Monoclonal antibodies against microorganisms

The recent spread of hybridoma technology among laboratories has promoted the development of monoclonal antibodies against a wide variety of infectious disease agents. While monoclonal antibodies theoretically represent an excellent (perhaps superior) alternative to conventional antisera as diagnostic, therapeutic or laboratory reagents, traditional antisera may be preferable to monoclonal antibody in some circumstances because of the fixed affinity and specificity as well as the limited functional capacities of some antibodies. The acceptance of monoclonal antibodies by the clinical microbiologist and physician must await proof of their reliability, safety and efficacy.     

Monoclonal antibodies against glycophorin A

Two mouse IgM monoclonal antibodies, 177.1 and 179.3, are directed against glycophorin A, the major sialoglycoprotein of human erythrocytes. Both antibodies agglutinate blood group M and N erythrocytes equally well, both before and after treatment with neuraminidase or trypsin, but fail to agglutinate erythrocytes treated with papain. Antibody 179.3 agglutinates MiVII(K.T.) cells, whose glycophorin A probably contains some alterations in amino acid sequence between residues 46-56, but antibody 177.1 does not agglutinate these cells. Neither antibody agglutinates En(a-)G.W. cells, which lack glycophorin A completely. The hemagglutinating activity of antibody 177.1 is inhibited by purified glycophorin A and its chymotryptic glycopeptides CH1 (amino acid residues 1-64) and CH3 (amino acid residues 35-64), whereas the hemagglutinating activity of 179.3 is inhibited weakly by glycophorin A but not by chymotryptic peptides. These antibodies both are classified as anti-En(a-)FS but apparently bind different epitopes.     

Monoclonal antibodies against Trichomonas vaginalis

Spleen lymphocytes obtained from mice immunized with Trichomonas vaginalis ATCC 30001 were fused with P3-X63-Ag8-653 mouse myeloma in order to produce hybridoma-secreting antibodies against T. vaginalis associated antigens. Six hybridoma cloned cell lines were established; three produced IgG1, two produced IgG2a, and one produced IgM monoclonal antibody. These six monoclonal antibodies showed binding to seven isolated strains of T. vaginalis but did not bind to Giardia lamblia. Three of those monoclonal antibodies did not bind to Tritrichomonas foetus. These anti-trichomonal monoclonal antibodies should prove to be of great value as diagnostic and research reagents.     

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.     

Autoimmune gastritis: tolerance and autoimmunity to the gastric H+/K+ ATPase (proton pump).

The alpha and beta subunits of the gastric H+/K(+)-ATPase (proton pump) have been identified as the major molecular targets of parietal cell autoantibodies associated with pernicious anaemia and with murine experimental autoimmune gastritis (EAG) induced by neonatal thymectomy. Recent studies with EAG suggest that the mechanisms of peripheral tolerance and autoimmunity to extrathymic autoantigens are mediated by subsets of "regulator" and "effector" CD4+ T cells, respectively. The persistence of "effector" CD4+ autoreactive T cells in the periphery may be a direct consequence of the delayed developmental expression of the target autoantigen. We hypothesize that cytokines produced by the "regulator" T cells prevent the clonal expansion of the "effector" autoreactive T cells, and that neonatal thymectomy induces organ-specific autoimmunity in genetically susceptible individuals by the reduction of the "regulator" T cell population.     

Monoclonal antibodies against a plant virus

Summary Eight stable hybridoma clones derived from fusion of spleen cells of Tobacco Mosaic Virus (TMV) immunized mice with murine myeloma cells were grown in mass culture. They were obtained by 2 subsequent limiting dilution cloning cycles and subcultivation. Five of these clones secreted monoclonal antibodies which reacted with TMV nucleocapsid but not with capsid monomers and the monoclonal antibodies secreted by 3 other clones reacted with the TMV capsid monomer but not with the nucleocapsid. The specificity was determined by Enzyme Linked Immunosorbent Assay (ELISA) adjusted for the detection of antibodies in hybridoma culture supernatants.With 2 Figures     

Monoclonal antibodies against hepatitis A virus

Three monoclonal antibodies (K2-4F2, K3-2F2, and K3-4C8) of the immunoglobulin G2a class were raised against hepatitis A virus. The specificity of these antibodies was confirmed by immune electron microscopy, solid-phase radioimmunoassay, and in vitro neutralization in cell culture. Binding studies suggested that they all recognize closely related antigenic determinants. These monoclonal antibodies should prove to be of great value as diagnostic and research reagents.     

Monoclonal antibodies against metallothioneins and metalloproteins

Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.     

Monoclonal antibodies against BLV-transformed sheep cells

Summary Hybrid cell lines were prepared by the fusion of BALB/c myeloma, P3X63Ag 8.653 cells with the spleen cells of BALB/c mice that were immunized with SF-28 cells, sheep fibroblasts transformed with BLVin vitro. Five clones of hybrid progeny produced cytotoxic IgG₁ antibodies against SF-28 cells. The tested monoclonal antibodies showed cytotoxic activity against 4 out of 6 BLV-transformed sheep fibroblasts as well as SF-28 cells but failed to react with other cells, including BLV-producing FLK cells, bovine lymphosarcomas and normal sheep cells. These monoclonal antibodies showed suppressive effect on colony formation of SF-28 cells in soft agar culture.     

Monoclonal antibodies against diagnostic Anisakis simplex antigens

Five monoclonal antibodies (UA2, UA3, UA5, UA6, and UA8) specific for Anisakis simplex are described. All are IgG1/κ monoclonal antibodies, except for UA2, which is an antibody IgM/κ. The molecular weights of the major components recognized in immunoblotting are 48 and 67 kDa (UA2); 139 kDa (UA3 and UA5; same epitope); 35, 38, and 139 kDa (UA6); and 205 kDa (UA8). UA2 was the only monoclonal antibody to recognize both components of an excretion-secretion antigen preparation and antigens in the excretory cell and esophageal glands of third-stage A.␣simplex larvae; antigens in the excretory cell were also recognized by UA3 and UA6. Cross-reactivity studies using a hyperimmune polyclonal rabbit serum reacting with various ascaridoid nematodes indicated that the antigens captured by our monoclonal antibodies were specific for A. simplex. Finally, comparative studies of our monoclonal antibodies and An2 (the only monoclonal antibody currently available for serodiagnosis of human anisakiasis), based on the calculation of multiples of normal activity for human anisakiasis sera, indicated that our monoclonal antibodies (and particularly UA3) recognized antigens that are good candidates for serodiagnostic purposes. Received: 13 February 1997 / Accepted: 16 March 1997     

Monoclonal antibodies against rat immunoglobulin kappa chains

Monoclonal antibodies directed against rat kappa light chains have been generated by immunizing SJL/J mice with soluble rat immunoglobulin, followed by fusion of immune spleen cells with the P3-X63-Ag8 myeloma cell line. Monoclonal antibodies from three of these hybridoma cell lines, MAR 18.5, 80.2, and 103.6, have been extensively characterized. MAR 18.5, 80.2, and 103.6 antibodies are of the gamma 2a kappa isotype, and bind strongly to protein A, allowing easy purification. Monoclonal antibody from clone 18.5 binds equally well to Ig of both RI-1a and RI-1b allotypes, whereas 80.2 and 103.6 antibodies selectively bind to RI-1b. These monoclonal antibodies can be FITC conjugated for use as a second antibody in indirect immunofluorescence assays, or radiolabeled for use in radio immunoassays requiring a specific antirat kappa antibody. The antiallotype specific monoclonal antibodies also may be of use in the study of rat immunoglobulin genetics.     

Monoclonal antibodies against Neisseria meningitidis lipopolysaccharide.

A cell line producing monoclonal antibodies directed against a lipopolysaccharide component of Neisseria meningitidis group A has been established. These antibodies reacted with only one of three lipopolysaccharide serotyping strains of group A meningococci by coagglutination, enzyme-linked immunosorbent assay, and Western blotting techniques. A Western blot analysis showed that a NaOH digest of lipopolysaccharide was detectable by the serotype-specific antibody. The monoclonal antibodies cross-reacted with a group B meningococcal strain in an enzyme-linked immunosorbent assay. The immunoblotting analysis also showed that these antibodies reacted with the lipopolysaccharides of a group B meningococcus as well as Haemophilus influenzae type B, but not with the lipopolysaccharides of several strains of Salmonella typhi, Escherichia coli, Streptococcus pneumoniae, and Neisseria gonorrhoeae.     

Monoclonal antibodies against rat leukocyte surface antigens

Summary: Monoclonal antibodies have proven to be powerful tools for studying the properties of leukocyte surface antigens and the cells that express them. In the past decades many monoclonal antibodies (mAb) for identifying the different rat leukocyte surface antigens have been described. A list of mAb is provided in Table 1 below. The rat leukocyte surface antigens are divided into different sections, including rat CD antigens (a), rat leukocyte surface antigens without CD designation (b), rat major histocompatibility complex (MHC) antigens (c), rat T-cell receptors (d) and rat immunoglobulins (e). The molecular and functional characteristics of rat leukocyte surface antigens are discussed in more detail in some of the other chapters of this issue (e.g. Van den Berg et al., p. 45). A more extensive overview of the properties of leukocyte surface antigens is provided by Barclay et al. (1).     

Monoclonal antibodies against mouse splenic stromal cells

A panel of rat monoclonal antibodies directed against mouse splenic stromal cells were isolated. These monoclonal antibodies were Immunohistochemically divided into four groups which reacted with non-lymphoid cells of the murine spleen; (1) in the white pulp, (11) at the marginal zone, (111) in the red pulp, and (IV) on the endothelium of splenic blood vessels. These monoclonal antibodies were studied Immunohistochemically In lymphoid organs by means of light and electron microscopy. Monoclonal antibodies SS-4 (group I) reacted with fibroblastic reticulum cells that were distributed only in the white pulp of the spleen and In the follicular areas of lymph nodes. The SS-4 staining cell, In clustered splenic stromal cells, formed colonies which Included a small number of Thy-1 positive lymphocytes. Therefore, we concluded that SS4 staining stromal cells comprise the lymphoid cornpartment. In contrast, monoclonal antibodies SS-1, SS-3 and SS-5 (group II) reacted with dendritic shaped cells in the marginal zone of the spleen. Examination of splenic extra-medullary hematopolesis in mice rescued by bone marrow transplantation after lethal irradiation revealed that SS-3 and SS-5 reacted with dendritic shaped stromal cells in clonal nodules of engrafted marrow in the red pulp. SS-3 and SS-5 staining cells could not be observed in physiologic hematopoiesis of non-transplanted mice. It was suggested that SS3 and SS-5 staining stromal cells are Involved in primitive hematopoiesls. Monoclonal antibodies SS2, SS-6 and SS-7 (group 111) mainly reacted with dendritic cells and macro-phages in the red pulp. Monoclonal antibodies SS-8 and SS-9 (group IV) reacted with endothelial cells of blood vessels and sinuses. These findings of heterogeneity in mouse splenic stromal cells are further evidence that specific micro-envlronments are composed by speclalired stromal cells.     

Monoclonal antibodies against Vibrio cholerae lipopolysaccharide.

A cell line producing monoclonal antibodies directed against the core region of Vibrio cholerae lipopolysaccharide has been established. These antibodies were inhibited by lipopolysaccharide preparations of both O-group 1 vibrios and some non-O-group 1 vibrios as detected in enzyme-linked immunosorbent assay-inhibition experiments. Coagglutination experiments with monoclonal and polyclonal antibodies adsorbed to protein A-carrying staphylococci were performed. All V. cholerae strains tested, regardless of serotype, were agglutinated when mixed with staphylococci coated with the monoclonal antibodies, whereas staphylococci coated with group-specific (O1) polyclonal antibodies only agglutinated with O-group 1 vibrios.     

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.