IgG Repertoire Research Articles

VH gene usage and CDR3 analysis of B cell receptor in the peripheral blood of patients with PBC

We have analyzed the IgM, IgG and IgA BCR repertoire in PBC patients by means of quantitative RT-PCR and CDR3-spectratyping with immunoscope technology. PBMC from 35 PBC patients and 18 normal controls were analyzed. Quantitative B cell repertoire analysis of IgM from healthy donors showed the preferential usage of VH3a, VH3b and VH4 families. Very similar VH family usage was observed in IgM B cells from PBC patients. CDR3 spectratyping of IgM BCR rearrangements showed a Gaussian distribution for dominant VH families in control donors, and similar diversity was found for the VH3b family in PBC patients. In contrast, VH3a and VH4 families showed oligoclonal expansions in some patients. Quantitative B cell repertoire analysis of IgG and IgA did not reveal any difference in VH chain distribution in PBC patients as compared to the control donors. Immunoscope profiles of CDR3 length distribution showed several peak expansions in B cells from control donors, particularly for the VH3a and VH4 families. CDR3 length distribution profiles of IgG and IgA from PBC patients were oligoclonal too, with expansions throughout the various VH chains. However, no common expansions within the CDR3 region were found intraindividually between IgG, IgA and IgM, and between patients. In conclusion, immunoscope technology does provide, for the first time, a sensitive and rapid method for detailed immunoglobulin gene usage analysis in peripheral B cells from PBC patients. This study failed to demonstrate preferential B cell rearrangements in the blood of patients with PBC, but this technology may be more successful if applied to the analysis of compartmental B cells (i.e. liver infiltrating B cells).

The IgE repertoire in PBMCs of atopic patients is characterized by individual rearrangements without variable region of the heavy immunoglobulin chain bias

Patients with atopic diseases are characterized by high levels of specific IgE production. However, little is known about the composition of their B-cell repertoires. We sought to analyze the complete PBMC-derived IgE repertoire and to compare clonal expansions between different patients. We have analyzed the IgE-bearing B-cell receptor repertoire in highly atopic patients (>1000 IU/mL) using quantitative RT-PCR, complementarity determining region 3 spectratyping, and sequence analysis. Three representative patients were additionally followed during anti-IgE therapy. Atopic patients exhibited 100 to 1000 times more IgE-specific transcripts than control individuals. These patients used a variable region of the heavy immunoglobulin chain (VH) epsilon repertoire highly similar to their IgM and IgG repertoires, with preference of VH3b, VH4, VH3a, and VH1 segments. Each patient harbored individual clonal expansions, most probably as correlation of allergen-specific IgE production. Common expansions within the complementary determining region 3 shared by several individuals with similar sensitization patterns were found in spectratyping analysis. However, these antigen-driven expansions showed differences on the sequence level. In omalizumab-treated patients the clinical improvement was paralleled by a clear increase in the ratio of IgG/IgE transcripts. The IgE repertoire in atopic patients follows the VH use patterns seen for other immunoglobulins and seems to preferentially recruit individual rearrangements rather than public expansions. The detailed analysis of the IgE B-cell repertoire is highly suitable to follow changes in IgE uses during different therapy modalities.

The Postnatal Maturation of the Immunoglobulin Heavy Chain IgG Repertoire in Human Preterm Neonates Is Slower than in Term Neonates

During the perinatal period the development of the IgH chain CDR3 (CDR-H3) repertoire of IgM transcripts is maturity-dependent and not influenced by premature exposure to Ag. To study whether maturity-dependent restrictions also predominate in the perinatal IgG repertoire we compared 1000 IgG transcripts from cord blood and venous blood of extremely preterm neonates (24-28 wk of gestation) and of term neonates from birth until early infancy with those of adults. We found the following. First, premature contact with the extrauterine environment induced the premature development of an IgG repertoire. However after preterm birth the diversification of the IgG repertoire was slower than that after term birth. Second, the IgG repertoire of preterm neonates retained immature characteristics such as short CDR-H3 regions and overrepresentation of D(H)7-27. Third, despite premature exposure to the extrauterine environment, somatic mutation frequency in IgG transcripts of preterm infants remained low until they reached a postconceptional age corresponding to the end of term gestation. Thereafter, somatic mutations accumulated with age at similar rates in preterm and term neonates and reached 30% of the adult level after 6 mo. In conclusion, class switch was inducible already at the beginning of the third trimester of gestation, but the developing IgG repertoire was characterized by similar restrictions as those of the developing IgM repertoire. Those B cells expressing more "mature" H chain sequences were not preferentially selected into the IgG repertoire. Therefore, the postnatal IgG repertoire of preterm infants until the expected date of delivery differs from the postnatal repertoire of term neonates.

Antibody Repertoire Development in Fetal and Neonatal Piglets. XIII. Hybrid VH Genes and the Preimmune Repertoire Revisited

The expressed porcine VH genes belong to the VH3 family (clan), four of which, VHA, VHB, VHC, and VHE, alone comprise approximately 80% of the preimmune repertoire. However, so-called "hybrid" VH genes that use CDR1 of one VH gene and the CDR2 of another are frequently encountered. We studied > 3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (VHZ and VHY), together with VHF and the four genes mentioned above, constitute the major VH genes and these account for > 95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major VH genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of VH repertoire diversification and 2) establish a library of 29 putative porcine VH genes. One-third of these genes are chimeric genes and their sequences suggest that the porcine VH genome developed by duplication and splicing from a small number of prototypic genes.

Autoantibodies against alpha B-crystallin, a candidate autoantigen in multiple sclerosis, are part of a normal human immune repertoire

Human T-cell responses to the stress protein alpha B-crystallin in multiple sclerosis (MS)-affected brain samples are dominant when compared to other myelin antigens. The establishment of the apparent autoimmune repertoire against this antigen has been suggested to involve cross-priming during viral infection. Yet, another possibility would be that determinant spreading during ocular inflammation could generate a response to alpha B-crystallin, since it is also a major component of the eye. In this study, we compared serum IgG, IgA and IgM repertoires against a range of eye lens-derived ocular antigens using sera from healthy control subjects and MS patients with or without uveitis. This comparison revealed that among ocular antigens, alpha B-crystallin is the dominant target antigen for serum autoantibodies in both MS patients and healthy controls. Uveitis generally did not affect the antibody reactivity profile. These data provide further support for the notion that a normal adult human immune system is selectively reactive to alpha B-crystallin and they indicate that this responsiveness is unlikely to result from determinant spreading following ocular inflammation.

23 The Repertoire of Igg Expressing B-Cells Develops Much Slower in Preterm Than in Term Infants

Background: Even very immature preterm infants in the first weeks of life develop a repertoire of B cells with IgG-type antigen receptors (1). Yet, the different kinetics of the postnatal evolution of B effector cells in preterm an term neonates are unknown. Hypothesis: The variable (V) region gene diversity of the IgG repertoire and the somatic diversity increases slower in preterm than in term neonates. Study design: Crossover observational study. Methods: Heavy chain V-region genes coding for the antigen binding site of the IgG-type B-cell receptor were amplified, cloned and sequenced. The diversity of the V-region gene repertoire was determined from the percentage of different amplificates in the total number of amplificates. In addition, the number of somatic mutations per V-region gene was calculated. Results: We analyzed peripheral blood samples of 8 preterm infants (GA 24–27 weeks, postnatal age 0–14 weeks) and of 16 term infants (postnatal age 0–16 weeks). In preterm infants V-region gene diversity increased slowly with postnatal age (r=0.82; p<0.000) from 30% at birth to 47% at 4 weeks, and to 82% at 12 weeks. In contrast, in term neonates already at two weeks of age diversity was higher than 80%. Furthermore the frequency of somatic mutations per V-region gene increased faster in preterm (r=0.712; p<0.004) than in term infants (r=0.83; p<0.000). Per postnatal week the accumulation of somatic mutations was three times higher in term than in preterm infants (0.12% per week vs 0.04% per week). Conclusion: In the first weeks of life both preterm and term infants respond to environmental antigens. However, preterm infants are much slower in developing an IgG repertoire. (1) Bauer K et al. Diversification of Ig heavy chain genes in human preterm neonates prematurely exposed to environmental antigens. J Immunol 2002;169:1349–1356

Generation and selection of an IgG-driven autoimmune repertoire during B-lymphopoiesis in Igmicro-deficient/lpr mice.

Class switch recombination (CSR) is a well-regulated process that occurs in peripheral lymphoid tissue, and is thought of as an important factor constructing the memory repertoire. We have recently shown that CSR normally occurs during bone marrow (BM) development, and these isotype-switched B cells are negatively selected by Fas signaling. This novel pathway of B cell development may generate a primary repertoire driven by gamma-heavy receptors, the nature of which is yet unknown. To study this gammaH-driven repertoire we used mice lacking IgM-transmembrane tail exon ( micro MT), where B cell development is limited by their ability to undergo CSR. We already showed that lack of Fas signaling rescues development of a significant population of isotype-switched B cells and production of high titers of non-IgM serum antibodies in micro MT mice deficient in Fas ( micro MT/lpr), thereby providing a mouse model allowing the assessment of gammaH-driven repertoire. Using a tissue array and phage display epitope library we report here that IgG repertoire in micro MT/lpr mice is oligo-monoclonal, bearing self-tissue reactivity. This is supported by analysis of the Vkappa utilization in peripheral B cells from micro MT/lpr mice, which revealed a strikingly restricted repertoire. In contrast, micro MT/lpr B cells that are grown in non-selective BM cultures utilize a wide repertoire. These results suggest that the Fas pathway is an important regulator in the generation and selection of an autoimmune gammaH-driven repertoire in vivo.

Single-cell repertoire analysis demonstrates that clonal expansion is a prominent feature of the B cell response in multiple sclerosis cerebrospinal fluid.

Single-cell RT-PCR was used to sample CD19(+) B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominantly polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for approximately 70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.

Autoreactive Antibody Repertoire Is Perturbed in Atherosclerotic Patients

In patients with clinical symptoms of coronary atherosclerosis, T cells are activated and directed to autologous proteins contained in the active plaques, suggesting that autoimmune responses may play a role in atherosclerosis progression. Organ-specific autoimmune diseases are sometimes accompanied by broad alterations of serum autoreactive antibody repertoires. We thus investigated antibody repertoires at a global level, using a technique of immunoblotting that allows for the quantitative screening of antibody reactivities in complex antibody mixtures toward a large panel of antigens derived from homologous tissue extracts, followed by multiparametric statistical analysis of the data. We analyzed the autoreactive IgG repertoire in 20 patients with documented coronary atherosclerosis and in 20 matched healthy controls. Total proteins from atherosclerotic carotid specimens and normal arterial tissues (target organs) and from kidney, liver, and stomach (non-target control organs) were used as panels of antigens. Patients had a significantly perturbed antibody repertoire and an enhanced autoreactivity of IgG to target and non-target organs, as compared with controls. Reactivity of purified IgG to plaque and normal artery proteins was greater in patients, but reactivity of IgG in the whole serum toward normal arterial tissue was lower than in controls; this suggests that, in patients, autoreactivity toward normal arteries is regulated by serum factors. Our data indicate that atherosclerotic patients develop a perturbed humoral immune response directed toward arterial proteins, which impacts on the overall autoreactive repertoire. These findings further substantiate that autoimmune processes take place in atherosclerosis and most likely influence disease progression.

Diversification of Ig heavy chain genes in human preterm neonates prematurely exposed to environmental antigens.

Preterm neonates are exposed to extrauterine environmental Ags during the time period that corresponds to the last trimester of normal intrauterine development. To study whether this precocious exposure to Ags accelerates the Ig repertoire diversification, we compared IgH chain genes of preterm neonates (gestational age, 25-29 wk) during their first postnatal months with those of term neonates. Preterm infants approaching their expected date of delivery after 8-13 wk of extrauterine life used a similar V(H), D(H), and J(H) gene segment repertoire as term neonates born after intrauterine development. Furthermore, the length increase of the NDN region between V(H) and J(H) by 0.25 nt per gestational week (r = 0.556, p < 0.0001) was not accelerated. Thus, the generation of the V(H) region gene repertoire is developmentally controlled and independent of environmental influences. However, exposure to extrauterine Ags induced class switch and somatic mutations in IgH chain genes within 2 wk after premature birth and IgG transcript diversity and mutational frequency increased with the duration of extrauterine life. Three-month-old preterm infants expressed a heterogeneous IgG repertoire at their expected date of delivery with V(H) region genes carrying significant numbers of somatic mutations with evidence for Ag selection. Term neonates, however, had no such IgG repertoire. We conclude that restrictions in the neonatal Ig V(H) region gene repertoire persist until term despite exposure to environmental Ags. Yet, many weeks before term the immune system of the preterm neonate can already support germinal center reactions in response to environmental Ags.

Long-term persistence of oligoclonal serum IgM repertoires in patients treated with allogeneic bone marrow transplantation (BMT).

Immunoglobulin gene rearrangements in patients treated with BMT have restricted repertoire diversity. Clonal variability remains low for 3 months and reconstitution of the humoral immune system appears to follow a wave-like pattern. In the present study we analysed serum IgM and IgG repertoires in 44 patients from 1 week to 3 years after transplantation. We applied a quantitative immunoblot technique in combination with a newly developed method for estimation of repertoire diversity in complex mixtures of antibodies. Our results demonstrate that 60% of BMT patients have severely reduced diversity in the IgM repertoire during and after the first year post-BMT, compared with healthy controls. In contrast, the majority of patients have a polyclonal IgG repertoire, similar to that of healthy controls. Serum IgM repertoires remain oligoclonal even though the serum concentration of total IgM is within normal range around 6 months post-BMT. During the first years after transplantation IgM as well as IgG repertoires are less diverse in patients receiving a BM graft from a sibling donor compared with those receiving a graft from an HLA-matched unrelated donor. Patients in the latter group show a higher incidence of infections and minor antigen mismatches which may promote the development of a diverse immunoglobulin repertoire post-BMT.

Isolation and functional characterization of recombinant GAD65 autoantibodies derived by IgG repertoire cloning from patients with type 1 diabetes.

The generation of human monoclonal autoantibodies is critical for understanding humoral immune responses in autoimmunity. In this study, we isolated the first human recombinant antibodies to glutamate decarboxylase (rGAD65ab) by IgG repertoire cloning, phage display of Fab fragments, and biopanning from two patients at onset of type 1 diabetes. We demonstrate that natural Ig heavy- and light-chain pairings of autoantibodies can be isolated by the recombinant approach and have a major selection advantage over other rGAD65ab. Among eight rGAD65ab, three (rGAD65ab A-C) displayed all functional and structural properties of known disease-related GAD65ab, including reactivity in the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), islet cell antibody (ICA) test, and variable gene usage. Dominant epitope recognition was directed to the previously defined epitope EP-1 in the middle of GAD65, corroborating its immunodominance in the molecule. New features, such as assay-dependent GAD65 reactivity and new epitope recognition, were observed in two rGAD65ab (D and E). These antibodies were positive in the GAD65 ELISA and ICA test but not in the GAD65 RIA, providing the first examples for ICA with incongruent results in solid-phase and fluid-phase assays. In conclusion, phage display-derived antibodies reflected well the natural autoantibody response in type 1 diabetes and may allow further characterization of assay-dependent features of GAD65ab and the recognition of epitopes in solid- but not fluid-phase assays.

Striatal antibodies in children with Tourette's syndrome: multivariate discriminant analysis of IgG repertoires

Antineuronal antibodies have been postulated to be the underlying pathophysiology in TS and other neuropsychiatric disorders. Serum antibodies from 20 children with TS, and 21 control subjects against human striatum, globus pallidus, muscle, and HTB-10 cells were assayed by Western blot techniques. A MANOVA differentiated between TS and control blots, and a discriminant analysis demonstrated which variables contributed most to differences between groups. Prominent differences between TS and control blots were identified using striatal epitopes in contrast to similar patterns shown between groups for globus pallidus, muscle and HTB-10 tissue, supporting striatal autoimmune involvement in TS pathophysiology.

Intracellular Stability of Anti-caspase-3 Intrabodies Determines Efficacy in Retargeting the Antigen

Although intracellular antibodies (intrabodies) are being explored as putative therapeutic and research reagents, little is known about the principles that dictate the efficacy of these molecules. In our efforts to address this issue, we generated a panel of five intrabodies, directed against catalytically inactive murine caspase-3, by screening single-chain antibody (Fv) phage display libraries. Here we determined criteria that single-chain Fv fragments must fulfill to act as efficient intrabodies. The affinities of these intrabodies, as measured by surface plasmon resonance, varied approximately 5-fold (50-250 nm). Despite their substantial sequence similarity, only two of the five intrabodies were able to significantly accumulate intracellularly. These disparities in intracellular expression levels were reflected by differences in the stability of the purified protein species when analyzed by urea denaturation studies. We observed varied efficiencies in retargeting the antigen murine caspase-3, from the cytosol to the nucleus, mediated by intrabodies tagged with an SV40 nuclear localization signal. Our results demonstrate that the intrinsic stability of the intrabody, rather than its affinity for the antigen, dictates its intracellular efficacy.

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama

Functional heavy chain immunoglobulins have, so far, only been found in camels and llamas. Antigen-specific fragments of these heavy chain IgGs (V HH) are of great interest in biotechnology because they are very stable and can be produced at high level by the yeast Saccharomyces cerevisiae. The work described in this paper was conducted to determine whether llamas ( Lama glama) are a practical source of antigen-specific V HH fragments. Llamas were immunised with various types of antigens and the antibody responses were examined during the course of immunisation. Both, conventional and heavy chain IgG antibodies were produced in response to each of the antigens. The heavy chain IgG repertoire displayed a recognition pattern different to that of conventional llama IgGs, resulting in the expansion of the accessible epitope repertoire. Llamas have a lower proportion of heavy chain IgG antibodies in their serum than have camels. To enable the specific and efficient isolation of V HH genes from peripheral blood B-cells, the long and short-hinge sequences of Lama glama heavy chain IgGs were determined, revealing the presence of a novel subclass of short-hinge heavy chain IgG. Long and short-hinge specific PCR primers were designed to be used in the construction of llama V HH libraries. We conclude that, using the techniques described, antigen-specific V HH antibody fragments are readily accessible from the llama, thus providing highly valuable binding molecules for a variety of applications.

Altered control of self-reactive IgG by autologous IgM in patients with warm autoimmune hemolytic anemia

Warm autoimmune hemolytic anemia (WAIHA) is characterized by an accelerated clearance of red blood cells (RBCs) associated with the presence of anti-RBC immunoglobulin (Ig)G autoantibodies. In the present study, we analyzed the self-reactive IgG and IgM antibody repertoires of patients with WAIHA using a technique of quantitative immunoblotting on a panel of whole tissue extracts as sources of self-antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that self-reactive antibody repertoires of IgG purified from plasma and of IgG purified from RBC eluates do not differ between healthy donors and patients with WAIHA, whereas autoreactive repertoires of IgM from patients exhibit broadly altered patterns of reactivity as compared with those of healthy controls. We further demonstrate that IgG purified from eluates of RBCs of healthy donors induces agglutination of RBCs in an indirect Coombs assay to a similar extent as IgG purified from eluates of RBCs of patients with WAIHA. The capability of IgG to induce agglutination of RBCs is suppressed in unfractionated eluates of healthy donors' cells, whereas it is readily found in unfractionated eluates of patients' RBCs. IgM is an essential factor in controlling the ability of IgG in unfractionated RBC eluates to induce agglutination of RBCs. These observations indicate that anti-RBC IgG autoantibodies of patients with WAIHA share extensive similarity with natural antiRBC autoantibodies of healthy donors and suggest that defective control of IgG autoreactivity by autologous IgM is an underlying mechanism for autoimmune hemolysis in WAIHA. (Blood. 2000;95:328-335)

Altered control of self-reactive IgG by autologous IgM in patients with warm autoimmune hemolytic anemia

AbstractWarm autoimmune hemolytic anemia (WAIHA) is characterized by an accelerated clearance of red blood cells (RBCs) associated with the presence of anti-RBC immunoglobulin (Ig)G autoantibodies. In the present study, we analyzed the self-reactive IgG and IgM antibody repertoires of patients with WAIHA using a technique of quantitative immunoblotting on a panel of whole tissue extracts as sources of self-antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that self-reactive antibody repertoires of IgG purified from plasma and of IgG purified from RBC eluates do not differ between healthy donors and patients with WAIHA, whereas autoreactive repertoires of IgM from patients exhibit broadly altered patterns of reactivity as compared with those of healthy controls. We further demonstrate that IgG purified from eluates of RBCs of healthy donors induces agglutination of RBCs in an indirect Coombs assay to a similar extent as IgG purified from eluates of RBCs of patients with WAIHA. The capability of IgG to induce agglutination of RBCs is suppressed in unfractionated eluates of healthy donors' cells, whereas it is readily found in unfractionated eluates of patients' RBCs. IgM is an essential factor in controlling the ability of IgG in unfractionated RBC eluates to induce agglutination of RBCs. These observations indicate that anti-RBC IgG autoantibodies of patients with WAIHA share extensive similarity with natural antiRBC autoantibodies of healthy donors and suggest that defective control of IgG autoreactivity by autologous IgM is an underlying mechanism for autoimmune hemolysis in WAIHA. (Blood. 2000;95:328-335)

Generation of a large complex antibody library from multiple donors

We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1–5×10 7 independent clones giving a final combined library of 4×10 9 members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.