DNA immunization followed by a single boost with cells: a protein-free immunization protocol for production of monoclonal antibodies against the native form of membrane proteins

Recent advancements in antibody-based therapies require the development of an efficient method for generation of monoclonal antibodies (MAbs) against the native form of membrane proteins. We examined DNA immunization followed by a single boost with cells as a protein-free immunization protocol for production of MAbs. Mice immunized with plasmid cDNAs encoding human CD30 or Ret tyrosine kinase were given a single boost with cells expressing the corresponding antigen prior to cell fusion. A total of nine cell fusion experiments revealed that the cell boost is necessary for efficient generation of hybridomas and the DNA-cell boost method gave good yields of specific MAbs (5–59 MAbs from one mouse). All IgG isotypes except IgG3 were generated, although IgG2a was the dominant isotype. All the MAbs reacted with native antigens expressed on cells in a fluorescence-activated cell sorter (FACS) analysis as well as with recombinant CD30 or Ret protein genetically fused with human Fc in an enzyme-linked immunosorbent assay (ELISA). The affinities of the anti-CD30 MAbs to CD30-Fc protein ranged from 0.9 to 12.4 nM K_ds, which were comparable to existing MAbs to these proteins, which range from 3.0 to 13.0 nM. Western blot analysis and topographical epitope mapping experiments based on the mutual competition of pairs of the anti-CD30 MAbs revealed that about 40% of the epitopes were linear epitopes and that each epitope was topographically classified into one of six groups. The large number of MAbs that react with high affinities to a variety of epitopes on the native form of antigens indicates that the method presented in this paper could be generally useful for generating MAbs to other membrane proteins.     

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.     

Characterization of the CD30L binding domain on the human CD30 molecule using anti-CD30 antibodies

CD30 and its counter-receptor CD30 ligand (CD30L) are members of the TNF-receptor/TNFalpha superfamily and function to regulate lymphocyte survival and differentiation. Several monoclonal antibodies (MAbs) have been developed against CD30 and, based on mutual inhibition assays, are grouped into three nonoverlapping serologic clusters. However, the relationship between the epitopes recognized by the antibodies comprising each cluster and the binding domain for CD30L is not known. Using a soluble CD30L/CD8alpha chimeric protein, we assessed the ability of anti-CD30 MAb to inhibit the binding of CD30L to CD30 expressed by the CD30+ Karpas 299 cell line. CD30L binding by CD30 is blocked by MAb that recognize epitopes belonging to cluster Group A (like Ber-H2, Ber-H8, and HRS-4) as well as cluster Group C (like HeFi-1 and M44). Cluster Group B antibodies, including M67 and Ki-1, do not affect CD30L binding to CD30. The pattern of CD30L binding inhibition shows only limited correspondence to the functional capacity of some anti-CD30 MAb to trigger CD30 signaling. Finally, we demonstrate that the anti-CD30L MAb M81 also completely inhibits CD30/CD30L interaction. This information is useful for applying these MAbs in functional studies to further investigate the CD30/CD30L system and for designing assays for soluble CD30L.     

Characterization of the opsonic and protective activity against Staphylococcus aureus of fully human monoclonal antibodies specific for the bacterial surface polysaccharide poly-N-acetylglucosamine

Carbohydrate antigens are important targets of the immune system in clearing bacterial pathogens. Although the immune system almost exclusively uses antibodies in response to foreign carbohydrates, there is still much to learn about the role of different epitopes on the carbohydrate as targets of protective immunity. We examined the role of acetyl group-dependent and -independent epitopes on the staphylococcal surface of polysaccharide poly-N-acetylated glucosamine (PNAG) by use of human monoclonal antibodies (MAbs) specific for such epitopes. We utilized hybridoma technology to produce fully human immunoglobulin G2 (IgG2) MAbs from B cells of an individual post-Staphylococcus aureus infection and cloned the antibody variable regions to produce an IgG1 form of each original MAb. Specificity and functionality of the purified MAbs were tested in vitro using enzyme-linked immunosorbent assays, complement deposition, and opsonophagocytic assays. We found that a MAb (MAb F598) that bound the best to nonacetylated or backbone epitopes on PNAG had superior complement deposition and opsonophagocytic activity compared to two MAbs that bound optimally to PNAG that was expressed with a native level (>90%) of N-acetyl groups (MAbs F628 and F630). Protection of mice against lethality due to S. aureus strains Mn8 and Reynolds further showed that the backbone-specific MAb had optimal protective efficacy compared with the acetate-specific MAbs. These results provide evidence for the importance of epitope specificity in inducing the optimal protective antibody response to PNAG and indicate that MAbs to the deacetylated form of PNAG could be immunotherapeutic agents for preventing or treating staphylococcal infections.     

Treatment of type 1 diabetes with anti-CD3 monoclonal antibody

Anti-CD3 monoclonal antibodies (MAbs) were developed as a way of inducing immune suppression of T cells. More recent studies have indicated that anti-CD3 MAbs can affect immune responses by inducing immune regulation. We recently reported that a single course of treatment with a non-FcR binding anti-CD3 MAb, hOKT3gamma1(Ala-Ala), can lead to preservation of insulin production in patients with new-onset Type 1 diabetes for even beyond 1 yr after treatment. The sustained insulin production was accompanied by improvement in glucose control and reduced use of insulin. Our studies of the mechanism of the non-FcR binding anti-CD3 MAb indicate that the MAb delivers an activation signal to T cells resulting in disproportionate production of interleukin-10 (IL-10) relative to interferon-gamma(IFN-gamma) in vitro compared with FcR binding anti-CD3 MAb, and detectable levels of IL-10, IL-5, but rarely IFN-gamma or IL-2 in the serum after treatment. In addition, the drug induces a population of CD4+IL-10+ CCR4+ cells in vivo. Preclinical data suggest that anti-CD3 MAb induces a population of regulatory T cells that can prevent or lead to reversal of Type 1 diabetes. The induction of cells with a regulatory phenotype may account for the ability of anti-CD3 MAb to induce immune regulation.     

Epitopes and functional responses defined by a panel of anti-Fas (CD95) monoclonal antibodies

Fas (CD95) is a cell surface glycoprotein that mediates apoptotic cell death when cross-linked with agonistic anti-Fas monoclonal antibodies (MAbs) or the endogenous Fas ligand. In this study, we investigated the in vitro biological properties of a panel of anti-human Fas MAbs. We found that five anti-Fas MAbs of IgG1 subclass (B.E28, B.G30, B.L25, DX2, and B.G34) induced marked apoptotic cell death in Fas-expressing leukemia cells, although this killing was delayed when compared to the cytolytic effect mediated by the prototypic anti-Fas MAb of IgM subclass (clone CH-11). On the other hand, four clones (ZB4, B.G27, B.D29, and B.K14) efficiently blocked apoptotic cell death induced by the CH-11 MAb or Fas ligand. The ability of these MAbs to inhibit cell death appeared to correlate with their relative affinity for the Fas molecule. Furthermore, different clones recognized the same epitope and elicited different effects (induction or inhibition of cell killing); conversely, different clones elicited the same effect but recognized different epitopes. These results suggest that the different biological effects of anti-Fas MAbs would not be mediated in an epitope-restricted manner. The relative binding affinity might correlate to some extent with the biological properties of the MAb.     

Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria.

Three monoclonal antibodies (MAbs) were generated from splenocytes of a BALB/c mouse immunized with heat-killed Mycobacterium tuberculosis. All three MAbs bound to surface epitopes of M. tuberculosis as shown by whole-cell enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoelectron microscopy. One immunoglobulin M (IgM) MAb bound to lipoarabinomannan, the second IgM MAb bound to mycolyl-arabinogalactan-peptidoglycan complex, and the third MAb, an IgG3, bound to a surface epitope of an uncertain nature. The MAbs demonstrated different cross-reactivity patterns with other mycobacteria. Two of the MAbs were used to develop a modified ELISA spot assay for the detection of mycobacteria.     

Epitopes on the S1 subunit of pertussis toxin recognized by monoclonal antibodies.

To identify the neutralizing epitopes on the S1 subunit (A promoter) of pertussis toxin, we characterized anti-S1 monoclonal antibodies (MAbs) X2X5, 3CX4, and 6FX1. We confirmed by immunoblot analysis that these MAbs bind to the S1 subunit and not to the B oligomer of pertussis toxin and that they recognize different epitopes by a competitive binding enzyme-linked immunosorbent assay. These MAbs had differential abilities to neutralize the lymphocytosis-promoting factor activity of pertussis toxin in mice: 3CX4 and 6FX1 had partial neutralizing abilities, while MAb X2X5 had none. With these MAbs, the epitopes on the S1 subunit were examined by using trypsinized S1 peptides, recombinant truncated S1 molecules, and synthetic peptides. The non-neutralizing MAb X2X5 bound in immunoblots to tryptic peptides of various sizes as small as 1.5 kilodaltons; the neutralizing MAbs 3CX4 and 6FX1 bound only to a 24-kilodalton tryptic peptide band. Immunoblot studies with recombinant truncated S1 molecules demonstrated that amino acid residues 7 to 14 and 15 to 26 play an important role in the binding of neutralizing MAbs and the non-neutralizing MAb, respectively. The binding of these MAbs was not dependent upon the presence of C-terminal amino acid residues 188 to 234. To further define B-cell epitopes, the binding of the MAbs we tested to synthetic peptides representing the entire S1 subunit were examined. Neutralizing MAbs 3CX4 and 6FX1 bound to none of these peptides, further suggesting that these MAbs recognize conformational epitopes. The non-neutralizing MAb X2X5 bound to peptides 11 to 26 and 16 to 30, demonstrating that the major antigenic determinant recognized by this MAb is a linear epitope located within residues 16 to 26.     

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity of each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity ( approximately 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant.     

Comparisons of rotavirus VP7-typing monoclonal antibodies by competition binding assay.

Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes.     

Monoclonal antibodies against human basic fibroblast growth factor

Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF. Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b. All these MAbs bound the complete form of hbFGF produced in E.coli. Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E. coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs. MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF. MAb52 and MAb98 recognized the one located between the amino acid residue no. 14 and 40. None of MAbs bound bovine acidic FGF (aFGF). Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed. This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF. Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column.     

Characterisation of Putative Monoclonal Anti-G3M(U) and Anti-G3M(G) Reagents and their Antigenic Determinants

Monoclonal antibodies (MAbs) against IgG3 allotypic markers were evaluated in haemagglutination inhibition and IgG3 capture ELISA. MAbs PNF69C and 200D1 exhibited G3m(u) and G3m(g) specificity respectively. In HAI target epitopes detected by MAbs were remarkably stable to physiochemical degradation. Western blotting revealed that MAb 200D1, bound to intact IgG3 heavy chain disease protein and not its pFc' fragment; a result consistent with the CH2 domain location of the G3m(g) allotope. The G3m(u) allotope is also located within this domain. Suprisingly anti-G3m(u) MAb PNF69C bound to the pFc' of IgG3-related protein, HW, and to the pFc' of IgG1-related protein, PR, in Western blot.     

Anti-idiotypic antibodies as probes for the determination of idiotopes shared by human and monoclonal murine antibodies

Murine monoclonal antibodies (MAbs) against rye grass Group I (rye I) allergen were previously produced (290A-167, 348A-6 and 539A-6) and are further characterized herein. By competitive binding to polystyrene-bound allergen, it was shown that the three MAbs are directed against three different non-overlapping epitopes on rye I. Human rye I-specific IgG isolated from the serum of one rye grass sensitive patient could recognize the same or closely related epitopes than those recognized by MAbs. Antisera were then raised in rabbits against purified F(ab')2 fragments of human rye I-specific IgG and F(ab')2 fragments of 539A-6 MAb. The antisera were rendered idiotype specific by adsorption with insolubilized non-specific human IgG from the same donor, insolubilized normal murine immunoglobulins and finally with rye I. As shown by competitive binding to polystyrene-bound allergen, rabbit anti-idiotypic antibodies (anti-ID antibodies) produced against F(ab')2 fragments of human rye I specific IgG could inhibit the reaction between two MAbs (290A-167 and 539A-6) and the relevant allergen. Furthermore, human rye I specific IgG could inhibit the binding of 125I-labelled 539A-6 MAb to its specific anti-ID antibody to a significant degree. Human autoanti-idiotypic antibodies were also shown to inhibit the reaction between the three anti-rye I MAbs and the antigen. These observations suggest that cross-reactivity exists between idiotypic determinants of human rye I-specific IgG and the three murine MAbs, which implies structural similarity in the V genes coding for the variable region of the antibody from these two different species.     

Antigenic differences between metastatic cells in bone marrow and primary tumours and the anti-MUC1 humoral immune response induced in breast cancer patients

The dissemination of a malignant neoplasia is a complex process, which requires a set of molecules that remains unknown. It has been suggested that mucins and their carbohydrate-associated antigens may be implicated in tumour spreading which may be also influenced by an anti-MUC1 immune response. In this pilot study, we report the pattern of carbohydrate and peptidic MUC1-associated epitopes on carcinoma cells isolated from bone marrow (BM), taking into account primary tumour histopathologic features. We also bring information about the anti-MUC1 humoral response in these patients. Seventeen patients with invasive breast carcinoma were included. A sample of the primary tumour, a serum sample and a BM aspirate were obtained from each patient. Clinical features studied were tumour size, number of metastatic nodes, histological type and disease stage. Standard immunohistochemistry was performed with antigenic retrieval using different monoclonal antibodies (MAbs): anti carbohydrate antigens: Lewis x (KM380), sLewis x (KM93), Lewis y (C14) and Tn, anti-MUC1 peptide core MAbs: C595, HMFG2 and SM3, anti-cytokeratins, anti-protoncogenes ErbB2 and ErbB3 (IgG) MAbs and also anti-CD34 and anti-CD45 MAbs. ELISA techniques were employed to study circulating MUC1 as well as free and complexed anti-MUC1 antibodies. Immunohistochemical results showed that carbohydrate antigenic expression increases in BM neoplastic cells compared to the original tumours. However, we were not able to demonstrate that a humoral immune response to MUC1 has been induced in these patients. Finally, the employed procedures allow the selective immortalisation of micrometastatic carcinoma cells since short-term cell lines were established.     

Subcellular Distribution of Monoclonal Antibody Defined Epitopes on Immunodominant Mycobacterium tuberculosis Proteins in the 30-kDa Region: Identification and Localization of 29/33-kDa Doublet Proteins on Mycobacterial Cell Wall

Two different groups of monoclonal antibodies (MAbs) directed to different epitopes on 30-kDa region proteins of Mycobacterium tuberculosis were isolated; MAbs 5F9, 5D5 and 5D2 reacted with a single 33-kDa protein band, whereas MAb 3A8 reacted with a distinct 29/33-kDa doublet when analysed by immunoblotting. The present paper describes the distribution of MAbs defined epitopes in the 29-33-kDa region proteins in well-characterized subcellular fractions: cytosol, plasma membrane, cell wall as well as culture filtrate of M. tuberculosis. MAbs 5F9, 5D5 and 5D2 reactive epitopes were found in cytosol, whereas 3A8 epitope is distributed in all cellular compartments of the mycobacterium as well as in the culture filtrate. Localization of these epitopes by indirect immunofluorescence and immunogold-labelling demonstrated that only 3A8 epitope is present on the cell surface of the mycobacterium. Both immunoblotting and ELISA showed that only MAb 3A8, and not 5F9, 5D5 and 5D2, reacted with secreted BCG 85 antigen complex of Mycobacterium bovis BCG. Furthermore, using an MAb 3A8-coupled affinity column, we purified antigen 3A8 from the cytosol fraction of M. tuberculosis. All these MAbs reacted with antigen 3A8 with varying degrees of intensity, thus suggesting that they are directed to a single protein. Absence of 5F9, 5D5 and 5D2 epitopes in the cell wall, culture filtrate and to a single protein. Absence of 5F9, 5D5 and 5D2 epitopes in the cell wall, culture filtrate and BCG-85 complex suggests that these epitopes might have been lost during the processing of the same 33-kDa protein on its way out from cytosol to the cell wall or when the protein is secreted out into the culture filtrate. Our results demonstrate, for the first time, direct evidence of the presence of a 30-kDa region protein not only in secreted antigens but also in the cell wall and on the cell surface of the mycobacterium.     

Epitope mapping of monoclonal antibodies capable of neutralizing cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli

Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.     

Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.     

Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.     

Characterization of monoclonal antibodies against apolipoproteins A-I and A-II. Epitope expression in LpA-I and LpA-I:A-II particles

Two monoclonal antibodies (MAbs) against apolipoprotein A-I (apo A-I), 6B9 and FF9B10, and one MAb against apolipoprotein A-II (apo A-II), 3F5, were characterized. To establish the epitope of apo A-I recognized by these antibodies, different experimental approaches were performed. First, competition between MAbs and the related epitopes on the same antigen was performed using double-determinant tests with previously characterized MAbs. Second, competition of different synthetic peptides of apo A-I in solution with apo A-I immobilized to solid phase was carried out. The MAbs against apo A-I (6B9 and FF9B10) appear to recognize discontinuous epitopes located in the amino-terminal region of the apo A-I. In competition experiments MAb 3F5 did not recognize central- or carboxy-terminal peptides of apo A-II. Furthermore, apo A-II was stronger recognized when it was included in HDL or LpA-I:A-II than in its purified form. So the epitope for 3F5 is better expressed in the lipoprotein structure. Finally, to establish the epitopes expression in different antigens in solution, competition of purified apo A-I, apo A-II, LpA-I, and LpA-I:A-II particles or HDL3, with apo A-I or HDL immobilized to solid phase, was carried out. The results showed that both MAbs against apo A-I reacted with poor affinity against free apo A-I, with high and similar affinities against Lp A-I and Lp A-I:A-II lipoparticles and with the highest affinity against HDL3. The MAb 3F5 against apo A-II recognized only LpA-I:A-II and not LpA-I lipoparticles.     

Application of Monoclonal Antibodies in Functional and Comparative Investigations of Heavy-Chain Immunoglobulins in New World Camelids

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the γ chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.