Monoclonal antibodies against isotypic and isoallotypic determinants of human IgA1 and IgA2: fine specificities and binding properties

We have analysed and compared the fine specificity and behavior in various immunoassays of 10 mouse monoclonal antibodies, from three independent laboratories, directed against IgA1, IgA2 or non-IgA2m(2). The following observations were made. (1) Although all of the monoclonal antibodies were specific for a particular IgA subclass or isoallotype in a radioimmunoassay, three of them were not specific when tested in indirect immunofluorescence on plasma cells derived from pokeweed-activated peripheral blood lymphocytes. In this highly sensitive system, contrary to direct immunofluorescence previously performed using formalin-fixed lymphoid tissue, the anti-IgA1 69.114 reacted with some of the IgA2 plasma cells, the anti-IgA2 DLDB7 reacted with some of the IgA1 plasma cells and the anti-IgA2 16.512 dimly reacted with all IgM plasma cells. (2) Among the eight anti-IgA subclass antibodies, seven were directed against the CH2 domain of IgA whereas the anti-IgA1 1-155-1 recognised an epitope destroyed by Streptococcus sanguis IgA1 protease and localised in the hinge region of IgA1. The two anti-isoallotype antibodies were directed against epitope(s) probably localised in the 65 C-terminal amino acid residues of the alpha-CH3 domain. All of the 10 antibodies were able to react with endogeneously produced surface IgA on B-cells. (3) Using monoclonal anti-IgA subclass antibodies in radioimmunoassay may be hazardous in the absence of knowledge of their affinity constants and of careful control experiments: some of the antibodies were not sensitive in radioimmunoassays designed to measure the serum titer of specific IgA1 and IgA2 antibodies. Moreover, major differences were observed between the different monoclonal reagents with respect to the influence of the size of IgA on a solid-phase sandwich radioimmunoassay. While three of the anti-IgA1 underestimated dimeric IgA relative to monomeric IgA, the fourth anti-IgA1 and all the anti-IgA2 overestimated dimeric IgA relative to monomeric IgA, by a factor sometimes close to 7.     

Monoclonal antibodies to amoxicillin express different idiotypes determined by anti-idiotype antibodies production

Penicillins are β-lactam antibiotics able to generate several antigenic determinants that are recognized by the immune system. To study the differences in the antigen binding site of two monoclonal antibodies (Mab) specific to amoxicillin, polyclonal rabbit anti-idiotypic antibodies were produced. One Mab, AO3.2 (IgG2a), specific to a structure formed by the acyl-side chain structure and a part of the nuclear region of amoxicillin. The second one, AO6.2 (IgE), is specific to the side chain of amoxicillin, although it also recognizes the side chain of other penicillins (penicillin G and ampicillin). These antibodies were used to immunize rabbits in order to produce polyclonal anti-idiotipic antibodies, which were purified in several steps by affinity chromatography. The specificity and cross-reactivity studies were made by ELISA and ELISA inhibition. The results suggest that the anti-Id antibodies produced are the internal image of the antigen, since the binding to their specific idiotype is blocked mainly by the original hapten (amoxicillin): in 98% of the cases with anti-id-1 (induced against AO3.2) and in 59% with anti-id-2 (induced against AO6.2). The absence of cross-reactivity of each anti-idiotypic antibody with the different Mabs specific to amoxicillin shows that the idiotypes induced by the same hapten have differences that are reflected by the nonrecognition of these anti-idiotypes. We conclude that such a small molecule as amoxicillin can present several antigenic determinants that induce a panel of antibody specificities especially directed against the side chain.     

Pattern of anti-cytomegalovirus IgM antibodies determined by immunoblotting

Summary In order to improve the knowledge of the humoral immune response to CMV infection, we developed an immunoblotting technique which allowed a better analysis of the changes in the pattern of anti CMV-polypeptides IgM. We examined 234 sera belonging to 27 renal allograft recipients developing a primary or recurrent CMV infection and 12 non infected recipients. Thus we found that 11 main anti CMV-polypeptides IgM antibodies were present in over 25% of the infected patients. They reacted with proteins whose molecular weights ranged from 32K to 205 K.We showed that anti-p 45–47 IgM antibodies were present in 100% of CMV infected recipients and never in the non-infected population. They appeared very early in the course of the infection (5.43 weeks post-graft for primary infection and 5.00 weeks for recurrent ones) and, therefore, constitute a good marker of active infection. Two other CMV-specific IgM antibodies (anti-p 60–64 and anti-p 100) were found exclusively in the course of primary infections. Anti-p 60–64 IgM was observed at a high frequency (57.1%) and with a mean delay of 6.57 weeks post-graft. Therefore, the anti-p 60–64 IgM detection could be helpful for the diagnosis of primary infection. In almost 100% of both primary and recurrent infections, we observed anti-p 140 and anti-p 38 IgM antibodies. Only about 50% of non-infected patients had low levels of anti-p 140 and anti-p 38 IgM.The follow-up of recurrent infections showed that the anti CMV-polypeptides IgM antibodies appeared earlier than in primary infection. When we compared anti-p 45–47 IgM detection by immunoblotting and anti-CMV IgM detected by ELISA we observed that immunoblotting permitted the diagnosis 2.5 weeks earlier for primary infection, and 1 week earlier for recurrent infection, than ELISA. In addition, the detection of anti-p 45–47 IgM antibodies also occurred earlier than virus excretion.     

Monoclonal antibodies against human haptoglobin

Three monoclonal antibodies: 2.36.71.41, 7.60.66.55, and 18.4.40. 80 to human haptoglobin 2-1 were produced, purified and characterized. The affinity constants ranged within 0.3-2.4 x 10(8) M-1. The monoclonal antibodies 7.60.66.55 and 18.4.40.80 reacted with beta subunit of haptoglobin, showed similar epitope affinities and epitope densities on main haptoglobin types. However, the epitope on the haptoglobin molecule for the monoclonal antibody 18.4.40.80 occupied somewhat more surface than that for the antibody 7.60.66.55. The monoclonal antibody 2.36.71.41 was able to bind both alpha and beta chains of haptoglobin. In ELISA affinity reactions this antibody achieved with haptoglobin 2-2 the plateau phase at absorbance values 15% higher than with haptoglobin 2-1, and 60% higher than with haptoglobin 1-1. End-point titration of the monoclonal antibody 2.36.71.41 against three haptoglobin types showed differences in titer, indicating distinct epitope densities.     

Monoclonal antibodies with different specificities against cytokeratins. An immunohistochemical study of normal tissues and tumors.

Monoclonal antibodies against cytokeratins, isolated from human callus, were prepared. Here we describe the characterization of three of these monoclonal antibodies (Clones 77, 78, and 80) with special emphasis on the staining characteristics of a number of normal epithelial tissues and a series of tumors. On thin sections and in immunoblotting experiments our monoclonal antibodies showed different specificities. In immunoblots Clone 80 stained more bands in preparations of cytokeratin from callus, cultured keratinocytes, and a metastasis of a lung carcinoma than Clones 77 and 78. In this last cytokeratin preparation Clones 77 and 78 each stained a separate band not stained by Clone 80. In normal tissues Clone 80 stained all types of epithelia except myoepithelium and podocytes of glomeruli. Clone 78 stained mainly squamous epithelium. Clone 77 stained differentiated squamous epithelium, transitional epithelium, ductal epithelium, and parenchymatous epithelium. These results confirm the heterogeneity of cytokeratins. In neoplasms Clone 77 was positive with adenocarcinoma and squamous cell carcinoma. Clone 78 stained squamous cell carcinoma and well-differentiated adenocarcinoma. Clone 80 stained all epithelial tumors, including anaplastic carcinoma, and is therefore useful in tumor diagnosis.     

Virus-specific serum IgG, IgM, and IgA antibodies in cytomegalovirus mononucleosis patients as determined by immunoblotting technique

The immune response to individual human cytomegalovirus (CMV) structural polypeptides was studied in paired sera from 15 adult CMV mononucleosis (CMV-MN) patients and healthy controls by immunoblotting technique (IB). IgM and IgG antibodies to at least 11 structural polypeptides with molecular weights of 28K, 49K, 55K, 57K, 66-70K, 82K, 87K, 110K, 150K, 205K, and 235K were detected in the patients' sera in the serum sample obtained in the acute phase of the disease. IgA antibodies to polypeptides with molecular weights of 66-70K, 82K, 110K, and 150K were also detected in these sera. In healthy seropositive adults, IgG antibodies with the same molecular weight polypeptides, excluding the 205K and 235K polypeptides, were detected as in convalescent CMV-MN patients. A prominent reactivity of IgM and IgA antibodies to the 66-70K and 150K polypeptides was noted in the acute sera from all the CMV-MN patients examined, but not in a number of late convalescent sera. The potential implications of these findings in the development of specific serological tests are discussed.     

Monoclonal antibodies to distinct epitopes on human IgA and their use to IgA determination

Several monoclonal antibodies (MAbs) against human IgA have been obtained which specifically bind to human myeloma and polyclonal IgA. Three of these MAbs have been purified and studied further. They recognize both IgA subclasses and define three distinct epitopes on the IgA molecule. These MAbs were used to develop a solid-phase radioimmunoassay (RIA) in which one MAb was immobilized and the other two were labeled with 125I. The assay has a sensitivity in the nanogram range. A good correlation was found (r = 0.97, P less than 0.001) when the solid-phase RIA was compared with a commercially available immunodiffusion technique for the determination of IgA levels in serum samples.     

Monoclonal antibodies against human antithrombin III.

Three monoclonal antibodies identified as D8, B11 and C5 of different specificities have been produced against human antithrombin III (AT). The apparent dissociation constants (Kd app) of the AT-antibody interaction were determined by ELISA method: Kd app (D8) = 2.4 nmole, Kd app (B11) = 13 nmole, Kd app (C5) = 24 nmole. All three antibodies reacted with isolated AT on immunoblots obtained with "native" PAGE. The D8 antibody also reacted with plasma and serum AT while B11 antibody reacted with serum thrombin-antithrombin (TAT) complexes as well.     

Monoclonal antibodies of four different specificities for neutralization of type 1 polioviruses.

Four independent hybridoma clones have been established that produce neutralizing antibodies specific to type 1 poliovirus. Each clone produced antibody which neutralized a distinct set of type 1 test strains: (i) all 15 strains tested; (ii) the inducer strain only; (iii) predominantly wild strains; or (iv) all vaccine-related and some wild strains.     

Monoclonal antibodies against human erythrocyte membrane antigens and the antigens recognized by these antibodies

Six monoclonal antibodies (KOR-E1-E6) were raised against human erythrocyte membrane antigens. Aggregating reactions of normal human erythrocytes with or without enzyme treatment and specific antigen deficient (null type) erythrocytes were used for detection of the antigens. SDS-polyacrylamide gel electrophoresis with Western blotting and immunoperoxidase methods were also used to confirm the results. The antigen recognized by KOR-E1 and KOR-E5, which was sensitive to protease, trypsin, and neuraminidase, and only expressed on human erythrocytes, was identified as Pr1h. The antigen recognized by KOR-E2 and KOR-E6 was identified as the EnaTS portion of glycophorin A, because the antigen was sensitive to protease and trypsin, but resistant to neuraminidase, and was not present on En(a-) erythrocytes. The antigen recognized by KOR-E3 that was protease-, trypsin-, and neuraminidase resistant, and absent on En (a-) erythrocytes, was identified as Wrb antigen. As KOR-E4 reacted with all erythrocytes examined, the antigen it recognizes could not be determined.     

Monoclonal antibodies against Enterocytozoon bieneusi of human origin

Enterocytozoon bieneusi is clinically the most significant among the microsporidia infecting humans, causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. The lack of immune reagents is largely due to the absence of methods for laboratory propagation of E. bieneusi. We recently described a procedure for the concentration and purification of spores from diarrheic stool of infected humans. Purified spores were used to immunize mice for production and screening of monoclonal antibodies (MAbs) against E. bieneusi. The eight immunoglobulin M MAbs generated and fully characterized did not cross-react with other human microsporidia or with other microorganisms normally present in stool. One of the MAbs, 2G4, reacted with E. bieneusi spores in stools from monkeys and humans, without background fluorescence, which makes it an ideal diagnostic reagent. It also recognizes intracellular stages of the parasite and will be suitable for determining tissue distribution of E. bieneusi in infected hosts. At least two immunodominant antigens of E. bieneusi of 33,000 and 35,000 Da exist, which were recognized by rabbit and mouse antisera. The availability of MAbs against E. bieneusi will simplify considerably the diagnosis of this infection in humans and will provide tools for epidemiologic investigations regarding the true prevalence of the infection in various human and mammalian populations and the environmental sources of infection.     

Serum antibodies in rickettsia patients as determined by immunoblotting technique.

The Western-blot technique (WB) was used to determine which polypeptides of Israeli spotted fever (ISF) isolates and other spotted fever group rickettssia (SFGR) reference isolates (G212, S484, A828) and two reference strains. R. Rickettsii (Sheila Smith strain) and R. conorii (Boutonneuse fever), were used as antigen sources for the WB. Immunoperoxidase assay (IPA) seropositive (titer greater than 80) and seronegative (titer less than) sera were examined with the separated polypeptides of the above strains. WB analysis of the rickettsial polypeptide-serum reactions showed that R. conorii and the three isolates of ISF reacted identically with the sera, except that in the three ISF strains a 175 kD protein was present. It was also observed that all of the IPA seropositive sera examined reacted with the following polypeptides: 18kD, 20kD, 22kD (28kD to 37 kD LPS group), while each seropositive and seronegative serum reacted differently with polypeptides 23kD, 42kD, 45kD, 46kD, 52kD, 55kD, 70kD, 82kD, 105kD, 125kD, 155kD and 175kD. Using this technique, no heat labile polypeptides (preelectrophoretic treatment: 100 degrees C for 2 min vs 37 degrees C for 20 min) were observed in SFGR strains used in this study. Our results indicate that the immunoblot technique shows no difference between R. conorii and ISF antigens except the existence of 175kD protein antigen in the latter.     

Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315–43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1. 80H.3. and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (lgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (lgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence. i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited BFU-GM and CFU-E. Antigens recognized by 80H.3. 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.     

Monoclonal antibodies against differentiation antigens on human macrophages

Human macrophages matured in vitro from blood monocytes without additional exogenous activation were used to produce monoclonal antibodies (mAbs). Ten of them were selected for high avidity and the best discrimination between mature macrophages and a panel of other cells including freshly prepared monocytes, B cells, T cells, granulocytes and thrombocytes. Five mAbs (MAX. 1, MAX. 2, MAX. 3, MAX. 11, MAX. 24) were found to detect macrophage differentiation antigens. One mAb (MAX. 26) detects an antigen which is shared by mature macrophages and T cells.     

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.     

Monoclonal antibodies to the amino- and carboxyl-terminal domains of human transferrin.

Seven high affinity antibodies to human serum transferrin which recognize at least four different epitopes are described. Apparent dissociation constants (Kd's) have been determined for the binding of the antibodies to human transferrin in the presence and absence of iron. Small differences in reactivity were found. Five of the antibodies bind to the isolated amino-terminal half-molecule of human transferrin. Two of the antibodies appear to be to the C-terminal lobe since they bind to holo-transferrin but do not recognize the N-terminal half-molecule. Immunoblotting shows that six of the antibodies recognize both reduced and nonreduced transferrin. In addition, all of the antibodies bind with sufficiently high avidity to transferrin to make them useful as probes in studies in which binding of transferrin to the specific transferrin receptor is examined.     

Monoclonal antibodies to baculovirus structural proteins: determination of specificities by Western blot analysis

Conventional mouse hybridoma technology was utilized to produce a panel of monoclonal antibodies which reacted with baculovirus proteins. Using an enzyme-linked immunosorbent assay (ELISA), the hybridomas which were raised against polyhedrin from Autographa californica nuclear polyhedrosis virus (AcNPV) and Choristoeura fumiferana nuclear polyhedrosis virus (CfNPV) were found to cross-react differentially with polyhedrins and granulins from several species of baculoviruses. Hybridoma antibodies which reacted against the nonoccluded form (NOV) of AcNPV in an ELISA test expressed different specificities for the occluded form of the virus (OV), a mutant strain of AcNPV, and CfNPV. Four hybridoma clones produced antibody which neutralized the infectivity of AcNPV NOV. One hybridoma antibody reacted strongly with the uninfected Spodoptera frugiperda host cell line. Using Western blot analysis, it was shown that hybridoma antibodies against polyhedrin reacted differentially with the complete polypeptide and protease-generated fragments of polyhedrin. The polypeptide specificity of 19 of 28 hybridoma antibodies which reacted with OV and NOV of AcNPV was assigned using Western blot analysis.