Assay restriction profiles of three monoclonal antibodies recognizing the G3m(u) allotype. Development of an allotype specific assay

Three monoclonal antibodies raised against a purified human IgG3 paraprotein were found to exhibit a restriction profile for IgG3/G3m(u) and pan-IgG specificity which was dependent on the assay system. When adapted to an IgG3 subclass capture ELISA, all three McAbs discriminated between paraproteins expressing G3m(u) and antithetical markers G3m(st). One of the antibodies (PNF69C) was selected and conditions were optimised for Gm typing purposes. Using this system G3m(u) could be detected on captured IgG3 derived from human sera. This system may prove useful in the elucidation of Gm allotype profiles.     

Determination of homozygosity or heterozygosity for the G2m(n) allotype by a monoclonal, precipitating antibody

Monoclonal antibody SH 21 was found to be a good anti-IgG2m(n) reagent both for the conventional typing (HA inhibition) and for precipitation in polyethylene glycol containing gel. Antibody SH 21, together with monoclonal antibody HP 6014 (anti-IgG2), was used for the discrimination between G2m(+n) homozygotes and heterozygotes in a double diffusion assay, where the two antibodies and a serum sample were placed in corners of a triangle. In the case of a homozygote SH 21 was able to stop the diffusion of IgG2 to a precipitation line, but some IgG2 of a heterozygote was able to diffuse beyond the precipitate, forming a crossed precipitation pattern. The validity of the typing was checked with serum samples from 54 families. All the 33 'obligatory heterozygotes' (G2m(+n) parent(s) of a G2m(-n) child, or the other way round) exhibited the crossed precipitate. The frequency of the G2mn allele in a Finnish population of 297 unrelated adults was 0.409.     

A new assay uses monoclonal anti-Rh(D) antibodies to determine rheumatoid factor specificity: reactivity to a monoclonal antibody of the Gm allotype G3m(21) is more frequent in rheumatoid patients negative for G3m(21)

A new method has been developed to determine the specificities of polyclonal rheumatoid factors (naturally occurring antibodies which react with human Fc gamma) (RF) found in sera from patients with rheumatoid arthritis. In this method, monoclonal anti-Rh(D) antibodies of known IgG isotype and allotype are bound to erythrocytes and then act as the target IgG antigen for RF in a direct haemagglutination test. Using two monoclonal anti-D antibodies of the IgG3 isotype and G3m(21) allotype, which were cloned from different donors, we found that a large number of rheumatoid sera reacted with both these G3m(21) proteins. In contrast reactivity of rheumatoid sera with polyclonal anti-D of the G3m(21) allotype in the direct haemagglutination test was rare. A strong correlation was found between reactivities to both G3m(21) monoclonal anti-D antibodies but not with a monoclonal anti-D antibody carrying the alternative allele, namely G3m(5). Haemagglutination inhibition experiments using human paraproteins of known IgG isotype and allotype provided some additional evidence that this method can detect RF with specificity for the G3m(21) allotypic determinant or a related allotypic determinant in polyclonal rheumatoid sera. When each patient's autoantibody response was related to their Gm phenotype, we found that the frequency of reactivity for G3m(21) monoclonal anti-D antibodies was significantly greater in patients negative for G3m(21) than in patients positive for the G3m(21) allotype. IgM preparations from patients' sera were dissociated at acid pH but no 'hidden' antibodies were found. We suggest trans-placental sensitization as one of several possible interpretations of this finding.     

Epitope mapping and structural analysis of the anti-Der p 1 monoclonal antibody: insight into therapeutic potential

Group 1 allergen from Dermatophagoid pteronyssinus (Der p 1) belongs to the papain-like cysteine protease family and is a major cause of allergic rhinitis and asthma. An anti-Der p 1 monoclonal antibody, mAb W108, was selected and isolated from Der p-specific IgG2b-producing hybridoma clones. Two-dimensional electrophoresis and immunoblotting showed that mAb W108 reacted with four components of Der p extracts with a molecular mass of 35 kDa and pI values varying from 4 to 6; it also reacted with IgE antibodies in the sera of Der p-sensitive patients. In the competitive assay and using azocasein as a substrate, we found that mAb W108 inhibited not only the binding of Der p 1, but also its cysteine protease activity in a dose-dependent manner. The two peptide segments of Der p 1 identified by mAb W108 (aa 151–197 and 286–320) were parts of inter-connecting loops located in the substrate-binding cleft and on the surface of the domain comprising mainly β-sheets. From the predicted interaction between the amino acid sequence in the CDR3 of mAb W108 and Der p 1-binding epitopes, the possible binding sites for mAb W108 to Der p 1 may sterically hinder the IgE epitope and the active site of cysteine protease activity. Administration of mAb W108 in the Der p-sensitized murine model of asthma alleviated allergen-induced airway inflammation and the Th2 cytokine immune response, suggesting its therapeutic potential. These findings can provide new insights into understanding IgE-mediated disease and the design of modified allergen vaccines for future allergen-specific immunotherapy.     

Practical method for production of monoclonal antibody to human IgG allotype G1M F and its applications in ELISAs and dot immunobinding

An anti-G1M F monoclonal antibody was produced by immunization of mice with a single dose of F(ab')2 fragments of normal IgG1-enriched IgG and subsequent fusion of their lymph node cells with P3U1 myeloma cells. Antibody specificity was tested by an ELISA in microtiter plates coated with allotype positive or negative IgG. The usefulness of the antibody as a G1M F typing reagent in inhibition and direct immobilization-type ELISAs and dot immunobinding was demonstrated by re-typing of 100 GM-allotyping control sera. The advantages and disadvantages of these assay methods are discussed.     

Epitope mapping of dengue 1 virus E glycoprotein using monoclonal antibodies

Summary Ten monoclonal antibodies (MAbs) were raised against dengue 1 (DEN 1, Hawaii) virus E glycoprotein. Specificity of the MAbs was tested by ELISA and immunofluoresence. Eight were DEN 1 type-specific, one was DEN group-reactive (DGR) and one was flavivirus cross-reactive (FCR). Two of these type specific MAbs exhibited haemagglutination-inhibition (HI) and neutralized (N) DEN 1 virus in vivo (HS). These two MAbs showed 100% protection against a challenge of 100 LD₅₀ of DEN 1 virus in adult Swiss albino mice. The remaining six MAbs were HI negative, N negative and non-protective against challenge (NHS). Of these only three were reactive in the CF test. The DGR, FCR and one of the NHS MAbs (NHS-3) did not react with DEN 1 virus grown in Vero cells, whereas they reacted with DEN 1 virus grown in LLC-MK2 and C6/36 cells in immunofluorescence, probably indicating a difference in the synthesis/processing of viral proteins in these different cell lines. An epitope map of the E gp was drawn using a computer programme based on the additivity index values. The epitope map delineated five domains, a) S-I representing type-specific, HI positive, N positive and protecting MAbs. b) S-II representing type-specific, HI negative, N negative MAbs. c) S-III representing type-specific HI/N negative MAb, but distinct from S-II. d) DGR representing HI/N negative DEN group reactive MAb. e) FCR representing HI/N negative flavivirus cross-reactive MAb. Epitope analysis of a number of different DEN 1 strains isolated in India over a period of 30 years showed that the domains S-II and S-III which react with HI negative, DEN-1 specific MAbs were variable. The DGR domain and the S-I domains were conserved.     

Epitope mapping and characterization of a neutralizing monoclonal antibody against human adenovirus type 3

Human adenovirus serotype 3 (HAdV-3) has occurred as a global epidemic in recent years causing serious diseases such as pneumonia in pediatric and adult patients. Development of reliable diagnostic reagents and identification of neutralizing epitopes is important for the surveillance and control of infection. In this study, a neutralizing monoclonal antibody (MAb) MAb 1B6 was generated using the HAdV-3 virion. MAb 1B6 specially recognized the HAdV-3 virus particles and the HAdV-3 hexon protein, but not the virus particles or the hexon protein of HAdV-7 and HAdV-4 by western-blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). Analysis using a series of peptides from the hexon protein and chimeric adenovirus (Ad) particles of epitope mutants revealed that MAb 1B6 bound to the exposed region (amino acid positions 414–424 of hexon) in hypervariable region 7 (HVR7). ELISA demonstrated that MAb 1B6 could recognize the corresponding regions of other HAdV-3 genotypes that have some residues substituted. The identification of the neutralizing epitope and the generation of MAb 1B6 may be useful for clinical serotype-specific diagnosis, subunit vaccine construction for HAdV-3 infection, and virion structural analysis.     

Epitope mapping of the HIV-1 gag region with monoclonal antibodies.

A new type of immunochemical mapping of the human immunodeficiency virus type 1 (HIV-1) gag region was performed. By use of native HIV-1 viral lysates or the gag recombinant p24-15 antigen, a new set of monoclonal antibodies (Mabs) to the gag region proteins was generated. Synthetic HIV-1 peptides covering the entire gag region were used to specifically localize the continuous epitopes by direct binding to the Mabs and by blocking the Mab immunoreactivity. The identified immunogenic epitopes were localized between the gag amino acids (aa) 108-127, 203-217, 208-222, 248-282, 273-302, 288-307, 308-322, 331-354 and 408-422. These continuous epitopes formed seven immunogenic regions. One strongly p17-reactive Mab appeared to react with a discontinuous epitope, the components of which were 110 aa distant in the linear sequence: aa 23-27 and 128-132. The synthetic peptides appeared to be more congruent with the Mab-reactive sites in solution than when coated to a solid phase.     

Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the beta 19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies.     

Epitope analysis for human sperm-immobilizing monoclonal antibodies,MAb H6-3C4, 1G12 and campath-1

Human monoclonal antibody, MAb H6-3C4, possesses strong sperm immobilizing activity. MAb H6-3C4 has been suggested by several research groups to react with a carbohydrate moiety of male reproductive tract CD52 (mrtCD52). In the present study, we analysed the epitope on mrtCD52 for MAb H6-3C4 and found that it was polymorphic in Western blot analysis and disappeared after enzymatic removal of the N-linked carbohydrate moiety. Two other monoclonal antibodies (1G12, campath-1) with sperm-immobilizing activity recognized mrtCD52 in a polymorphic manner similar to MAb H6-3C4. Further analysis showed that 1G12 recognized a structure formed by the peptide and/or a glycosylphosphatidylinositol (GPI) anchor portion as does campath-1. Results of a lectin binding assay suggested the presence of O-linked carbohydrates on mrtCD52. Our results also indicated that the peptide portion of CD52 could serve as an epitope for sperm-immobilizing antibodies. It was concluded that the epitope of MAb H6-3C4 is similar to, but distinct from, those of 1G12 and campath-1, and that mrtCD52 contains different antigenic epitopes.     

Production and characterization of monoclonal antibodies to human casein. A monoclonal antibody that cross-reacts with casein and alpha-lactalbumin

Four hybridomas have been produced that secrete monoclonal antibodies to human beta-casein, a protein synthesized by fully differentiated breast epithelial cells. The antibodies have been characterized on immunoblots and have been shown to react with methanol: acetone-fixed sections of human lactating mammary gland. Immunoblots show that three of these antibodies react with casein whereas one, F20.10, also recognizes an epitope present on human alpha-lactalbumin. Computer analysis of the amino acid sequences of these two milk proteins reveals very little sequence homology, leading to the conclusion that the three-dimensional shape, rather than the primary sequence, is important in this cross-reactivity.     

Mouse monoclonal antibodies induced by anti-allotype antibody display internal images of the rabbit VHa1 allotype: direct visualization by immunoelectron microscopy

We have previously shown that injection of adult rabbits with anti-immunoglobulin (Ig) antibody (Ab) induces the expression of genetically unexpected Ig markers, i.e., a1 allotypic determinants. We now show that these rabbit Ig markers can also be induced in mice by a similar treatment; in the latter case the a1 determinants are located in the antigen-combining site and thus represent "internal images". Two monoclonal antibodies (mAb) were developed from mice treated with anti-allotype Ab. These mAb were reactive with all homologous and heterologous anti-a1 Ab but not normal Ig; efficiently inhibited the binding of rabbit a1 Ig to anti-a1 Ab; and elicited the production of anti-allotype Ab when injected into normal mice. To determine whether the a1-like determinants on these mAb were located in the antigen-combining site, immunoelectron microscopy was utilized to directly visualize Ab complexes. Complexes composed of intact Ab and anti-a1 Fab fragments yielded uniform binding patterns which were identical to those produced by anti-idiotypic reactions. In each case, an identical tip-to-tip binding configuration was observed with a single Fab fragment attached at an approximate 180 degree angle to the V region terminus of each Ab arm. In contrast, rabbit a1 Ig bound as many as two anti-a1 fragments per Ig arm; these fragments were attached laterally and at right angles to the intact molecule. These mAb thus provide the first direct evidence that Ab2 beta determinants are located in, or near, the antigen-combining site.     

Epitope mapping of anti-breast and anti-ovarian mucin monoclonal antibodies.

Anti-breast cancer antibodies (BC2, HMPV and 4B6) and an anti-ovarian cancer antibody (OM1) were found to react with mucins--indeed with the protein core encoded by the MUC1 gene. This gene contains a VNTR (variable number of tandem repeats) encoding a 60 bp (= 20 amino acids) repeat sequence and within this amino acid sequence SAPDTRPAP was predicted, by hydrophilicity analysis, to be the immunogenic peptide sequence. The four antibodies were shown to react with MUC1 VNTR encoded peptides in direct binding and inhibition studies. The precise reactivity of the 4 mAbs was mapped using ELISA in both solid and liquid phase, and demonstrated the epitopes to be: APDTR (BC2 and HMPV), PDTR (4B6) and DTRPA (OM1). By using the pepscan method, the epitopes were shorter (PDTR, DTR and DTRP). However when these short peptides (except DTR) were synthesized they did not react; flanking amino acids are needed for the epitopes. Clearly several different methods should be used to define the reactive epitope. Within (S)APDTR, major amino acid substitutions could be made--even of three to four amino acids without altering antibody binding, provided that P and R were not substituted. It was of interest that an anti-ovarian cancer antibody gave similar anti-peptide reactions to the anti-breast cancer antibodies; apparently MUC1 peptides in ovarian cancer are the same as in breast cancer.     

Preparation and characterization of monoclonal antibodies to rotavirus

By utilizing a strain of cultivable simian rotavirus (SA-11) as an immunizing antigen, we prepared 4 clones of mouse-mouse hybridoma, namely C127, C139, C172, and C214 which secreted monoclonal antibodies against the immunogen itself, SA-11 and also against other group A strains such as Wa and S2. Western blot analyses revealed that all of these antibodies are directed against VP6, a 42 kDa major inner capsid protein of group A rotavirus. Competitive experiments suggested that C127, C172 and C214 recognized three distinct epitopes on VP6, while C139 appeared to react with an epitope at or near the same epitope recognized by C172. We developed a two-step ELISA with excellent sensitivity and specificity for rotavirus detection by utilizing C127 and/or C214 as a capture antibody and rabbit anti-rotavirus conjugated with horseradish peroxidase as a probe. Also, when both monoclonal C127 capture antibody and polyclonal rabbit anti-rotavirus-HRP were incubated with rotavirus simultaneously in a one-step assay, equivalent sensitivity and specificity were observed. The data show that these generated anti-rotavirus antibodies can be utilized effectively as reagents for the detection of human rotaviruses in stool specimens.     

Production and characterization of monoclonal antibodies to ARVCF

We have generated the first monoclonal antibodies (MAbs) to Armadillo repeat gene deleted in velo-cardiofacial syndrome (ARVCF), a recently identified Armadillo repeat-containing protein closely related to the catenin p120ctn. Six ARVCF-specific MAbs were characterized for isotype, species cross-reactivity, and utility in assays including immunofluorescence, immunoprecipitation, and Western blotting. All six antibodies were isotyped as IgG1 and several cross-reacted with ARVCF from a variety of species including human, rat, dog, and monkey, but not mouse. Importantly, none of the ARVCF MAbs cross-reacted with p120ctn, despite the high homology between these proteins. MAbs 3B2 and 4B1 were consistently the best in all applications and will provide valuable tools for further study of the role of ARVCF in cells.     

Production and characterization of two monoclonal antibodies to human pancreatic trypsin 1

Two monoclonal antibodies (MAb) G6 and A8 directed against human trypsin 1 have been produced by hybridization of myeloma cells with spleen cells of OF1 immunized mice. Antibodies were screened by radioimmunoassay. Monoclonal antibodies were purified by affinity chromatography on Protein A-Sepharose, and we found that MAb G6 was of the IgG2b class and MAb A8 of the IgG2a class. Both MAbs had a high affinity for trypsin 1 with the respective affinity constants equal to 1.3 x 10(8) l/mol for G6 and 3.2 x 10(7) l/mol for A8. Epitope specificity was studied by western blotting, using human trypsinogens 1 and 2. Both MAbs gave a positive reaction with native trypsinogen 1 and no reaction with the same protein after reduction. Only MAb G6 reacted with trypsinogen 2 in the native form. Its affinity for trypsin 2 was found similar to that for trypsin 1 with a constant equal to 2.7 x 10(7) l/mol. Both antibodies appeared directed against conformational and not sequential epitopes.     

Production and characterization of monoclonal antibody to gentamicin

Splenocytes from BALB/c mice immunized with a gentamicin-hemocyanin conjugate were fused with X63-Ag8.653 murine myeloma cells to produce hybridomas that secreted monoclonal antibody to gentamicin. Sixteen positive clones were obtained. The monoclonal antibody chosen for a gentamicin immunoassay has been characterized with respect to class, subclass, type of light chain, electrophoretic homogeneity, and binding affinity. Gentamicin monoclonal antibody purified from mouse ascites fluid was analyzed by immunoelectrophoresis, double immunodiffusion, enzyme-linked immunosorbent assay (ELISA), and polyacrylamide gel electrophoresis (PAGE). The results show that the antibody is an IgG2a (kappa). Two bands were detected when the purified antibody was electrophoresed on polyacrylamide gels: a major band and a less mobile minor component (5.6% of the major band). Both were IgG2a (kappa). The major band contained antibody which bound 2.1 moles of the substrate-labeled gentamicin derivative, beta-galactosyl-umbelliferone sisomicin, per mole of IgG, whereas one mole of the minor band bound only 0.095 moles of the substrate-labeled conjugate. The antibody has an affinity constant of 1.22 X 10(10) M-1.     

Epitope mapping of monoclonal antibodies raised to recombinant Mengo 3D polymerase

The cDNA coding sequence of the RNA-dependent RNA polymerase (3D^pol) of Mengovirus was cloned and expressed in a bacterial system. Eleven monoclonal antibodies were raised against the recombinant Mengo 3D^pol (rM3D). All of them recognized the recombinant and the viral-induced form of the protein. The panel of monoclonals belonged to the IgG1 and IgG2a isotypes and were mapped to four different epitopes in the 3D molecule by competition assays. All monoclonals recognized Mengo 3D^pol in western blots and cross-reacted with the homologous polymerases of seven other cardioviruses but failed to react with 3D^pol from poliovirus type 1 and 3 or rhinovirus type 14 and 16.     

抗人滋养细胞单克隆抗体的制备及其免疫生物学特性鉴定

研制抗人滋养细胞单克隆抗体并分析其免疫生物学特性。方法采用滋养细胞免疫Ｂａｌｂ／ｃ小鼠;用细胞ＥＬＩＳＡ法鉴定ｍＡｂ的特异性;用补体依赖的溶细胞作用测定ｍＡｂ的细胞毒性。结论利用细胞免疫法所获该组ｍＡｂ所针对的抗原表位,可能是诱发免疫性流产的滋养细胞膜特异性抗原。     

Functional characterization of monoclonal antibodies to interferon-tau

Interferon tau (IFNtau) produces an array of biological effects, including antiluteolytic, antiviral, antiproliferative and immunomodulatory activities, without the consequent cytotoxicity associated with other type I IFNs. Four anti-IFNtau monoclonal antibodies (MAbs) have been characterized by determining regional epitopes and observation of their effects on IFNtau binding, antiviral and antiproliferative activity. Using an enzyme-linked immunoadsorbent assay (ELISA) developed against six overlapping synthetic peptides representing the entire linear sequence of IFNtau, three antibodies, HL-98, HL-100 and HL-127, were found to react with the carboxy terminal peptide, while HL-129 bound the penultimate amino terminal peptide. Binding studies indicated that MAbs directed against either region could effectively inhibit the binding of alkaline phosphatase labeled IFNtau to cells expressing type I IFN receptors. While only two of the MAbs significantly reversed IFNtau-induced growth inhibition, the antiviral activity of IFNtau was significantly inhibited by MAbs that bound the amino and carboxy termini, confirming the functional importance of these domains in the binding and subsequent activity of IFNtau.