Use of tetrameric antibody complexes to stain cells for flow cytometry

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.     

Epitopes in human growth hormone and chorionic somatomammotropin studied with monoclonal antibodies

The immune complexes formed by human growth hormone or human chorionic somatomammotropin and various monoclonal antibodies have been studied by gel filtration and polyacrylamide gel electrophoresis. Two of the monoclonal antibodies gave rise to complexes with molecular weights suggesting an antigen:antibody 1:1 ratio. When both antibodies were simultaneously incubated with human growth hormone the ratio estimated for the new complex was 1:2, indicating the existence of two nonoverlapping epitopes in the antigen. The other monoclonal antibodies exhibited a more intricate behavior: incubated separately with human growth hormone they gave rise to both types of the aforementioned complexes. A similar phenomenon could be demonstrated with human chorionic somatomammotropin. The study of the immunoreactivity of a synthetic peptide indicates that the involved epitopes are localized within the region limited by amino acid residues 44 and 128 of human growth hormone.     

Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.     

Interaction of monoclonal antibodies with horseradish peroxidase

Properties of four types of monoclonal antibodies to horse-radish peroxidase were investigated. The dissociation constants and molecular-weight composition of the immune complexes were determined. The antibodies are shown to be directed to different epitopes on the polypeptide chain. Results of the theoretical prediction of the epitope localisation are presented. The interaction between the antibodies and peroxidase isozymes were studied.     

Detection and localization of a lectin on Actinomyces viscosus T14V by monoclonal antibodies

A cell-associated lectin activity that mediates lactose-inhibitable adherence of Actinomyces viscosus T14V has been localized to a specific population of fimbriae by the use of monoclonal antibodies. Nine monoclonal antibodies were produced that reacted with only 1 of 2 immunoelectrophoretically distinct fimbrial components on T14V. The fibrillar morphology of this component was revealed by the immunoelectronmicroscopic examination of bacteria incubated with the monoclonal antibodies. The lectin activity associated with these structures was detected when isolated fimbriae were cross-linked with monoclonal antibodies to form immune complexes with agglutination activity for neuraminidase-treated human erythrocytes, a reaction that was inhibited by lactose. Although the 9 monoclonal antibodies differed in their fine specificities, they reacted only with strains of A. viscosus and A. naeslundii that exhibited lactose-inhibitable adherence. These findings indicate that the lectin activity common to these bacteria resides on fimbriae that are antigenically related to those of T14V.     

Comparative study of assays detecting circulating immune complexes and specific antibodies in patients infected with Toxocara canis

A sandwich ELISA method using previously described E/S antigen-specific monoclonal antibodies has been developed to detect circulating immune complexes in patients infected with Toxocara canis. This technique could be used for the study of the dynamics of the parasite-host relationship, as we believe the detection of immune complexes and/or soluble antigen to be an improvement over detection of antibodies only. In this parasitosis, antibodies may be present in residual levels for prolonged periods after active infection.     

Evaluation of chemotherapy in experimental toxocarosis by determination of specific immune complexes

Parasitism by the larval phase of Toxocara canis is a chronic process in which the larvae survive in the tissues, resulting in the constant stimulation of the immune system. As a result, the detection of specific antibodies may not reflect the active state of the parasite. We have studied the dynamics of the production of specific immune complexes by ELISA with the monoclonal antibody TC-1 in rabbits inoculated with single and multiple doses of T. canis eggs. We also compared this with the production of specific antibodies and their possible modification after treatment with mebendazole. The specific antibodies against excretory-secretory antigen were detected with peaks at 10 and 12 weeks depending on the dose and remained positive during the entire experiment (62 weeks). Treatment caused an increase in the level of detectable antibodies dropping to similar levels to the controls. Specific immune complexes were detected only in multiple doses, and were then positive during the entire experiment. From the beginning of treatment the values of immune complexes fell quickly, remaining at undetectable levels during the rest of the experiment. For this reason the detection of specific immune complexes is a valid technique for monitoring the efficiency of treatment.     

The influence of anti-(ricin toxin A chain) monoclonal antibodies on the pharmacokinetics of ricin toxin A chain and recombinant ricin A chain in mice

Summary Two monoclonal antibodies against ricin toxin A chain (RTA) have been examined for their effects on the blood survival and biodistribution of RTA and recombinant ricin A chain in mice. When admixed with the toxins at 1:1 molar ratios prior to intravenous injection, the antibodies prolonged blood survival and whole-body retention of both species of RTA, and this was due essentially to reduced renal clearance of the toxins. Immune complexes were identified by gel filtration chromatography and immune precipitation with anti-IgG antiserum in mixtures prior to injection and in the serum of mice injected with the mixtures. An irrelevant monoclonal antibody showed no complex formation, and no effect on biodistribution. These studies have shown that immune complexes formed between monoclonal antibodies and protein antigens of molecular mass up to at least 30 kDa survive in the circulation, rather than being cleared by the reticuloendothelial system. Such antibodies could be used to modulate the biodistribution of toxic molecules such as ribosome-inhibiting proteins like RTA. This might be exploited therapeutically, for example in the construction of bispecific antibodies against ribosomal inhibiting proteins and tumour-associated antigens.     

Monoclonal antibody characterization of Plasmodium falciparum antigens in immune complexes formed when schizonts rupture in the presence of immune serum

When Plasmodium falciparum parasites are cultured with some immune sera, merozoites are agglutinated by antibodies to form immune clusters of merozoites and prevent their invasion into erythrocytes. Within these immune clusters of merozoites, several antigens that are normally found in the soluble fraction after detergent extraction accumulate in relatively insoluble immune complexes. From mice immunized with these immune complexes, we obtained hybridomas secreting monoclonal antibodies (mAb) that react with various immune clusters of merozoites antigens, including mAb 3D5, which recognizes a 101-kDa antigen (p101) and mAb, 5E3, which recognizes a 113-kDa antigen (p113). Both mAb reacted with antigens at the surface of schizonts, in the vacuolar space, and at the surface of merozoites before their release from schizont-infected cells. Both p101 and p113 were synthesized by mature trophozoites and young schizonts. In pulse-chase experiments, p113 was processed to 100-, 70-, 55-, and 50-kDa products. Both p101 and p113 appeared in the culture medium when schizont rupture occurred in normal culture medium but were found in immune complexes when schizont rupture occurred in the presence of immune serum. Antibodies in immune complexes, when dissociated with acid and used to probe immunoblots, reacted with affinity-purified p101 and p113. Antigens such as these, which are accessible at the parasite surface and react with antibodies present in immune serum that inhibits parasite invasion, are logical candidates to study in the search for a vaccine against the erythrocytic stages of malaria.     

The effectiveness of different rat IgG subclasses as IgE-blocking antibodies in the rat basophil leukaemia cell model

The degranulation of mast cells in an allergic response is initiated by the aggregation of high-affinity IgE receptors (Fc epsilon RI) by IgE and antigen. Recently it has been shown that such degranulation can be inhibited by cross-linking Fc epsilon RI and low-affinity IgG receptors (Fc gamma RII) which are also expressed by mast cells. The ability of various monoclonal antibodies to block the degranulation of rat basophil leukaemia (RBL) cells sensitized with IgE antidinitrophenyl (DNP) antibodies has been investigated. Sensitized cells were challenged with immune complexes formed using varying concentrations of antigen, and of both high- and low-valency antigen. It is reported here that rat IgG1 antibodies, which are associated in the rat with a Th1-type response, act as highly effective blocking antibodies over a wide concentration range. Rat IgG2a antibodies, which are associated with a Th2-type response, were able only to inhibit degranulation when immune complexes were formed with very low concentrations of high-valency antigen (DNP32-HSA). Under these conditions, some inhibitory activity was seen with high-affinity murine IgA anti-DNP but not with low-affinity rat IgG2b anti-DNP antibody-containing immune complexes. In addition to this inhibitory activity, IgG2a antibodies were shown to be capable of inducing degranulation of cells via unoccupied Fc epsilon RI. These results demonstrate that blocking activity may arise via both inhibitory receptors and by masking of antigen.     

The epitopes of two complex-specific monoclonal antibodies, related to the receptor recognition site, map to the COOH-terminal end of human alpha 2-macroglobulin

The elucidation of the molecular structure of the receptor recognition site of human alpha 2-macroglobulin (alpha 2M) was approached by mapping the epitopes of two monoclonal antibodies (F2B2 and F12A3). These antibodies were shown to be complex-specific, defining neo-antigenic sites not detectable on native alpha 2M and thereby mimicking the specificity of the receptor expressed on macrophages and fibroblasts. The antibodies inhibited binding of alpha 2M-trypsin complexes to the receptor. The epitopes of both monoclonal antibodies are shown here to be located on the Mr 60,000 heat-induced fragment of partially reduced alpha 2M. Limited proteolysis of alpha 2M-methylamine with lysine-specific bacterial endoproteinase was examined by rate electrophoresis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to correlate the loss of the epitopes with the generation of defined fragments. 14C-Labeled alpha 2M-methylamine was used as an internal marker for the position of the thioesters. Finally, the epitopes were protected toward proteolysis by subjecting immune complexes of alpha 2M-methylamine with the monoclonal antibodies to proteolysis under the same conditions as uncomplexed alpha 2M-methylamine. The results obtained allowed us to map the epitopes of both the monoclonal antibodies to within a distance of about Mr 20,000 from the COOH-terminal end of human alpha 2M.     

Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1.

We characterized the structural forms of the human immunodeficiency virus env-encoded proteins with a panel of monoclonal and polyclonal antibodies. Western blot (immunoblot) assays with antibodies specific for gp41 invariably recognized a major component of 160 kilodaltons and a less intense component of 120 kilodaltons in viral lysates. We demonstrated that these species are noncovalently associated tetramers and trimers of gp41 which represent the native form of this protein in virions. These complexes were stable when boiled in the presence of low concentrations of sodium dodecyl sulfate but were dissociated to gp41 monomers at high sodium dodecyl sulfate concentrations. Moreover, two human monoclonal antibodies preferentially recognized the oligomeric complexes over monomeric gp41 in Western blots, indicating the presence of epitopes recognized by the human immune system on the gp41 multimers which are not efficiently expressed by the dissociated monomers. The demonstration of the existence of multimeric env complexes and the enhanced and altered antigenicity of such multimers may be relevant to the design of subunit and recombinant human immunodeficiency virus env vaccines.     

Immunoelectron microscopic analysis of the binding of monoclonal antibodies to molecular variants of human placental alkaline phosphatase

Three monoclonal antibodies with distinct antigenic specificities were examined by electron microscopy for their binding to three common genetic variants (SS, FS, and FF) of human placental alkaline phosphatase. In the reaction with the monoclonal antibody H5, all three variants of human placental alkaline phosphatase preferentially formed circular immune complexes composed of two antibodies and two enzyme molecules. In separate reactions with the F11 and B2 monoclonal antibodies, the SS variant formed circular complexes and the FS variant formed Y-shaped complexes composed of one antibody and two enzyme molecules, whereas the FF variant scarcely reacted. These results confirm immunochemical data showing that H5 binds to both S and F subunits with similar affinities, whereas F11 and B2 bind the S subunit with markedly higher affinity than they do the F subunit. Furthermore, the formation of circular complexes in the reaction of the mixture of the two antibodies, F11 and B2, with FS molecules suggests that these two antibodies bind to different sites on the S subunit. Therefore, the F and S subunits differ from one another at more than one site. This is the first indication that alleles of human placental alkaline phosphatase may result from more than just single point mutations in the gene encoding them.     

Both ends ofEscherichia coli ribosomal protein S13 are immunochemically accessiblein situ

To investigate the structure ofEscherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopesin situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13in situ.     

Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients

Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.     

Both ends of Escherichia coli ribosomal protein S13 are immunochemically accessible in situ.

To investigate the structure ofEscherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopesin situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13in situ.     

A monoclonal antibody that neutralizes poliovirus by cross-linking virions.

The neutralization of type 1 poliovirus by monoclonal antibody 35-1f4 was studied. The virions were rapidly linked by antibody into oligomers and larger aggregates, followed by slow redistribution of antibody between the immune complexes. The antibody content and infectivity of immune complexes were determined. Remaining single virions were fully infectious and free of antibody. The oligomers and larger aggregates did not significantly contribute to the residual infectivity, which therefore correlated with the number of remaining single virions. Papain digestion of neutralized poliovirus released fully infectious, antibody-free virions from the immune complexes. Anti-immunoglobulin antibodies reneutralized these virions. Polymerization was shown to occur even at virus concentrations of less than 10(3) PFU per ml.     

Expression of antibody interferes with disulfide bond formation and intracellular transport of antigen in the secretory pathway.

To determine whether antibodies would interfere with the folding of glycoprotein antigens in the endoplasmic reticulum lumen of living cells, hybridoma cells producing monoclonal anti-hemagglutinin (HA) antibodies were infected with influenza virus. The fate of the newly synthesized HA was determined using an established pulse-chase approach. When the monoclonal antibodies were against epitopes present on early folding intermediates, folding and intracellular transport of HA to the Golgi complex were severely disturbed. On the other hand, when the antibodies were specific for the native HA trimers, immune complexes were formed, but folding or transport of HA was not affected. The use of antibodies in this way provided in situ information about the protein folding process inside the endoplasmic reticulum lumen of cells without external perturbation of the folding chains or the folding compartment.     

Preparation of peroxidase: antiperoxidase (PAP) complexes for immunohistological labeling of monoclonal antibodies

The production of mouse peroxidase:antiperoxidase (PAP) complexes suitable for immunohistological use in conjunction with monoclonal antibodies is described. Three approaches were explored: 1) production of conventional polyclonal PAP complexes; 2) conversion of rabbit PAP to "pseudo-mouse PAP" by incubation with monoclonal mouse anti-rabbit immunoglobulin; 3) formation of PAP complexes from monoclonal mouse antiperoxidase. PAP complexes prepared by the latter technique gave the best immunohistological labeling reactions, being stable on storage and compatible with a wide range of human monoclonal antibodies. Gel filtration revealed that monoclonal PAP is of lower molecular weight than conventional PAP complexes (fulfilling theoretical predictions based on the monospecificity of monoclonal antibodies).     

Immunological characterization of rapeseed myrosinase

A purified 75-kDa myrosinase and a crude rapeseed myrosinase fraction were used as antigens to produce mouse anti-myrosinase monoclonal antibodies. The 75-kDa myrosinase was also used to produce a polyclonal rabbit antiserum. The antiserum and one monoclonal antibody reacted with three distinct rapeseed polypeptides of 75, 70 and 65 kDa (M75, M70 and M65, respectively). A second set of monoclonal antibodies reacted exclusively with the 75-kDa form of myrosinase, and a third set showed specificity towards two components of 52 and 50 kDa (myrosinase-binding proteins, MBP52 and MBP50, respectively). MBP52 and MBP50 lack inherent myrosinase activity, but are nevertheless capable of mediating immunoprecipitation of myrosinase due to their interaction with myrosinase. Gel chromatography and glycerol gradient centrifugation experiments resolved two myrosinase-containing fractions. One of these had an approximate molecular mass of 140 kDa and consisted of disulfide-linked dimers of the 75-kDa myrosinase. The other fraction was heterogeneous in size with molecular masses ranging from 250 kDa to approximately 1 MDa. The high-molecular-mass fractions contained complexes consisting of disulfide-linked 70-kDa and 65-kDa myrosinases and non-covalently bound 52-kDa and 50-kDa myrosinase-binding proteins.