Cross-reaction of a monoclonal antibody to human MHC class II molecules with rabbit B cells

Monoclonal antibodies, 21w4 and 44H10, against human MHC class II determinants, were analysed for their reactivity with rabbit lymphoid cells. Both MAb bind to all human B lymphoblastoid lines, irrespective of their HLA-DR phenotype. HLA-DR antigens, purified by affinity to 21W4 IgG Sepharose, can be precipitated with 44H10 MAb, indicating that both antibodies react with the same molecules. Competitive inhibition studies with purified MAb IgG show that 44H10 and 21w4 recognize different epitopes of HLA-DR molecules. The 21w4 MAb, previously shown to cross-react with cells of pig, mouse and sheep, does not react with rabbit lymphoid cells. The 44H10 MAb binds to lymphoid cell suspensions prepared from rabbit appendix, spleen and mesenteric lymph nodes. Its reactivity correlates with that of RABELA, a polyclonal antibody specific for rabbit B cells. Mesenteric lymph node and spleen cell suspensions, depleted of sIg+ cells, are devoid of 44H10+ cells, while similarly-treated appendix B cells still contain a subset of B cells which are sIg-, RABELA+ and 44H10+. These studies thus report on the presence of MHC class II determinants on rabbit B cells, cross-reacting with human HLA-DR.     

Mapping epitopes on the insulin molecule using monoclonal antibodies

A panel of 18 monoclonal antibodies (mAb) delta to insulin have been prepared and used to begin to map antigenic determinants on the insulin molecule. All 18 mAb were of the IgG class, with 14 IgG1, 2 IgG2a and 2 IgG2b. The affinities of these mAb for their immunizing insulin ranged from 1 X 10(6) to 3 X 10(8) 1/M. The epitope recognized by three of the mAb, 1, 7 and 16 involves the three residues of the A chain, A 8-10, the so called A chain-loop determinant. This A chain loop is one of the most evolutionarily diverse regions of insulins from different species. Another mAb, 10, has been hypothesized to recognize a nearby epitope composed of the A chain residues, A4 and A8 and a B chain residue, B29, that are adjacent on the surface of the insulin molecule. Four of the mAb bind to synthetic B chain. The epitopes recognized by these 4 mAb and the last 10 mAb are unknown but the mAb are grouped according to their ability to bind to different species of insulin or proinsulin. The results of an 18 X 18 matrix analysis of pairs of mAb binding simultaneously to insulin indicate that, despite the finding that some mAb see similar antigenic sites on the insulin molecule, each of the mAb recognizes a unique site on the insulin molecule. Finally, a lower estimate of the number of possible antibodies made to insulin has been calculated to be greater than or equal to 115, a number only 10-fold lower than the lower limit of antibodies made to dinitrophenyl (DNP) or (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP), following hapten protein immunization.     

Characterization of two porcine proinsulin reactive monoclonal antibodies by immunostaining of beta-cells in pancreatic sections of different species.

Murine monoclonal antibodies against porcine proinsulin were generated by somatic cell hybridization. As detected by radioimmunoassay, 2 monoclonal antibodies KSPI14D4 and KSPI13G10 showed a strong binding to 125I-labelled porcine proinsulin but not to insulin. The species specificity of these 2 monoclonals was found to be different as shown by indirect immunofluorescence using sections of Bouin-fixed pancreata of different species. The KSPI14D4 recognized the proinsulin of pig, mouse, man, cattle, rat, dog, and cat but not that of guinea pig, whereas the KSPI13G10 bound to porcine proinsulin only. From these results it is concluded that KSPI14D4 effectively recognizes a wide-spread epitope located in one of the insulin-C-peptide junctions of the proinsulin molecule, whereas KSPI13G10 is directed to a species-specific epitope of the porcine connecting peptide.     

Murine hybridoma monoclonal antibodies against insulin: cross-reactivity with insulins of three species and blocking of insulin binding to its receptor

Six hybridoma cell lines secreting monoclonal antibodies against pig insulin and cross-reacting with human and bovine insulins were obtained. Five of these monoclonal antibodies were IgG1, kappa, one IgG2b, kappa; their pI values were in the range of pH 6.3-7.4 and dissociation constants of the insulin-antibody complexes were 0.3-2 X 10(-8) mol/l, as determined by an immunoradiometric inhibition assay. All of these antibodies reacted with sterically closely related determinants and blocked the binding of 125I-pig insulin to the receptor on human MOLT-4 cell line.     

Protection against Brucella melitensis or Brucella abortus in mice with immunoglobulin G (IgG), IgA, and IgM monoclonal antibodies specific for a common epitope shared by the Brucella A and M smooth lipopolysaccharides.

Mice passively immunized prior to a challenge infection with immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) specific for a common epitope of both A- and M-dominant strains had viable Brucella abortus 544 or Brucella melitensis H38 counts in the spleen reduced to the same extent as did mice passively immunized with MAbs specific for either the A or the M epitope. The IgA MAb was not effective.     

Structurally defined epitopes of Haemophilus ducreyi lipooligosaccharides recognized by monoclonal antibodies.

By use of enzyme-linked immunosorbent assay and immunoblotting techniques, the migration patterns and binding epitopes of lipooligosaccharides (LOS) from 10 Haemophilus ducreyi strains were investigated with two monoclonal antibodies (MAbs), MAHD6 and MAHD7, raised against LOS from H. ducreyi ITM 2665. Closely related LOS, with defined structures, from Haemophilus influenzae, Bordetella pertussis, Aeromonas spp., and synthetic glycoproteins were also included in the analyses. The MAbs bound to conserved epitopes of LOS exposed on the surface of H. ducreyi. The MAb MAHD6 reacted with 8 of the 10 LOS from H. ducreyi but with none of the other Haemophilus or Bordetella spp. with structurally defined LOS. It is suggested that MAb MAHD6 binds to a LOS epitope (-DD-Hepp-1-->6-beta-D-Glcp-). This LOS epitope is not present in the hexasaccharide structure of LOS from H. ducreyi ITM 4747 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J. A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Because MAb MAHD6 reacts with the epitope mentioned above, it also discriminates between the two LOS structures, the hexasaccharide group and the nonasaccharide group, of H. ducreyi strains. MAb MAHD7 recognizes the common conserved inner core region of the LOS because it reacts with all H. ducreyi strains and with LOS with minor components in the inner core epitope structure. Rabbit polyclonal sera raised against the LOS from strains CCUG 4438 and CCUG 7470 were tested with the 10 LOS from the H. ducreyi strains. The antiserum to CCUG 7470 reacted with all H. ducreyi strains as did MAb MAHD7, whereas the antiserum to CCUG 4438 reacted with only its homologous strain and strain ITM 4747. Also, the LOSs of our reference strains CCUG 4438 and CCUG 7470 were structurally analyzed by use of sugar analyses and electrospray ionization-mass spectrometry. The hexasaccharide and nonasaccharide structures obtained from LOS of strains CCUG 4438 and CCUG 7470 were identical to the described LOS structures from H. ducreyi ITM 4747 and ITM 2665, respectively. In conclusion, the MAb MAHD6 recognizes an epitope present in the nonasaccharide LOS group, whereas the MAb MAHD7 recognizes a conserved epitope on LOS of H. ducreyi, which is present in all strains of H. ducreyi tested. Two major groups of oligosaccharides were distinguished by their LOS structures and the reactivity of monoclonal as well as polyclonal antibodies. The majority of H. ducreyi strains possess a nonasaccharide structure of LOS.     

A monoclonal antibody directed against an autoimmune epitope on the human beta1-adrenergic receptor recognized in idiopathic dilated cardiomyopathy

A monoclonal antibody (MAb M16) was obtained by immunizing Balb/C mice with free peptide H26R, corresponding to the second extracellular loop of the human beta1-adrenergic receptor (beta1AR), against which functional autoantibodies have been detected in patients with idiopathic dilated cardiomyopathy. The MAb was found to be of IgG2b type and directed against a conformational epitope, encompassing the sequence recognized by the human autoantibodies. BIAcore measurements yielded an equilibrium constant of 6.5 X 10(7) M1 with an association rate constant (kon) of 6.5 X 10(4) M(-1) sec(-1) and a dissociation rate constant (koff) of 1.0 X 10(-3) sec(-1). It immunoprecipitated only poorly the solubilized beta1AR of Sf9 cell membranes. Functionally, the MAb was capable of not only reducing the number of the maximal binding sites to the beta1-adrenergic receptor of transfected Sf9 cell membranes, but also of displaying a positive chronotropic effect on cultured neonatal rat cardiomyocytes. These properties, which the MAb shares with the human autoantibodies, makes it an interesting tool for passive transfer studies in mice.     

A monoclonal antibody defines a geographically conserved surface protein epitope of Babesia equi merozoites.

Babesiosis is a tick-borne hemoparasitic disease affecting horses worldwide. To investigate mechanisms of immunity to this parasite, the antibody response of infected horses to Babesia equi merozoite proteins was evaluated. Immunoprecipitation of B. equi merozoite antigens with sera from infected horses revealed 11 major proteins of 210, 144, 108, 88, 70, 56, 44, 36, 34, 28, and 25 kDa. Monoclonal antibody (MAb) 36/133.97, which binds to live merozoites, immunoprecipitated proteins of 44, 36, 34, and 28 kDa. When immunoprecipitations were performed with in vitro translation products of merozoite mRNA, MAb 36/133.97 immunoprecipitated proteins of 38, 28, 26, and 23 kDa which comigrated with proteins immunoprecipitated by sera from infected horses at 10(-3) to 10(-4) dilutions. In Western blot analysis, MAb 36/133.97 recognized proteins of 44, 36, 34, and 28 kDa, and a 28-kDa protein was identified by sera from infected horses at a dilution of 10(-4). MAb 36/133.97 bound to B. equi isolates from Florida and Europe. Furthermore, the binding of MAb 36/133.97 to merozoite proteins was inhibited by sera of infected horses from 19 countries. Collectively, these data indicate MAb 36/133.97 binds to a geographically conserved peptide epitope on multiple B. equi merozoite proteins, including a merozoite surface protein, and MAb 36/133.97 reacts with a B. equi protein immunodominant in infected horses.     

Monoclonal antibodies to human thyroglobulin: evaluation of immunoreactivity.

We have earlier reported production and characterization of monoclonal antibodies (MAbs) to human thyroglobulin (h-tg). In the present study H10 I MAb was evaluated for its immunoreactivity towards different forms of tg and various human thyroid tumours. The specificity of H10 I MAb was validated by the absence of cross reaction with tri-iodothyronine (T3) Thyroxine (T4) and human gamma globulins. Sodium-dodicyl-sulphate polyacrylamide gel electrophoresed (SDS-PAGE) immunoblot of h-tg on the nitrocellulose membrane revealed multiple immunoreactive bands on reaction with polyclonal antibody (PAb) in comparison with total lack of reactivity with H10 I MAb. The absence of immunoreactivity of H10 I MAb was demonstrated with SDS treated, Dithiothreitol (DT) treated and heat denatured tg using dot immunobinding technique. However, the H10 I MAb was able to react with tg treated with unfolding agents such as urea and guanidine hydrochloride. All the treated forms of tg were equally recognized by PAb. The immunoreactivity of the oxidized/reduced tg towards H10 I MAb was markedly reduced (60.0%) as compared to that obtained with native tg. It appears that H10 I MAb is directed towards conformational epitope involving sulphydryl bonds. Immunohistochemically, a comparable immunoreactivity between PAb and MAb was observed with normal thyroid tissues, follicular thyroid tissues, Hurthle cell carcinoma tissues and poorly differentiated thyroid tumor tissues using immunoperoxidase staining. The sections from papillary carcinoma tissue (thyroid as well as metastatic lymph node) exhibited intense immunoreactivity with PAb. Thyroglobulin present on these sections was not recognized by H10 I MAb. Nonetheless, H10 I MAb was able to detect tg in follicular differentiation wherever present. The absence of immunoreactivity of H10 I MAb in papillary carcinoma strongly suggests that this neoplasm produces tg which is antigenically different from the protein present in the normal tissue. The reactivity of H10 I MAb with metastatic lymph node of an unknown primary origin suggests its usefulness in the identification of prevalent metastasis of differentiated thyroid carcinoma other than papillary type.     

Detection of Neisseria gonorrhoeae by dot-enzyme immunoassay using monoclonal antibodies

A highly sensitive and specific dot-enzyme immunoassay for the detection of Neisseria gonorrhoeae was developed using a pool of monoclonal antibodies (MAbs). The MAbs were obtained following immunization of mice with lithium acetate extracted outer membrane (OM) preparations. Western immunoblotting experiments demonstrated that MAbs NG26 and NG38, both IgG2a, reacted with lipopolysaccharides (LPS) and with the major OM protein, P1, respectively, MAb NG28, an IgG3, did not react in Western immunoblotting, MAbs NG28 and NG38 failed to react with OM treated with proteolytic enzymes or with semi-purified preparation of LPS. MAb NG26 reacted with the same LPS preparation. Binding radioimmunoassay with live bacteria showed that all the MAbs adsorbed to cell surface-exposed antigenic determinants. The limit of detection of the dot-enzyme immunoassay was between 1 and 4 x 10(4) cfu per dot. Using a panel of 177 strains of N. gonorrhoeae, MAbs NG28 and NG38 recognized only P1A and P1B strains respectively. MAb NG26 reacted with P1A, P1B and non-typable strains. These MAbs did not react with other Neisseria species or other bacterial species. Using this pool, the dot-enzyme immunoassay had a sensitivity of 93.2% and a specificity of 100%.     

Aspergillus fumigatus: identification of 16, 18, and 45 kd antigens recognized by human IgG and IgE antibodies and murine monoclonal antibodies.

The immunochemical properties of antigens produced by Aspergillus fumigatus were investigated with biochemical purification techniques in conjunction with the production of murine monoclonal antibodies (MAbs) and binding studies with human IgG and IgE antibodies. A. fumigatus antigens were partially purified by gel filtration and hydrophobic interaction chromatography on phenyl-Sepharose. Two fractions that eluted with either 2 mol/L or 0.15 mol/L of NaCl demonstrated strong binding to human IgG and IgE antibodies. Immunoprecipitation analysis with IgG antibodies from six patients with different Aspergillus-related diseases demonstrated that the 2M and 0.15M fractions contained major antigens of molecular weight 18 kd (Asp f I) and 45 kd, respectively. The 125I-labeled 2M fraction was used to compare IgG antibodies to A. fumigatus in sera from 25 patients with Aspergillus-related diseases. IgG antibodies were significantly higher in patients with allergic bronchopulmonary aspergillosis (geometric mean, 437 U/ml) than in patients with asthma (geometric mean, 14 U/ml; p less than 0.001), but undetectable (less than 5 U/ml) in 43/48 control subjects. A good correlation was found between levels of IgG antibodies to the 125I-labeled 0.15M fraction and the 125I-labeled 2M fraction in sera from 106 patients with cystic fibrosis (r = 0.77; p less than 0.001). Five murine IgG MAbs and two IgM MAbs were raised against the 2M fraction, and immunoprecipitation with the IgG MAb demonstrated two distinct antigens within the 2M fraction, Asp f I, and a 16 kd antigen. The results of a solid-phase RIA with IgG MAb 4A6 demonstrated that approximately 85% of A. fumigatus-allergic patients with allergic bonchopulmonary aspergillosis had IgE antibodies to Asp f I. The three protein antigens defined in these studies are useful probes for investigating the immunopathogenesis of diseases associated with colonization by A. fumigatus.     

Construction and characterization of a H19 epitope point mutant of MDV CVI988/Rispens strain

A recombinant virus, CVI/rpp38, was developed from the Marek's disease virus (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by transfection of CVI988/Rispens-infected chick embryo fibroblasts (CEFs) with plasmid pHA25 DNA containing pp38 gene from GA strain of MDV. Monoclonal antibody (MAb) H19 which reacts with pp38 from GA but not with that from CVI988 was used to screen for recombinant viruses in transfected cell culture plates by immunofluorescent assay (IFA). A positive plaque was isolated, propagated, and purified from cell-free virus particles after sonication of infected CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in IFA but also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CVI988 virus. DNA sequence of the mutant virus showed a substitution of G at position 320 by a resulting in a change of an amino acid residue from arginine to glutamine. Comparison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 and CVI988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified H19 epitope in pp38. This mutant is potentially useful to further explore the biological function of pp38 and its H19 epitope.     

Identification of two distinct allergenic sites on ryegrass-pollen allergen, Lol p IV

Lol p IV is an important allergen of ryegrass pollen. For the immunochemical identification of antigenic and/or allergenic site(s), murine monoclonal antibodies (MAbs) were prepared against Lol p IV. The hybridoma cell-culture supernatants were screened for anti-Lol p IV antibodies by a combination of ELISA and Western immunoblot analyses. The MAbs were finally purified from ascites on a Mono Q ion-exchange column. In a competitive radioimmunoassay with Lol p IV as the solid phase and 125I-labeled MAbs, it was established that MAbs 90, 91, 92, 93, and 94, although they differed in their relative affinities, recognized in common with one another an epitope designated as antigenic site A, whereas MAb 12 recognized a different epitope referred to as site B. Sites A and B were also demonstrated to constitute allergenic determinants of Lol p IV. Differences in the repertoire of specificities of the human IgE antibodies directed to Lol p IV were also demonstrated. Interestingly, it was found that sera from both allergic as well as from nonatopic individuals had IgG antibodies to sites A and/or B.     

Monoclonal antibody identifies a highly conserved and immunodominant epitope of the human immunodeficiency virus transmembrane protein

A murine monoclonal antibody (MAb), 10E9, has been generated which identifies a conserved and immunodominant epitope of the human immunodeficiency virus (HIV) transmembrane protein, gp41. The MAb reacts with the protein backbone of the mature env gene product and also with polyprotein precursor, gp160. Human sera were tested for their ability to competitively inhibit the immunoreactivity of MAb 10E9. Of 100 serum samples obtained from patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC), all showed strong inhibition to the reaction. In contrast, sera obtained from normal donors or those with other viral infections failed to perturb the binding activity of MAb 10E9. The geographic diversity of the AIDS/ARC patients studied provides evidence that the 10E9 epitope of gp41 is highly conserved.     

Unique epitope of bovine immunodeficiency virus gag protein spans the cleavage site between p16(MA) and p2L

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are closely related bovine lentiviruses that are difficult to distinguish by presently available diagnostic methods. Recently, in our laboratory, a monoclonal antibody (MAb; MAb 10H1) against the BIV Gag protein identified a differential epitope, located at the 6.4-kDa N terminus of a 29-kDa Gag capsid protein, which was absent in JDV. To define the essential amino acids of the epitope, a series of primers within the 163 bp of DNA corresponding to the 6.4-kDa protein were designed. The full-length 163-bp DNA fragment and the smaller DNA fragments with deletions were amplified by PCR and then cloned into pQE32 vectors for protein expression studies. The expressed proteins were analyzed with MAb 10H1 by Western blotting. The differential epitope has been narrowed to a 26-amino-acid region (R121 to R146), which includes 6 residues of p16(MA) (where MA represents the matrix protein) and 20 residues of p2L. A synthetic peptide corresponding to the putative 26-amino-acid epitope blocked MAb 10H1 binding to the expressed peptide. These experiments revealed that the epitope spans the cleavage site between p16(MA) and p2L and presumably will be valuable in distinguishing the two viruses.     

A common antigenic epitope in influenza A virus (H1, H2, H5, H6) hemagglutinin]

Avian influenza A viruses belonging to hemagglutinin (HA) subtypes H5 and H6 were studied in the infectivity neutralization test and radioimmunoprecipitation assay (RIPA) with monoclonal antibody MAb C179. This MAb recognizes a conformational antigenic epitope in the stem region of HA formed by two regions (amino acid positions 318-322 in HA1 subunit and 47-58 in HA2), conserved in all H1 and H2 influenza viruses. MAb C179 reacts with HA of H5 viruses in RIPA and neutralizes these strains as efficiently as H2 viruses. C179 precipitates H6 subtype HA but does not neutralize the infectivity of these viruses. Comparison of amino acid sequences of H2, H5, and H6 strains showed identical epitope recognized by MAb C179 in H5 and H6 HAs, which differs from epitopes of H1 and H2 by two amino acids in the HA2 subunit. Causes of disagreement between immunoprecipitation of H6 HA by MAb C179 and neutralization of this serosubtype by this MAb are discussed.     

Unique Epitope of Bovine Immunodeficiency Virus Gag Protein Spans the Cleavage Site between p16MA and p2L

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are closely related bovine lentiviruses that are difficult to distinguish by presently available diagnostic methods. Recently, in our laboratory, a monoclonal antibody (MAb; MAb 10H1) against the BIV Gag protein identified a differential epitope, located at the 6.4-kDa N terminus of a 29-kDa Gag capsid protein, which was absent in JDV. To define the essential amino acids of the epitope, a series of primers within the 163 bp of DNA corresponding to the 6.4-kDa protein were designed. The full-length 163-bp DNA fragment and the smaller DNA fragments with deletions were amplified by PCR and then cloned into pQE32 vectors for protein expression studies. The expressed proteins were analyzed with MAb 10H1 by Western blotting. The differential epitope has been narrowed to a 26-amino-acid region (R121 to R146), which includes 6 residues of p16^MA (where MA represents the matrix protein) and 20 residues of p2L. A synthetic peptide corresponding to the putative 26-amino-acid epitope blocked MAb 10H1 binding to the expressed peptide. These experiments revealed that the epitope spans the cleavage site between p16^MA and p2L and presumably will be valuable in distinguishing the two viruses.     

Immune response against a cross-reactive epitope on the heat shock protein 60 homologue of Helicobacter pylori

We previously established a monoclonal antibody (MAb), designated H9, which reacts with the heat shock protein 60 (HSP60) homologue of Helicobacter pylori as well as with other bacterial and human HSP60s. To determine the importance of a cross-reactive epitope on H. pylori HSP60 in H. pylori immunopathogenesis, we performed (i) mapping of an epitope on H. pylori HSP60 recognized by the H9 MAb, (ii) analysis of immunoglobulin G responses of patients with or without H. pylori infection to its epitope region, and (iii) studies of the protective effect of immunization with its epitope region on H. pylori infection in mice. The epitope recognized by the H9 MAb was mapped to the sequence of amino acids 189 to 203 (VEGMQFDRGYLSPYF) on the H. pylori HSP60 molecule. It was confirmed that the synthesized peptide designated pH9 was recognized by the H9 MAb. Enzyme-linked immunosorbent assay analysis showed that patients with H. pylori infection (n = 349) had significantly lower titers of pH9 antibody than did uninfected patients (n = 200) (P < 0.001), but this was not the case with purified H. pylori HSP60 recombinant Escherichia coli GroEL, or recombinant human HSP60. In C57BL/6 mice immunized with the pH9 peptide with Freund's complete adjuvant (FCA), the number of H. pylori organisms colonizing the stomach was significantly lower than that in mice immunized with pCont plus FCA (P < 0.0001) or FCA only (P < 0.005). The results suggest that the immune response to the cross-reactive epitope (pH9 region) on H. pylori HSP60 is unique and might be associated with protection against H. pylori infection.     

Characterization of a monoclonal antibody recognizing a polymorphic epitope mainly on HLA-DPw2 and DPw4 molecules

A monoclonal antibody (MAb), 13.3.B4, was obtained from a murine hybridoma after fusing Sp2/0 myeloma cells with spleen cells from C3H mice immunized with mouse L cells transfected with the A1 and B1 genes of HLA-DPw4. In radiobinding-assays, MAb 13.3.B4 bound to HLA transfectants expressing DPw2 or DPw4 as well as DPw2 or DPw4 homozygous B-cell lines, while most cell lines expressing other DP determinants were negative. MAbs with known DP or other HLA class II-specificities were used to inhibit binding of MAb 13.3.B4 in a radioimmunoassay. Three MAbs demonstrated inhibition, but their pattern of reactivity with HLA homozygous B cell lines differed from that of MAb 13.3.B4. An evaluation of DNA sequence data showed that MAb 13.3.B4 reacts with all cell lines expressing DP beta-chains of type 2.1, 2.2, 4.1 or 4.2. No correlation between 13.3.B4-reactivity and expression of DP alpha-chain variants was found. The results indicate that MAb 13.3.B4 defines a polymorphic epitope which may be determined by the sequence gly-gly-pro-met at residues 84-87 of the DP beta-chain.     

Protection against candidiasis by an immunoglobulin G3 (IgG3) monoclonal antibody specific for the same mannotriose as an IgM protective antibody

We previously reported that a liposome-mannan vaccine (L-mann) of Candida albicans induces production of mouse antibodies that protect against disseminated candidiasis and vaginal infection. Immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1, specific for a C. albicans cell surface beta-1,2-mannotriose, protects mice against both infections. Another IgM MAb, termed B6, which is specific for a different cell surface mannan epitope, does not protect against disseminated candidiasis. The B6.1 epitope is displayed homogeneously over the entire cell surface, compared to a patchy distribution of the B6 epitope. To determine if protection is restricted to an IgM class of antibody, we tested an IgG antibody. MAb C3.1 was obtained from L-mann-immunized mice. By results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, capture enzyme-linked immunosorbent assay, and immunodiffusion tests, MAb C3.1 is an IgG3 isotype. By epitope inhibition assays, we determined that MAb C3.1 is specific for same mannotriose as MAb B6. 1. As expected by the results of the inhibition assays, immunofluorescence microscopy showed that the C3.1 epitope is distributed on the yeast cell surface in a pattern identical to that of the B6.1 epitope. Kidney CFU and mean survival times of infected mice pretreated with MAb C3.1 indicated that the antibody enhanced resistance of mice against disseminated candidiasis. Mice in pseudoestrus that were given MAb C3.1 prior to vaginal infection developed fewer vaginal Candida CFU than control animals that received buffered saline instead of the antibody. The finding that an IgG3 antibody is protective is consistent with our hypothesis that epitope specificity and complement activation are related to the ability of an antibody to protect against candidiasis.