Avidity, potency, and cross-reactivity of monoclonal antibodies to pneumococcal capsular polysaccharide serotype 6B

Many pneumococcal capsular polysaccharides (PSs) are similar in structure, and a pneumococcal antibody often binds to all of the PSs with a similar structure. Yet, these cross-reactive antibodies may bind to the structurally related pneumococcal capsular PSs with an avidity too low to be effective. If memory B cells producing such weakly cross-reactive antibodies are elicited with pneumococcal conjugate vaccines, the memory cells for low-avidity antibodies could compromise the subsequent immune responses to the cross-reactive PS (original antigenic sin). To investigate these issues, we produced 14 hybridomas secreting monoclonal antibodies (MAbs) to the capsular PS of Streptococcus pneumoniae serotype 6B by immunizing BALB/c mice with antigens containing 6B PS and studied their epitope, avidity, in vitro opsonizing capacity, in vivo protective capacity, and "antigen binding titer" by enzyme-linked immunosorbent assay (ELISA) of 6A and 6B capsular PSs. Six MAbs bound to the non-cross-reactive 6B-specific epitope, and seven MAbs bound to the cross-reactive epitope present in both 6A and 6B PSs One MAb (Hyp6BM6) revealed a novel epitope. This epitope was found on 6A PS in solution, but not on 6A PS adsorbed onto the plastic surface of the ELISA plates. The avidity of the MAb for 6A or 6B PS ranged from 7.8 x 10(6) M(-1) to 4.1 x 10(11) M(-1). No MAbs were weakly cross-reactive, since none of the cross-reactive MAbs showed any tendency toward having less avidity to 6A PS (the cross-reactive PS) than to 6B PS. Avidity influenced the results of several antibody assays. When all of the hybridomas were examined, avidity strongly correlated with the titer of a unit amount of MAb to bind antigen-coated ELISA plates (r = 0.91) or to opsonize pneumococci in vitro (r = -0.85). Because both assay results are avidity dependent, the ELISA and the opsonization assay results were strongly correlated (r = 0.91), regardless of avidity. Avidity also correlated with the potency of a MAb to passively protect mice against pneumococcal infections. When only the immunoglobulin G hybridomas were examined, little increase in opsonizing capacity and in vivo protective potency was observed above 10(9) M(-1). Taken together, an ELISA measuring antigen binding titer may be an adequate measure of the protective immunity induced with pneumococcal vaccines, and the absence of a partially cross-reactive MAb suggests that antigenic sin may not be significant in responses to vaccines against the S. pneumoniae 6B serotype.     

An analysis of the properties of monoclonal antibodies directed to epitopes on influenza virus hemagglutinin

Summary Monoclonal antibodies (MAbs) specific for the hemagglutinin (HA) of the H3 subtype of influenza A virus were grouped according to their inability to bind to particular MAb-selected neutralization escape mutants of the virus having an amino acid substitution in one of the five postulated antigenic sites on the molecule. Additional residues critical to the binding of the MAbs were deduced from their patterns of reactivity with a panel of field strains and receptor mutants of the H3 subtype. The relationship of these residues to the actual epitopes recognized by the MAbs was inferred from their location on the three-dimensional structure of the HA molecule. In this way it was generally possible to identify a number of residues that are critical to the integrity of the epitope recognized by each of the MAbs examined. It was found that: (1) Several of these epitopes appear to be discontinuous and some may depend on residues contributed by more than one monomer. For example, residue 205, in the interface between monomers of the HA, was found to affect the integrity of the epitopes for several MAbs, possibly by stabilizing the conformation of residues around the receptor-binding pocket and/or in site B on the adjacent monomer. The activity of these particular MAbs was greatly decreased if the virus was exposed to pH 5. (2) All the MAbs tested neutralized viral infectivity and inhibited hemagglutination, although the single MAb directed to site C, which is the most distant from the receptor-binding site, was the least efficient. (3) Hemagglutination inhibition, and particularly neutralization tests, were more discriminating than ELISA in discerning subtle differences between the corresponding epitopes recognized by MAbs on different field strains. (4) Efficiency of neutralization of infectivity did not correlate consistently with hemagglutination inhibiting efficiency; MAbs postulated to bind to epitopes close to the receptor-binding pocket were very efficient at inhibiting hemagglutination, whereas neutralization efficiency tended to be more influenced by the affinity of binding of the MAb. (5) A MAb binding to any particular epitope could affect the binding of a second MAb directed to an epitope within the same or even a different antigenic site. The observed effect was most commonly inhibition of binding, which was not always reciprocal; enhancement of binding was also observed with certain combinations of MAbs. The relative affinity of the MAbs, in addition to steric constraints, were shown to be important factors in the ability to compete for interaction with HA.     

A convenient method for epitope competition analysis of two monoclonal antibodies for their antigen binding

Choosing optimum pair of capturing antibody and detecting antibody when developing monoclonal antibody (MAb)-based, sandwich enzyme-linked immunosorbent assays is a time-consuming process requiring the coupling of individual antibodies to an enzyme like horseradish peroxidase or alkaline phosphatase. The MAbs required for the two-site sandwich ELISA should bind to distinct epitopes of the antigen, and their binding should not be mutually exclusive. To determine if two monoclonal antibodies would occupy distinct sites of their antigen in binding, and enzyme-linked immunosorbent assay was devised, which is easy-to-use and does not require any coupling of monoclonal antibodies to enzymes. Microplate wells are coated with rabbit polyclonal antibodies raised against the same antigen of MAbs. After blocking, a limited amount of the antigen is added for incubation with the rabbit antibodies. Mouse monoclonal antibody 1 (Mab 1) is added to saturation. A serial dilution of MAb 2 (for analysis) or MAb 1 (for control) is added subsequently. An enzyme-labeled, goat anti-mouse secondary antibody and its substrates are added for color development. Thus, the epitope competition of two MAbs for their antigen binding is easily determined by the measurement and comparison of color development between the two MAb additions.     

Production and characterization of monoclonal antibodies against different epitopes of Ebola virus antigens

Ebola virus (EBOV) causes hemorrhagic fever in humans and nonhuman primates with up to 90% mortality rate. In this study, Ebola virus like particles (EVLPs) and the aglycosyl subfragment of glycoprotein (GP(1) subfragment D) were used to generate monoclonal antibodies (MAbs) against different epitopes of the viral antigens. Such MAbs could be useful in diagnostics and potential therapeutics of viral infection and its hemorrhagic symptoms. Hybridoma cell fusion technology was used for production of MAbs. The MAbs were characterized using ELISA and Western blot analysis. Furthermore, five recombinant sub-domains of GP(1) subfragment D were produced, which were used as antigen in Western blot analysis for epitope mapping. Seventeen MAbs of different epitope specificities against EBOV antigens [virion protein (VP40), secreted glycoprotein (sGP), and GP(1) subfragment D] were developed. Based on epitope mapping studies, the anti-GP MAbs were categorized into six groups. The binding of the three anti-sGP MAbs with different epitope specificities were mostly between aa 157 and 221. The two anti-VP40 MAbs with the same or overlapping epitopes are potentially good candidates for developing antigen detection assays for early diagnosis of EBOV infection. The anti-GP MAbs with different epitope specificities as an oligoclonal cocktail could be tested for therapy.     

Epitope mapping of mouse monoclonal antibodies to the ppUL83 lower matrix phosphoprotein of human cytomegalovirus

Of nine mouse monoclonal antibodies (MAbs) directed against the lower matrix protein (pp65; ppUL83) of human cytomegalovirus (HCMV), all immunoprecipitated the 65-kDa protein. Only five were reactive by Western blotting, however, and four of these mapped to linear antigenic epitopes located between amino acids 184-195 (MAb C6), 343-357 (MAb C11), 448-462 (MAb C5), and 448-459 (MAb C13). The epitope specificity of the fifth antibody (MAb C3) and the four that recognised nonlinear sites could not be determined. Competition binding studies using HCMV antigen extracted from productively infected human embryonic lung fibroblasts (HELF), in an enzyme immunoassay (EIA), showed that three of the antibodies reactive with linear epitopes and two of those reactive with conformational epitopes (MAbs C3, C6, C11, C14, and C18), were unique in their binding specificities. MAb C4 competed with MAb C8 and MAb C5 competed with MAb C13 for binding to ppUL83. One of the linear epitopes identified, corresponding to amino acids SAFVFPTKDVAL (MAb C6), was an epitope described previously for CD8+ cytotoxic T lymphocytes.     

An artificial glycoconjugate containing the bisphosphorylated glucosamine disaccharide backbone of lipid A binds lipid A monoclonal antibodies.

Monoclonal antibodies (MAbs) against lipid A, the endotoxic component of lipopolysaccharide (LPS) of gram-negative bacteria, are presently discussed as therapeutic agents against lethal gram-negative infections; however, their binding specificities are controversial. We have isolated from the LPS of Escherichia coli J-5 the 1,4'-bisphosphorylated beta 1-->6-linked glucosamine disaccharide backbone of its lipid A moiety, which was covalently linked to bovine serum albumin. It was shown by solid-phase enzyme immunoassay that one antibody (MAb A6) bound equally well to the glycoconjugate and synthetic E. coli-type lipid A over a broad range of antigen concentrations whereas two other MAbs (IC3 and S1-15) bound better to the conjugate at low antigen concentrations and better to the lipid A when high concentrations of antigen were used. This proves in a direct way that there exist lipid A MAbs with different specificities which bind to epitopes in the hydrophilic backbone of lipid A and which do not require the presence of fatty acids.     

Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus

Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeirea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wall and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.     

Detection of Vibrio cholerae with monoclonal antibodies specific for serovar O1 lipopolysaccharide.

Six hybridoma cell lines, each of which produced a monoclonal antibody (MAb) against Vibrio cholerae O1 lipopolysaccharide (LPS), were established. Each MAb was active serologically by both enzyme-linked immunosorbent assay (ELISA) and the slide agglutination test. In the ELISA, each MAb was tested against 7 O1 and 9 non-O1 LPS preparations. Three MAbs reacted with both Inaba and Ogawa serovars (A antigen), two MAbs reacted with the Ogawa serovars only (B antigen), and one MAb reacted with the Inaba serovars only (C antigen). Each MAb was also tested in the ELISA against whole-cell preparations of 37 O1 and 52 non-O1 V. cholerae serovars, 20 heterologous Vibrio species, and 37 heterologous bacterial species. The MAbs reacted with V. cholerae O1 cells only, except for one anti-A antigen MAb which reacted weakly with five V. cholerae non-O1 serovars and Serratia marcescens. Each anti-A antigen MAb was labeled with fluorescein isothiocyanate (FITC) and tested by direct immunofluorescence against selected O1 and non-O1 serovars. Each MAb-FITC conjugate, when tested alone, exhibited O1-specific fluorescence; however, mixtures of the MAb-FITC dramatically enhanced fluorescence intensity on O1 cells. This finding was also visualized by immunoelectron microscopy on both thin-sectioned and negatively stained O1 cells by using an anti-mouse immunoglobulin-colloidal gold conjugate. These results suggest that the A antigen can be described by more than one epitope and that a superior serotyping reagent can be prepared from a defined mixture of MAbs.     

Development and characterization of monoclonal antibodies to chicken riboflavin carrier protein

Several monoclonal antibodies (MAbs) specific to chicken riboflavin carrier protein (cRCP) were developed and characterized. Of the several MAbs analyzed, four were directed against nonoverlapping epitopes as demonstrated by MAb inhibition assay. Many of these epitopes appeared to be in close proximity and only three were situated at distinct part of the molecule as revealed by sandwich assay. A combination of chemical modification, peptide cleavage by chemical and enzymatic methods, was used to analyze the possible antigenic structure recognized by these MAbs. An assembled epitope spanning the region 22-87 forms the antigenic site recognized by 4999.1; while MAb 5555.3 interacted with the C-terminal peptide 203-219.     

Induction of monoclonal antibody with predefined ELNKWA epitope specificity by epitope vaccine

Since the hybridoma technique to produce monoclonal antibodies (MAbs) was discovered, thousands of MAbs with predefined protein specificity have been produced, and a natural or recombinant protein as antigen is necessary for inducing MAbs in the conventional hybridoma technique. To induce epitope-specific MAbs, we suggest an epitope vaccine as a new technique to induce MAbs with predefined epitope specificity. ELDKWA was identified as an important neutralizing epitope on HIV-1 gp41. The MAb 2F5, recognizing ELDKWA epitope, has shown broad neutralizing activity to many HIV strains, including primary isolates, but the mutant in ELNKWA epitope results in escape 2F5-based neutralization. To produce MAbs recognizing this mutated epitope for consideration of passive immunotherapy against the mutant bearing the ELNKWA epitope, MAbs with predefined ELNKWA epitope specificity were induced by synthetic epitope-peptide instead of a natural or recombinant gp41 bearing this epitope. Three MAbs were identified to recognize ELNKWA epitope on the synthetic epitope-peptide, and interestingly could bind the recombinant gp41 with ELDKWA epitope in an ELISA assay and immunoblotting analysis.     

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays. © 1996 Wiley-Liss, Inc.     

Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue.

uPAR (CD87), the receptor for the urokinase-type plasminogen activator (uPA) facilitates tumor cell invasion and metastasis by focusing uPA proteolytic activity to the cell surface. As uPAR exists in various molecular forms, it is desirable to use well defined antibodies for analyses of uPAR antigen expression in human malignant tumors by immunological methods. Therefore, twelve monoclonal antibodies (MAbs) directed against uPAR were generated by using nonglycosylated, recombinant human uPAR (spanning amino acids 1 to 284), expressed in Escherichia coli, as the immunogen. The reaction pattern of these MAbs with the immunogen and a series of carboxyl-terminally truncated versions of uPAR demonstrated that at least six different epitopes of uPAR are recognized. All MAbs reacted under reducing conditions in immunoblot analyses with E. coli-expressed uPA and also with highly glycosylated, functionally intact, recombinant human uPAR expressed in Chinese hamster ovary (CHO) cells. Seven of the MAbs recognized CHO uPAR under nonreducing conditions as well. By flow cytofluorometric analyses, three of these MAbs were shown to bind to native human uPAR present on the cell surface of monocytoid U937 cells with MAb IIIF10 being the best. Saturation of uPAR with uPA on U937 cells completely blocked interaction of MAb IIIF10 with uPAR (mapped epitope, amino acids 52 to 60 of domain I of uPAR). In turn, preincubation of U937 cells with MAb IIIF10 efficiently reduced binding of uPA to uPAR, indicating that the epitope detected by MAb IIIF10 is located within or closely to the uPA-binding site of uPAR, and thus, this site may be a target to influence uPA/uPAR-mediated proteolysis in tumors. Binding of MAbs IID7 or IIIB11 (mapped epitope, amino acids 125 to 132 of domain II of uPAR) to uPAR is not affected when uPAR is occupied by uPA. As these MAbs reacted strongly with cellular uPAR antigen in formalin-fixed paraffin-embedded tumor sections, the domain-II-specific antibodies IID7 and IIIB11 may be useful for immunohistochemical studies of uPAR expression in tissue remodeling processes in tumor invasion. In conclusion, we have devised well defined and epitope-mapped MAbs to uPAR that are highly specific tools for detection and targeting of uPAR in tumor tissue.     

Functional analysis of Bacillus anthracis protective antigen by using neutralizing monoclonal antibodies

Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis. It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B. anthracis in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.     

Analysis of human monoclonal antibodies elicited by vaccination with a Cryptococcus neoformans glucuronoxylomannan capsular polysaccharide vaccine.

The Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) has been conjugated to tetanus toxoid (GXM-TT) as an investigational vaccine. GXM-TT elicits antibodies that are protective in C. neoformans-infected mice. In an effort to characterize the fine specificity and molecular structure of human GXM-TT-elicited antibodies, we generated two GXM monoclonal antibodies (MAbs) from peripheral blood lymphocytes of a volunteer GXM-TT recipient and studied serum GXM antibody idiotype expression in 10 additional vaccinees. The MAbs, 2E9 and 3B6, are the immunoglobulin M(lambda) isotype and bind capsular polysaccharides of C. neoformans serotypes other than the serotype A that was used for immunization. Neither antibody competes with murine GXM MAbs for antigen binding, suggesting that the human MAbs recognize a different epitope. The B-cell superantigen staphylococcal protein A binds both MAbs, and human immunodeficiency virus gp120 binds 2E9. MAb nucleic acid sequence analysis revealed that both antibodies use an identical V lambda 1a-J lambda genetic element with different, somatically mutated, members of the VH3 gene family and different DH and JH gene elements. The gene elements used by both MAbs occur in fetal B-lymphocyte repertoires, autoantibodies, and other polysaccharide antibodies. Post-GXM-TT vaccination GXM antibodies from 10 additional vaccinees expressed a shared idiotype defined by rabbit antiserum raised against MAb 2E9. Our data suggest that the human GXM antibody response is restricted and raise questions regarding the importance of specific variable-region elements and superantigens in the generation of human antibody responses to encapsulated pathogens.     

Preparation and selection of monoclonal antibodies for detecting hepatitis B surface antigen in blood sera

HV monoclonal antibodies (MAb) were produced in order to improve the quality of HBsAg detection and their specific characteristics were compared with those of other MAbs. MAbs were characterized by asymmetric interactions with the antigen when used as first or second antibodies. The reactivity of a panel of HV and X MAb to ad and ay subtypes was studied by enzyme immunoassay. Mutual blocking (epitope mapping) of MAb helped select antibody couples for the creation of highly effective test system for the diagnosis of the major HBsAg subtypes. The sensitivity and specificity of MAbs were evaluated on reference and control panels of HBsAg sera and on serum specimens from a random sampling of 300 blood donors. The sensitivity of the most specific MAb pairs was 0.1 ng/ml for HBsAg subtype ay and 0.25 ng/ml for subtype ad. The specificity of attested MAb was 98.5% in incubation with stirring and 97% in static incubation. The optimal combinations of attested MAbs were used in the manufacture of Recomnathep B test system in the sandwich format.     

The use of anti-IgG monoclonal antibodies in mapping the monocyte receptor site on IgG

Monoclonal antibodies (MAbs) directed against epitopes on the Cγ1, Cγ2, Cγ3 and Cγ2-Cγ3 interface regions of human IgG were used to attempt to localize the monocyte Fc receptor (FcR) binding site. The MAbs have been assayed for their capacity to inhibit the interaction between ¹²⁵I-labelled IgG (¹²⁵I-IgG) and human monocytes or human histiocytic lymphoma U937 cells. Two MAbs specific for epitopes on the N-terminal region of the Cγ2 domain, and one MAb recognizing an epitope in the Cγ2-Cγ3 inter-domain region inhibited binding of ¹²⁵I-IgG to monocyte FcRs. The remaining MAbs, against a C-terminal Cγ3 domain epitope, another Cγ2/Cγ3 region epitope and the Glm(f) allotope on the Cγl domain did not inhibit the interaction. The capacity of the MAbs to bind to their respective epitopes on cell surface FcR-bound IgG was also studied, using indirect radiobinding and immunofluorescence assays. All of the MAbs, except those with Cγ2 domain specificities, were able to detect FcR-bound IgG under these conditions. The results confirm the role of the Cγ2 domain in the interaction of IgG with monocytes and demonstrate that epitopes in the Cγ3 and Cγ2-Cγ3 regions are not involved in the binding site.     

Characterization and formulation of multiple epitope-specific neutralizing monoclonal antibodies for passive immunization against cryptosporidiosis

The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [alphaCSL]), 3H2 (alphaGP25-200), and 1E10 (alphaP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.     

Two monoclonal antibodies to two different epitopes of human growth hormone form a precipitin line when counterdiffused as soluble immune complexes

We have tested whether soluble immune complexes obtained by mixing human growth hormone (hGH) with one anti-hGH monoclonal antibody (MAb) can form a precipitin line when diffused against another MAb in a polyethylene glycol containing gel. By testing seven anti-hGH MAbs one against the other in this assay, we have found that 10 pairs of MAbs out of the 21 possible combinations formed a line. Apparently, the first MAb formed soluble hGH dimers that were linked by the second MAb into precipitating linear complexes. Since each precipitin line was formed by the cooperative reaction of two MAbs, this sequential reaction of MAbs may be used in methods for the positive selection of MAbs that are suitable for two-site immunoassays.     

Enhancement of anthrax lethal toxin cytotoxicity: a subset of monoclonal antibodies against protective antigen increases lethal toxin-mediated killing of murine macrophages

We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcgamma receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages.     

Bank vole monoclonal antibodies against Puumala virus envelope glycoproteins: identification of epitopes involved in neutralization

Summary Bank vole (Clethrionomys glareolus) monoclonal antibodies (MAbs) against the two envelope glycoproteins (G 1 and G 2) of the Puumala (PUU) virus were generated and characterized. Analyses of the MAbs' antigen and epitope specificities showed non-overlapping reactivities of one anti-G 1 and two anti-G 2 MAbs. A significant neutralizing activity was shown by the anti-G 1 and one of the anti-G 2 MAbs, suggesting the existence of at least one neutralizing domain on each of the two glycoproteins. The two neutralizing MAbs reacted with eight PUU-related (serotype 3) virus strains, but did not react with Hantaan, Seoul, or Prospect Hill viruses in an immunofluorescence assay, indicating reactivity with epitopes unique for PUU virus. The non-neutralizing anti-G 2 MAb also reacted with Seoul virus, revealing the presence of a conserved G 2-epitope common for PUU and Seoul viruses, not involved in neutralization. Competitive binding of the MAbs and sera from nephropathia epidemica patients indicated that the defined neutralizing and non-neutralizing epitopes of the glycoproteins were immunodominant also in humans. In another experiment, magnetic beads coated with two MAbs were bound with the virus glycoproteins and used for selective enrichment of cells secreting anti-glycoprotein antibodies.