Heterohybridoma Cell Lines Research Articles

Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1.

Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314-330 (EP3) covering the central conserved sequence of this domain. Anti-hepatitis C virus-positive blood donors were screened for anti-EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA-confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti-EP3-producing heterohybridoma cell lines: Ul/F30 and Ul/F31 produced IgM-kappa, whereas Ul/F32 and Ul/F33 secreted the isotypes IgG1-lambda and IgG1-kappa, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies Ul/F32 and Ul/F33 have dissociation constants to the peptide of approximately 10(-9) M, binding to recombinant E1 protein expressed in COS-7 cells was different. Only Ul/F33 detected envelope protein of approximately 24-35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1.

Enhanced outgrowth of EBV-transformed chronic lymphocytic leukemia B cells mediated by coculture with macrophage feeder cells

AbstractB-cell chronic lymphocytic leukemia (B-CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes that produce mAbs often reactive with microbial or autoantigens. Long-term culture of B-CLL clones would permit the collection and characterization of B-CLL mAbs to study antigen specificity and of B-CLL DNA to investigate molecular mechanisms promoting the disease. However, the derivation of long-term cell lines (eg, by EBV), has not been efficient. We have improved the efficiency of EBV B-CLL transformation of CpG oligonucleotide-stimulated cells by incubating patient peripheral blood mononuclear cells in the presence of an irradiated mouse macrophage cell line, J774A.1. Using this approach, peripheral blood mononuclear cells isolated from 13 of 21 B-CLL patients were transformed as documented by IGHV-D-J sequencing. Four clones grew and retained CD5 expression in culture for 2 to 4 months. However, despite documentation of EBV infection by expression of EBNA2 and LMP1, B-CLL cells died after removal of macrophage feeder cells. Nevertheless, using electrofusion technology, we generated 6 stable hetero-hybridoma cell lines from EBV-transformed B-CLL cells, and these hetero-hybridomas produced immunoglobulin. Thus, we have established enhanced methods of B-CLL culture that will enable broader interrogation of B-CLL cells at the genetic and protein levels.

ORIGINAL ARTICLE: Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample

SummaryObjective Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes.Design, patients and measurements Two heterohybridoma cell lines secreting TSHR autoantibodies were isolated using standard techniques from the lymphocytes of a patient with hypothyroidism and high levels of TSHR autoantibodies (160 units/l by inhibition of TSH binding). The ability of the two new monoclonal antibodies (MAbs; K1‐18 and K1‐70) to bind to the TSHR and compete with TSH or TSHR antibody binding was analysed. Furthermore, the effects of K1‐18 and K1‐70 on cyclic AMP production in Chinese hamster ovary cells (CHO) cells expressing the TSHR were investigated.Results One MAb (K1‐18) was a strong stimulator of cyclic AMP production in TSHR‐transfected CHO cells and the other (K1‐70) blocked stimulation of the TSHR by TSH, K1‐18, other thyroid‐stimulating MAbs and patient serum stimulating type TSHR autoantibodies. Both K1‐18 (IgG1 kappa) and K1‐70 (IgG1 lambda) bound to the TSHR with high affinity (0·7 × 1010 l/mol and 4 × 1010 l/mol, respectively), and this binding was inhibited by unlabelled K1‐18 and K1‐70, other thyroid‐stimulating MAbs and patient serum TSHR autoantibodies with stimulating or blocking activities. V region gene analysis indicated that K1‐18 and K1‐70 heavy chains used the same V region germline gene but different D and J germline genes as well as having different light chains. Consequently, the two antibodies have evolved separately from different B cell clones.Conclusions This study provides proof that a patient can produce a mixture of blocking and stimulating TSHR autoantibodies at the same time.

A Human Monoclonal Autoantibody to the Thyrotropin Receptor with Thyroid-Stimulating Blocking Activity

Human monoclonal autoantibodies (MAbs) are valuable tools to study autoimmune responses. To date only one human MAb to the thyrotropin (TSH) receptor (TSHR) with stimulating activity has been available. We now describe the detailed characterization of a blocking type human MAb to the TSHR. A single heterohybridoma cell line was isolated from the peripheral blood lymphocytes of a patient with severe hypothyroidism (TSH 278 mU/L) using standard techniques. The line stably expresses a TSHR autoantibody (5C9; IgG1/kappa). Ability of 5C9 to bind and compete with 125I-TSH or TSHR antibodies binding to the TSHR was tested using tubes coated with solubilized TSHR. Furthermore, the blocking effects of 5C9 on stimulation of cyclic AMP production was assessed using Chinese hamster ovary (CHO) cells expressing the wild-type human TSHR or TSHRs with amino acid mutations. 5C9 IgG bound to the TSHR with high affinity (4 x 10(10) L/mol) and inhibited binding of TSH and a thyroid-stimulating human monoclonal autoantibody (M22) to the receptor. 5C9 IgG preparations inhibited the cyclic AMP-stimulating activities of TSH, M22, serum TSHR autoantibodies and thyroid-stimulating mouse monoclonal antibodies. Furthermore 5C9 reduced the constitutive activity of wild-type TSHR and TSHR with some activating mutations. The effect of different amino acid mutations in the TSHR on 5C9 biological activity was studied and TSHR Lys129Ala or Asp203Ala completely abolished the ability of 5C9 to block TSH-mediated stimulation of cyclic AMP production. The availability of 5C9 provides new opportunities to investigate the binding and biological activity of TSHR blocking type autoantibodies including studies at the molecular level. Furthermore, monoclonal antibodies such as 5C9 may well provide the basis of new drugs to control TSHR activity including applications in thyroid cancer and Graves' ophthalmopathy.

A Human Monoclonal Antibody that Binds Serotype A Botulinum Neurotoxin

Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

A NOVEL GENE FROM BRUGIA SP. THAT ENCODES A CYTOTOXIC FATTY ACID BINDING PROTEIN ALLERGEN RECOGNIZED BY CANINE MONOCLONAL IgE AND SERUM IgE FROM INFECTED DOGS

Brugia pahangi infection of dogs is a well characterized model of human lymphatic filariasis in which sera consistently show IgG or IgE reactivity to a 35-kDa antigen. Using dog lymph node B cells, we previously established a heterohybridoma cell line producing canine monoclonal IgE (cmAb 2.39) that activates and degranulates canine mast cells, and specifically recognizes a 35-kDa B. pahangi antigen. By affinity purification and sequencing of the native protein from B. pahangi adults, a 19-amino acid sequence was obtained; the derived nucleotide sequence showed homology to a Brugia malayi and 2 related Onchocerca volvulus expressed sequence tag (EST) clones from the Filarial Genome Project database. Consensus primers amplified a 244-bp product from adult and infective larval stage cDNA libraries of B. malayi, O. volvulus, and Wuchereria bancrofti, but not from those of nonfilarial nematodes. The B. malayi EST clone only showed nucleotide sequence homology to O. volvulus EST sequences. A 684-bp region from the open reading frame was expressed as a glutathione S-transferase fusion protein designated BmAl-1. CmAb 2.39, as well as serum IgE from dogs infected with B. pahangi and canine filarial heartworm, Dirofilaria immitis, recognized BmAl-1 on enzyme-linked immunosorbent assay and Western blots. BmAl-1 showed high binding affinity for a fatty acid; however, a search for sequence homology with known fatty acid binding proteins indicated that BmAl-1 is a unique fatty acid binding protein. This 35-kDa protein seems to be highly conserved in different stages and species of filarids, and it represents a previously unknown allergen that is possibly involved in the pathogenesis of filarial disease.

Expression of neutralizing recombinant human antibodies against Varicella Zoster virus for use as a potential prophylactic.

Chickenpox is a highly infectious disease that can be life-threatening to certain groups such as the newborn of nonimmune mothers and immunocompromised patients. At present, prophylactic treatment of individuals at risk involves the use of a polyclonal antibody preparation derived from the pooled sera of hyperimmune donors. While this product is effective, there are problems associated with maintaining supply, which depends on the availability of donors, and the variation of potency between batches. An effective human monoclonal preparation would be of value by providing a well-characterized and standardized preparation available on demand. In this study recombinant human anti-varicella zoster virus (VZV) monoclonals were generated from the mRNA of unstable anti-VZV secreting heterohybridoma cell lines, and characterized according to their molecular weight, isoelectric point, glycosylation, binding to C1q, and efficacy at neutralizing VZV in vitro. In one antibody (AEVZ 5.3) the VH region was grafted from the IgG1 parent antibody onto an IgG3 backbone to determine the effect of isotype on neutralization in vitro. Antibodies were expressed from NSO cells at concentrations of 3-24 microg/mL and contained the expected heavy and light chain fragments and N-linked glycan structures. Both AEVZ 5.1 and AVEZ 4 antibodies were IgG1 and recognized the viral coat protein glycoprotein E; both showed complement-independent and complement-enhanced neutralization. Changing the isotype of AEVZ 5.1 from IgG1 to IgG3 (AEVZ 5.3) further enhanced VZV neutralization in the presence of complement, but reduced its neutralization capacity in the absence of complement. Complement enhancement was consistent with our findings that the IgG3 form could bind more molecules of C1q. The results demonstrate the successful use of recombinant methods to generate stable, functional monoclonal antibodies. Modifications of the original antibodies were made with the aim of improving functionality. The resulting cell lines could be used for large-scale production of well-characterized antibodies for therapeutic use.

Human anti-Dia monoclonal antibodies for mass screening

The use of monoclonal antibodies (mabs) to blood group antigens is constantly increasing for routine typing. Two heterohybridoma cell lines, HMR15 and HMR22, were established by Epstein-Barr virus transformation of peripheral blood lymphocytes from a blood donor with anti-Dia. HMR15 mab directly agglutinated Di(a+) red cells, and HMR22 mab agglutinated Di(a+) red cells exclusively by the indirect antiglobulin test. Reactivities of both HMR15 and HMR22 mabs were specific for Dia and had good correlation with the reactivity of a commercial, polyclonal antiserum. The binding of monoclonal antibodies to antigen-positive red cells was mutually blocked by each other as well as by polyclonal anti-Dia. Immunoprecipitates by HMR22 mab with a Di(a+) preparation showed a 120 kDa band that was stained by anti-band 3. Dia typing of 2427 blood donors with the mabs and polyclonal typing serum detected 244 Di(a+) individuals (10.1%). No discrepancy was observed between the mabs and the polyclonal antiserum. HMR15 and HMR22 mabs are useful Dia typing reagents and can substitute for commercial antiserum.

A human monoclonal antibody encoded by the V4-34 gene segment recognises melanoma-associated ganglioside via CDR3 and FWR1

A heterohybridoma cell line producing the human monoclonal antibody (MoAb) MDT.1 has been established. The heavy chain of MoAb MDT.1 is encoded by the VH gene segment V4-34 (previously designated VH4-21), and the light chain is encoded by the V kappa 1-L12a gene segment, both in germline configuration. MDT.1 has reactivity against lipid A, double- and single-stranded DNA, red blood cell associated i antigen, and ganglioside antigens. In a panel of tumour cell lines, MDT.1 reacted specifically with melanoma cells and other tumour cells of neuroectodermal origin. Cellular recognition appears to be via tumour-associated ganglioside antigens, and may involve the minimal essential epitope NeuNac alpha 2-->3Gal beta 1-->-4Glc-. Binding to ganglioside antigen is inhibited by the monoclonal anti-idiotypic antibody 9G4. Since the 9G4 idiotope is located in framework region 1 (FWR1) of V4-34-encoded antibodies, this region is likely to be involved, either directly or indirectly, in ganglioside binding. The complementarity-determining region 3 (CDR3) of MDT.1 is arginine rich, with five out of 12 residues being arginine and these residues are candidates for interaction with the negatively charged ganglioside. The ability of MoAb MDT.1 to recognise ganglioside antigens is associated with potentially useful anti-tumour activity.

Analysis of changes during subclone development and ageing of human antibody-producing heterohybridoma cells by Northern blot and flow cytometry

The economic importance of obtaining high-producing subclones for large scale production of pharmaceutical proteins is self-evident. However, few papers have studied the changes that occur during subclone development. This information would be important for further improvement of screening and subcloning protocols. We have therefore compared subclones of a human-mouse heterohybridoma cell line producing a human antibody against HIV-1. Three subclones with low, medium and high specific production rates were selected for this study and their light and heavy chain mRNA content, the intracellular content of light and heavy chain and the specific secretion rates compared. In addition the long time stability of antibody expression in the highest producing subclone was analysed for one year. For the three subclones a correlation between the intracellular content in light chain and the secretion rate was found, while the intracellular content in heavy chain was the same for all three subclones. These results indicate that the assembly in the endoplasmic reticulum (ER) is one of the major rate limiting factors in antibody production. During long time cultivation of the heterohybridoma cell line a continuous decrease in light and heavy chain production was seen without the appearance of a non producing sub-population.

Variation in IgG1 heavy chain allotype does not contribute to differences in biological activity of two human anti-Rhesus (D) monoclonal antibodies

Background: Pooled human anti-Rhesus D antiserum is currently administered for the prevention of RhD alloimmunization. Increased demand, and decreased supply, of donated pooled antiserum has led to the investigation of the suitability of human monoclonal anti-RhD antibodies for use in its place. However, it is unclear which biological properties of monoclonal antibodies are important for function in RhD-positive foetal red cell clearance and the prevention of alloimmunization. Various antibodies behave differently in a number of in vitro assays of biological function. Objectives: To compare the function and structure of two human anti-RhD IgG1 monoclonal antibodies which differ in their ability to promote red cell lysis in vitro. In particular to examine whether the functional differences correlate to differences in the IgG1 heavy chain constant region (allotype). Study design: We report here the cloning, characterization and re-expression in stable myeloma cell transformants of cDNAs coding for two such antibodies, secreted by the heterohybridoma cell lines ESD-1 (THERAD 03) and LHM 70/45.3 (THERAD 06). The cDNAs were then recombined to exchange portions of the Fc encoding regions and the recombinant antibodies were assayed in vitro to determine RhD-positive red cell-dependent activity. Results: Recombinant THERAD 03 and 06 antibodies behaved identically to the parent antibodies. The `inactive' THERAD 06 did not have biological activity reconstituted by exchange with the THERAD 03 Fc regions, nor was THERAD 03 activity abolished by the reciprocal Fc region exchange. Conclusions: Human monoclonal anti-RhD antibodies can be cloned and re-expressed in stable cell lines, and exhibit identical properties to the parent antibodies. Differences in biological activity cannot be attributed to differences in IgG1 heavy chain allotype.

Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins

The isolation and characterization of human monoclonal antibodies (humAbs) against the hepatitis C Virus (HCV) glycoproteins E1 and E2 are described. B-cells from blood donors with anti-HCV were transformed with Epstein-Barr virus. The supernatants of the resulting lymphoblastoid clones were screened by ELISA with an extract of cells infected with a recombinant vaccinia virus RMPA95 expressing the envelope proteins E1 and E2 of an HCV genotype 1a virus (H strain). Positive clones were fused to the heteromyeloma cell line K6H6/B5. Fifteen heterohybridoma cell lines have been established. The specificity of the isolated humAbs was determined both by ELISA and Western blot assays. Several recombinant extracts expressing either the E1 or E2 protein or truncated forms were used in an attempt to map the epitopes on the viral glycoproteins. Some of the humAbs were used successfully for immunofluorescence investigation of transfected cells. Seven specific anti-E2 humAbs, which react with the envelope protein 2 of genotype 1a and 1b isolates, were characterized.

Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins

The isolation and characterization of human monoclonal antibodies (humAbs) against the hepatitis C Virus (HCV) glycoproteins E1 and E2 are described. B-cells from blood donors with anti-HCV were transformed with Epstein-Barr virus. The supernatants of the resulting lymphoblastoid clones were screened by ELISA with an extract of cells infected with a recombinant vaccinia virus RMPA95 expressing the envelope proteins E1 and E2 of an HCV genotype 1a virus (H strain). Positive clones were fused to the heteromyeloma cell line K6H6/B5. Fifteen heterohybridoma cell lines have been established. The specificity of the isolated humAbs was determined both by ELISA and Western blot assays. Several recombinant extracts expressing either the E1 or E2 protein or truncated forms were used in an attempt to map the epitopes on the viral glycoproteins. Some of the humAbs were used successfully for immunofluorescence investigation of transfected cells. Seven specific anti-E2 humAbs, which react with the envelope protein 2 of genotype 1a and 1b isolates, were characterized. J. Med. Virol. 55:28–34, 1998. © 1998 Wiley-Liss, Inc.

Human monoclonal antibodies to cytomegalovirus. Characterization and recombinant expression of a glycoprotein-B-specific antibody.

Human monoclonal antibodies (mAb) to human cytomegalovirus (HCMV) were established from spleen cells of a HCMV-positive donor. The antibodies (gamma 3, lambda) secreted from a stable heterohybridoma cell line were further characterized by immunoprecipitation and immune-fluorescence microscopy using HCMV infected cells and recombinant cell lines expressing HCMV glycoprotein B. The antibody reacted with the entire glycoprotein B or the extracellular domain expressed as glycoprotein-B--beta-galactosidase fusion protein in the native state, but the antibody was not neutralizing HCMV. Denatured and reduced forms of glycoprotein B were not recognized by this antibody, however, native glycoprotein B on the surface of infected cells was detected efficiently. The genes encoding the Fab part of the antibody were cloned and expressed in Escherichia coli. Recombinant Fab fragments specifically binding the extracellular domain of glycoprotein B could easily be isolated from the periplasmic space. Recombinant antibodies provide the opportunity to modify effector functions and to add tags to diagnostic antibodies for more efficient detection of CMV-infected cells.

HUMAN LYMPHOCYTOTOXIC MONOCLONAL AUTOANTIBODIES FROM A HIGHLY SENSITIZED RENAL DIALYSIS PATIENT

A panel of 5 human monoclonal autolymphocytotoxic antibodies (IRM-3, IRM-4, IRM-7, IRM-8, and IRM-10) of the IgM class was established from a highly sensitized renal dialysis patient (IRM), by the generation of mouse-human heterohybridomas. This panel was screened for reactivity against foreign and autoantigens by ELISA, and for reactivity against different tissue sections and HEp-2 slide preparations by indirect immunofluorescence. Cytotoxicity screening of heterohybridoma supernatants gave broad panel reactivity profiles, being cytotoxic against B cells from patient IRM and also against most B cells tested and less reactive with chronic lymphocytic leukemia B cells; T cells were the least sensitive target. Immunoblotting showed that monoclonal IRM displayed some heterogeneity in their binding profiles, although all of them recognized a cellular structure of 26 kDa. None of the heterohybridoma cell lines exhibited cytoplasmic nor surface staining with an anti-CD5 mAb. Results obtained showed that all the autolymphocytotoxic mAbs generated were also able to react against certain nuclear and cytoplasmic self-structures as well as foreign compounds. Monoclonal antibody IRM-7 and, to a lesser degree, IRM-10 exhibited multispecific properties similar to those observed for polyreactive or natural antibodies.

Influence of cell‐ and media‐derived factors on the integrity of a human monoclonal antibody after secretion into serum‐free cell culture supernatants

To investigate the effects of factors secreted by different cell lines on human monoclonal antibody (MAb) integrity, 600 mg of a human MAb, which specifically binds to human erythrocytes, were produced in a perfusion process. After purification by protein A affinity chromatography, the MAb was used for integrity testing in supernatants of several cell lines to investigate their potential to degrade the antibody in the extracellular environment. One insect cell line (IPLB-SF-21 AE) and four mammalian cell lines [CHO K1, BHK-21 (C13), C1271, P3-X63-Ag8.653], all of them commonly used for the production of recombinant proteins, and the human-human-mouse heterohybridoma cell line itself (H-CB-hahE), were adapted to serum-free culture media. For integrity testing all cell lines were cultivated in spinner flasks using serum-free media supplemented with 30 mug mL(-1) of purified MAb. MAb integrity was assayed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, both followed by Western blotting, and an antigen binding assay. None of the mammalian cells showed any detectable effects on antibody stability and integrity during exponential growth, whereas isoelectric focusing of monoclonal antibody taken from IPLB-SF-21 AE culture supernatants revealed a new band indicating a partial modification of the MAb by secreted factors of these cells. This observation did not correlate with the total proteolytic activity, which was measured in all supernatants and found to be lowest in the insest cell cultures. For mammalian cell cultures, it could be concluded from these findings that shifts of the antibody microheterogeneity pattern, which can be found normally as a result of variations in different production parameters, are not caused by extracellular factors once the product has been secreted into the supernatant. In addition to their well-known advantages in posttranslational modifications (e.g., formation of complex type N-glycans), mammalian cells appear to be more suitable as expression systems for human monoclonal antibodies to be used in vivo when compared with baculovirus-infected insect cells. (c) 1995 John Wiley & Sons, Inc.

The effects of oncogene transfection on growth and antibody production of human-mouse heterohybridomas

Human-mouse heterohybridomas producing human monoclonal antibodies show slower growth rates and lower peak cell densities than murine hybridomas. In order to improve the growth properties we transfected a heterohybridoma cell line with expression plasmids containing the oncogenes v-src, c-Ha-ras and SV40largeT. The plasmids were transferred by electroporation. Growth promoting activities of the plasmids were proven in NIH3T3 cells whereby a doubling of the maximum cell densities of this cell type was observed. The oncogene products were analyzed by means of northern blotting and immunofluorescence. After transfection of c-src and c-ras, a heterohybridoma cell line was derived which showed improved growth characteristics compared to the original cell line. Although specific antibody production was lower, antibody concentrations which accumulated in batch culture were higher due to increased maximum cell densities.

Cloning of human anti‐GM1 antibodies from motor neuropathy patients

Patients with multifocal motor neuropathy frequently have elevated titers of serum antibodies reactive with GM1 ganglioside. Although these antibodies may cause the syndrome, this has yet to be proven directly. As part of our studies on the nature and pathogenic potential of anti-GM1 antibodies, we have cloned B cells from the peripheral blood of 3 patients with multifocal motor neuropathy and generated four stable heterohybridoma cell lines secreting human monoclonal IgM anti-GM1 antibodies. In this report we describe the basic properties of these monoclonal antibodies in comparison with the patient's sera from which they were derived. The antibodies all differ in their pattern of reactivity with GM1 and other Gal(beta 1-3)GalNAc-containing glycoconjugates. They have widely varying thermal ranges and their reactivities are strongly influenced by the presence of accessory lipids. Affinity purification of the patient's sera with GM1 led to the identification of previously unrecognized paraproteins that were resolvable above the background of polyclonal anti-GM1 IgM. Our data demonstrate considerable heterogeneity in the immune response to GM1 both within individual sera and between different patients, which is likely to be of importance to their role in disease pathogenesis.

Flow cytometric analysis of the stability of antibody production by human × human × mouse heterohybridoma subclones

Flow cytometry has been utilized to evaluate the stability of antibody production by unstable subclones of a human × human × mouse heterohybridoma. Heterogeneity of cell-associated immunoglobulin heavy chain expression was demonstrated in different subclones and an increased frequency of cells containing low levels of heavy chain was found to correlate with low antibody productivity. However, the majority of cells were not completely devoid of heavy chains suggesting that the genetic information for the γ chain was not lost. In contrast, the gene encoding the κ light chain was shown to be absent from the subclones expressing low levels of heavy chain and these subclones also contained substantially reduced levels of heavy chain mRNA, suggesting that the production of this protein was controlled at the level of transcription or mRNA stability. In conclusion, the correlation of staining intensity as observed by flow cytometry and antibody productivity makes flow cytometry a suitable technique for the rapid evaluation of heterohybridoma cell lines used for antibody production.

Human Monoclonal λ Light Chain Protein Exhibits Specific Binding to the Variable Region of Monoclonal Anti-IgE Antibody

A monoclonal heterohybridoma cell line (B11-12) was developed by fusing EBV-transformed lymphocytes from a patient with hyper-IgE syndrome and SHM-D33 heteromyeloma cells. The resultant heterohybridoma secreted only human λ L chains exhibiting specific binding to mouse monoclonal Ab to human IgE (mAb-aIgE). B11-12 bound to four separate mAb-aIgE, but not to mAb of the same isotypes directed to other antigens. The mAb-aIgE differed in isotype (IgG1; IgG2b) demonstrating that B11-12 was recognizing an idiotypic, but not an isotypic determinant on the mAb-aIgE. Human IgE did not inhibit B11-12 binding to mAb-aIgE, suggesting the interaction between B11-12 and mAb-aIgE involved an idiotope outside the combining site of the mAb-aIgE. Human sera, including serum from the donor of the lymphocytes used to produce heterohybridoma B11-12, demonstrated the capacity to inhibit B11-12 binding to mAb-aIgE. This, along with the inability of these same sera to directly bind B11-12, suggests that the serum contained reactivity similar to B11-12. We propose that B11-12 demonstrates specificity resembling an anti-Id to anti-human IgE Ab.