specifically binds to kappa chain). The IgG(kappa) was reduced with dithiothreitol (DTT) and the unbound fraction (UF) (the free heavy (H) chain) and the bound fraction (BF) (the free kappa chain and the non-reduced IgG(kappa)) were separated using Protein L-Sepharose. The Fab fragment was separated by Protein A-Sepharose after papain hydrolysis of TSAb-IgG, then separated into two Fab(kappa) and Fab(lambda) forms by Protein L-Sepharose. The Fd fragment (or fragment containing Fd) was prepared from Fab(kappa) by DTT reduction followed by Protein L column. The free H chain fraction showed TS and TBI activity, but neither anti-thyroglobulin (Tg) nor anti-thyroid peroxidase (TPO) antibody activities. The free light (L) chain was not biologically active. Similar TS and TBI activities were found not only in IgG(kappa) and IgG(lambda) but also in the Fab(kappa) and Fab(lambda) fractions. Fd fragment that was not contaminated with free kappa chain had TS and weak TBI activities. The thyroid stimulating activity in 5 TSAb-IgG samples was found in IgG(kappa), IgG(lambda), Fab(kappa), Fab(lambda), H chain and Fd separated by papain digestion and reduction. These results showed that TSAb was polyclonal and that the Fd fragment was important as the biologically active site.

Cytolytic and cytostatic properties of an anti-human Fc gammaRI (CD64) x epidermal growth factor bispecific fusion protein.

A bispecific fusion protein (H22-EGF) that binds simultaneously to the epidermal growth factor receptor (EGF-R) and to the high affinity receptor for the Fc portion of human IgG, Fc gammaRI (CD64), has been successfully constructed and expressed. For this construction, genomic DNA encoding the Fd fragment of humanized anti-Fc gammaRI mAb, H22, which binds Fc gammaRI at an epitope that is distinct from the Fc binding site, was fused to cDNA encoding human epidermal growth factor (EGF), a natural ligand for EGF-R. The resulting H22Fd-EGF-expressing vector was transfected into a myeloma cell line that was transfected previously with a vector containing DNA encoding the H22 kappa-light chain. SDS-PAGE analysis of purified H22-EGF demonstrated that the fusion protein was secreted predominantly as H22Fab'-EGF monomer (approximately 55 kDa), even though a free Cys residue exists in the hinge region of the H22 Fab' component. Using a novel bispecific flow cytometry-binding assay, we demonstrated that the purified bispecific fusion protein, H22-EGF, was able to bind simultaneously to soluble Fc gammaRI and EGF-R-expressing cells. H22-EGF inhibited the growth of EGF-R-overexpressing tumor cells and mediated dose-dependent cytotoxicity of these cells in the presence of Fc gammaRI-bearing cytotoxic effector cells. These results suggest that this fusion protein may have therapeutic utility for EGF-R-overexpressing malignancies.

Separation method of IgG fragments using protein L

Protein L (IgG K-chain-binding bacterial protein) showed a precipitate line (pseudo-immuno-reaction) with IgG and F(ab')2 fragment, but did not show any line with the Fab fragment, the Fc fragment and free k-chains in the micro-Ouchterlony method. The IgG and Fab fraction obtained from pa-pain-digested IgG (from the sera of patients with chronic thyroiditis), followed by Protein A-Sepharose, were separated by Protein L-c affinity chromatography. The unbound fraction (UF) consisted of IgG(λ) or Fab(λ) and the bound fraction (BF) consisted of IgG(κ) or Fab(κ) were obtained. Anti-thyroglobulin and anti-thyroid peroxidase antibody activities were found equally in both the UF and the BF. When Fab(κ) was reduced with dithiothreitol (DTT), the Fd fragment in the UF could be separated from the free κ-chain and the unreduced Fab(κ) in the BF with a Protein L-Sepharose column. A separation method of human IgG fragments such as free κ-chain, combined forms of κ-chain [Fab or F(ab')2], and the Fd region, using Protein L, is described.

Recombinant human Fab antibody fragments to HIV-1 Rev and Tat regulatory proteins: Direct selection from a combinatorial phage display library

A human Fab phage display library has been produced from peripheral blood lymphocytes of an individual who was asymptomatic after 10 years of infection with human immunodeficiency virus type-1 (HIV-1). The library was panned against the HIV-1 Rev and Tat regulatory proteins and several clones, producing Fab binding to these proteins, were isolated (3 to Rev and 4 to Tat) with binding constants varying from 10 −6M to 10 −8M. DNA sequencing demonstrated two unique anti-Rev Fab clones, but the four anti-Tat Fab comprised only two unique IgG 1 heavy chain Fd fragments, illustrating redundancy of light chains. Peptide mapping of the epitopes recognized by these Fab indicated that three of the anti-Tat Fab were directed to the functional domain between amino acid residues 22–33 of the Tat molecule, and that binding was inhibited by reduction of this cysteine-rich region with dithiothreitol. The anti-Rev Fab were directed to sites adjacent to the Rev basic nucleolar localization sequence (residues 52–64) and to the Rev activation domain (residues 75–88). Binding constants were of a similar order to that of an anti-Rev single-chain Fv fragment (SFv) used successfully for intracellular immunization, and as such intracellular effects with the human anti-Tat and anti-Rev Fab are not precluded. These newly described human antibody fragments to HIV-1 regulatory proteins may be critical moieties for gene therapeutic protocols, to control HIV-1 replication in human cells.

Evidence for distinct contributions of heavy and light chains to restriction of antibody recognition of the HIV-1 principal neutralization determinant.

We have used phage Ab display technology to analyze two mAbs to HIV-1 envelope proteins gp120 and gp41. From the data obtained we are able to demonstrate that the recognition of the principal neutralization determinant of different strains of HIV-1 by neutralizing mAb M77 is restricted by its heavy and light chains in different ways. Native M77 is able to recognize and neutralize HIV-1 strain IIIB through binding to the gp120 V3 loop. M77 is unable to recognize strains of HIV-1 that differ on either the left or right side of the V3 loop tip. A chain-switched Fab fragment containing the M77 Fd fragment and a different light chain was able to recognize HIV-1 strains that differ from IIIB on the left side but not the right side of the V3 loop tip.

V-region and class specific RT-PCR amplification of human immunoglobulin heavy and light chain genes from B-cell lines.

We have designed and tested primers that amplify complete human kappa and lambda light chain genes, and human Fd fragments from gamma, mu and alpha heavy chain genes. These primers were tested for efficiency and specificity on monoclonal sources of human immunoglobulin RNA, obtained from human B-cell lines of known immunoglobulin gene expression. Analysis of the sequences derived from these B-cells confirms the specificity of the PCR primers and the extent of somatic mutation seen in different B-cell malignancies supports existing concepts for differing aetiologies in the tumours concerned.