Monoclonal antibodies against bacterial glucosamine 6-phosphate synthase: production and use for structural studies.

Fifteen mouse x rat hybridoma cell lines producing rat monoclonal antibodies (MAbs) directed to Escherichia coli Glucosamine 6-P Synthase (GlmS) were established and characterized. Most of them (13/15) are IgG2a while 2 were typed as IgG1. Their Kaff ranged from 1.5 x 10(6) to 9.6 x 10(8) M-1 as determined by Beatty et al. (1). The epitopes recognized by these MAbs were assigned to one of the two catalytical domains of the enzyme (CT1 and CT2) as demonstrated both by ELISA and Western-blotting using purified GlmS proteolytic fragments. The binding of the MAbs on either the native or denatured forms of GlmS, CT1 and CT2 was further analyzed by competitive immunoassay and most of the MAbs were found to bind preferentially to the denatured proteins. The study of the antigenic topography of GlmS by competitive radioimmunoassay demonstrated the existence of at least 10 independent epitopes on GlmS, divided into three groups. The first one (3/15) includes MAbs whose binding was not inhibited by any of the other MAbs. The second group (9/15) is comprised of MAbs that exhibit reciprocal binding inhibitory activity while the third group includes MAbs (3/15) presenting asymmetric inhibitory activity. Finally, since most of the isolated antibodies (10/15) bind to the 27 kDa amino-terminal glutamine binding domain (CT2), the capacity of these MAb to interfere with the associated glutaminase activity was analyzed.     

Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.     

Identification, quantitation, and purification of cockroach allergens using monoclonal antibodies

A panel of murine IgG monoclonal antibodies (MAbs) was raised against German cockroach (CR) (Blattella germanica) extract and selectively screened to identify MAb directed against allergen(s) recognized by IgE antibodies. Sera from 28 CR-allergic patients were used as sources of IgE antibodies to detect allergens "presented" by the MAb. Four clones (10A6, 3G12, 8F4, and 1D4) produced MAb to allergen(s) that bound IgE antibodies. Quantitative radioimmunoassays were used to compare levels of the MAb-defined allergens in CR extracts. MAb 10A6 reacted with a cross-reacting allergen that was detected in 9/14 CR species, including Blattella, Periplaneta, Blatta, Leucophea, and Supella spp, at concentrations of 100 to 10,000 U/ml. In contrast, MAb 3G12, 8F4, and 1D4 were Blattella specific. The allergen defined by MAb 8F4 was purified by MAb affinity chromatography and size-exclusion by high-performance liquid chromatography. It is a 36 kd heat-sensitive protein, isoelectric point, 5.2 to 5.4. Allergen 10A6 was partially purified by isoelectric focusing and high-performance liquid chromatography. It is a heat-stable, acidic protein (isoelectric point 3.15). Based on comparison of their properties with properties of previously described CR allergens, the allergens defined by MAb 10A6 and 8F4 have been provisionally designated Blattella germanica allergen I (Bla g I) and Blattella germanica allergen II (Bla g II), respectively. Assays of six commercial CR skin test extracts demonstrated a 200-fold difference in Bla g I levels (4.7 to 1085 U/ml) and only two extracts that contained detectable Bla g II (248 and 324 U/ml). The results demonstrate that MAb can be used to identify and define CR allergens and that the strategy of the use of MAb as a first step in allergen analysis and purification can be very effective, especially for poorly characterized allergen extracts.     

Evaluation of the anti-LipL32 monoclonal antibodies potential for use in leptospirosis immunodiagnostic tests

LipL32 is the major lipoprotein in the membrane of pathogenic leptospira. In this work, we report on the production of monoclonal antibodies (MAbs) against recombinant LipL32 (rLipL32) and on the evaluation of their potential for use as reagents in diagnostic tests for leptospirosis. The MAbs were all of the IgG(2b) isotype and reacted specifically with native LipL32 in pathogenic serovars only. MAbs reacted in the same region of the rLipL32 molecule and their affinity constant was between 5x10(7) M(-1) and 6x10(6) M(-1). These results suggest that although the MAbs cannot be used together, they are well suited for diagnostic tests of leptospirosis based on LipL32 detection.     

Production of monoclonal antibodies specific to major allergens of Cryptomeria japonica pollen.

Monoclonal antibodies (MAbs) directed against Cryptomeria japonica pollen antigen (CPAg) were prepared. CPAg was precipitated with these MAbs and both MAbs CPA7 (IgG2a) and CPA9 (IgG1) recognized two major glycoproteins (m = 41 kDa and 46 kDa) of CPAg. MAb CPA7 or CPA9 was coupled to CNBr activated sepharose and affinity purification of the major allergen of CPAg from the crude extract was performed. The affinity purified CPAg bound IgE antibody present in patients with Cedar pollinosis.     

Characterization of monoclonal antibodies physically adsorbed onto polystyrene latex particles.

The physicochemical and immunochemical properties of monoclonal antibodies (MAbs), adsorbed onto polystyrene latex particles, have been investigated. Both native and pH 2 pretreated MAbs were compared before and after immobilization. It was found that the antigen binding capacity of the immobilized, acidic pretreated MAbs was significantly higher than for the immobilized, native IgG molecules. This enhanced antigen binding capacity appeared to be due to an improved molecular orientation following adsorption of the monomeric, pH 2 treated IgG fraction. Additionally, experiments using F(ab')2 fragments demonstrated that the Fc portion of the MAb molecule is of major importance for achieving the enhanced binding capacity. Binding studies showed that the (apparent) affinity of native and pH 2 pretreated MAbs were similar; the K(a) values of the immobilized MAbs were higher than those of MAbs in solution.     

Equilibrium binding characteristics of monoclonal antibodies recognizing melanoma cell surface antigens

The equilibrium binding characteristics of a panel of six monoclonal antibodies (MAb) recognizing melanoma cell surface antigens (125 kdal cell surface melanoma associated glycoprotein antigen, 125kD-MAA; high molecular weight melanoma associated antigen, HMW-MAA; and a non-protein melanoma associated antigen, NP-MAA) were investigated using the cell lines SK-MEL-2, SK-MEL-5, and M21. The MAbs displayed equilibrium association constant (K) values ranging from 10(7) M-1 to 10(10) M-1 and maximum MAb binding values (Qmax) from 2 x 10(4) to 2 x 10(6) MAb molecules bound per cell. High trypsin concentrations were shown to have deleterious effects on Qmax values obtained for antibodies recognizing the 125kD-MAA, and even low trypsin concentrations affected Qmax values obtained for MAbs recognizing the HMW-MAA (although a complete linear recovery of HMW-MAA antigen was observed in 20-25 hours). Significant changes in Qmax were also noted for different cell passages. Except for MAb 43.2, little variation in K was observed when different cell lines were used. Linear Scatchard plots were obtained for all MAbs except 43.2 in which case concave down behavior was observed suggesting the existence of positive cooperativity between the binding sites of this MAb.     

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.     

Anti-idiotypic antibodies as probes for the determination of idiotopes shared by human and monoclonal murine antibodies

Murine monoclonal antibodies (MAbs) against rye grass Group I (rye I) allergen were previously produced (290A-167, 348A-6 and 539A-6) and are further characterized herein. By competitive binding to polystyrene-bound allergen, it was shown that the three MAbs are directed against three different non-overlapping epitopes on rye I. Human rye I-specific IgG isolated from the serum of one rye grass sensitive patient could recognize the same or closely related epitopes than those recognized by MAbs. Antisera were then raised in rabbits against purified F(ab')2 fragments of human rye I-specific IgG and F(ab')2 fragments of 539A-6 MAb. The antisera were rendered idiotype specific by adsorption with insolubilized non-specific human IgG from the same donor, insolubilized normal murine immunoglobulins and finally with rye I. As shown by competitive binding to polystyrene-bound allergen, rabbit anti-idiotypic antibodies (anti-ID antibodies) produced against F(ab')2 fragments of human rye I specific IgG could inhibit the reaction between two MAbs (290A-167 and 539A-6) and the relevant allergen. Furthermore, human rye I specific IgG could inhibit the binding of 125I-labelled 539A-6 MAb to its specific anti-ID antibody to a significant degree. Human autoanti-idiotypic antibodies were also shown to inhibit the reaction between the three anti-rye I MAbs and the antigen. These observations suggest that cross-reactivity exists between idiotypic determinants of human rye I-specific IgG and the three murine MAbs, which implies structural similarity in the V genes coding for the variable region of the antibody from these two different species.     

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

Novel monoclonal antibodies that bind to wild and fixed rabies virus strains

Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2bκ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays.     

Monoclonal IgM antibodies from cytomegalovirus-infected mice recognize the GlcNAc-containing receptor determinant of murine CMV as well as neutralizing anti-CMV IgG antibodies.

This study examines monoclonal antibodies derived from mice at different time points after infection with attenuated murine cytomegalovirus (MCMV). The antibodies obtained from mice at 3 weeks p.i. were of IgG type (designated V-antibodies) and several could neutralize the virus. Those obtained at 5 weeks p.i. were of the IgM class (designated R-antibodies), bound to uninfected (MEF, mouse embryo fibroblast) cells, and thereby blocked MCMV plaque formation. In ELISA, the IgM monoclonals (R-antibodies) bound to GalB1-3GlcNAc and GalB1-4GlcNAc, the receptor determinants for MCMV. Similarly, these GlcNAc-containing residues blocked the binding of purified IgM monoclonal antibodies (MAbs) to MEF. The R- and V-series of antibodies showed mutual binding activities; for example, IgM MAb R-2D8 bound specifically to four (V-8C4, V-1C7, V-8C7, V-9C5) of six neutralizing IgG MAbs in ELISA. The same neutralizing IgG MAbs bound to antireceptor IgM antibodies in an immunoblot assay. This suggests that the IgM monoclonals directed against the known cell surface receptor determinant are anti-idiotypic antibodies against neutralizing antiviral IgG antibodies. The neutralizing antiviral IgG MAbs bound to 60- and 66-kDa MCMV polypeptides on Western blots, suggesting that these viral polypeptides may be important in MCMV binding to this receptor. The R-series might represent anti-idiotype antibodies capable of down-regulating antiviral V-antibodies and may also represent a mechanism for the induction of IgM autoantibodies directed at cell surface glycolipids present in autoimmune CMV-associated neuropathies.     

Monoclonal antibodies against Escherichia coli heat-stable toxin (STa) and their use in a diagnostic ST ganglioside GM1-enzyme-linked immunosorbent assay.

Seven monoclonal antibodies (MAbs) against heat-stable enterotoxin (ST) from a human Escherichia coli isolate were prepared and evaluated for their usefulness in an ST immunodetection assay, the ST ganglioside GM1-enzyme-linked immunosorbent assay (ELISA). This assay is based on the ability of STa, as present in, for example, culture filtrates from ST-producing E. coli, to inhibit specific anti-ST antibody from binding to solid-phase-bound ST ganglioside (GM1-bound ST-cholera B subunit). Four of the MAbs were of immunoglobulin G1 (IgG1), one was of IgG2b, and two were of IgM isotype. All the IgG1 MAbs could be completely inhibited by addition of free ST; 0.2 to 0.4 ng of purified ST inhibited binding of these MAbs by 50%. The non-IgG1 MAbs were, in contrast, not inhibited by 200-fold-higher amounts of purified ST, probably because they were directed against linkage epitopes or were of low affinity or both. When the IgG1 MAbs were tested in the ST GM1-ELISA, ST could be detected in culture filtrates from stock human E. coli isolates with 100% sensitivity and specificity. ST in filtrates from fresh stool cultures was demonstrated with higher sensitivity with the MAbs ST GM1-ELISA than with the conventional infant mouse test. Both subtypes of STa, STaI and STaII, could be detected by the ST GM1-ELISA by using either IgG1 MAb in the immunodetection step, whereas infant-mouse-active ST from Yersinia enterocolitica failed to react.     

Characterisation of Putative Monoclonal Anti-G3M(U) and Anti-G3M(G) Reagents and their Antigenic Determinants

Monoclonal antibodies (MAbs) against IgG3 allotypic markers were evaluated in haemagglutination inhibition and IgG3 capture ELISA. MAbs PNF69C and 200D1 exhibited G3m(u) and G3m(g) specificity respectively. In HAI target epitopes detected by MAbs were remarkably stable to physiochemical degradation. Western blotting revealed that MAb 200D1, bound to intact IgG3 heavy chain disease protein and not its pFc' fragment; a result consistent with the CH2 domain location of the G3m(g) allotope. The G3m(u) allotope is also located within this domain. Suprisingly anti-G3m(u) MAb PNF69C bound to the pFc' of IgG3-related protein, HW, and to the pFc' of IgG1-related protein, PR, in Western blot.     

Production and characterization of highly specific anti-methotrexate monoclonal antibodies

Spleen cells of a Biozzi HR mouse immunized with a bovine serum albumin-methotrexate conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-three monoclonal antibodies (MAbs), selected by indirect ELISA, were produced and partially characterized. Using methotrexate (MTX) and eight structurally related compounds, binding specificities of the MAbs were assessed by inhibition enzyme immunoassay. All the MAbs had very low affinity for folic acid and its analogs and for the major MTX metabolite 7-hydroxymethotrexate. Using a computer cluster analysis program based on the binding specificities, the MAbs were divided into three groups. The thirteen MAbs in group I recognized primarily the pteridine portion of the MTX molecule; the eight group II MAbs recognized the benzene ring as well as the pteridine structure. The two MAbs in group III poorly distinguished between the different parts of the MTX molecule. The apparent equilibrium association constants of the anti-MTX MAbs in groups I, II, and III ranged from 7 x 10(9) to 3 x 10(8) M-1 (except for 1 MAb), from 5 x 10(7) to 6 x 10(6) M-1 (except for 2 MAbs), and from 1 x 10(6) to 3.5 x 10(5) M-1, respectively.     

Characterization of the opsonic and protective activity against Staphylococcus aureus of fully human monoclonal antibodies specific for the bacterial surface polysaccharide poly-N-acetylglucosamine

Carbohydrate antigens are important targets of the immune system in clearing bacterial pathogens. Although the immune system almost exclusively uses antibodies in response to foreign carbohydrates, there is still much to learn about the role of different epitopes on the carbohydrate as targets of protective immunity. We examined the role of acetyl group-dependent and -independent epitopes on the staphylococcal surface of polysaccharide poly-N-acetylated glucosamine (PNAG) by use of human monoclonal antibodies (MAbs) specific for such epitopes. We utilized hybridoma technology to produce fully human immunoglobulin G2 (IgG2) MAbs from B cells of an individual post-Staphylococcus aureus infection and cloned the antibody variable regions to produce an IgG1 form of each original MAb. Specificity and functionality of the purified MAbs were tested in vitro using enzyme-linked immunosorbent assays, complement deposition, and opsonophagocytic assays. We found that a MAb (MAb F598) that bound the best to nonacetylated or backbone epitopes on PNAG had superior complement deposition and opsonophagocytic activity compared to two MAbs that bound optimally to PNAG that was expressed with a native level (>90%) of N-acetyl groups (MAbs F628 and F630). Protection of mice against lethality due to S. aureus strains Mn8 and Reynolds further showed that the backbone-specific MAb had optimal protective efficacy compared with the acetate-specific MAbs. These results provide evidence for the importance of epitope specificity in inducing the optimal protective antibody response to PNAG and indicate that MAbs to the deacetylated form of PNAG could be immunotherapeutic agents for preventing or treating staphylococcal infections.     

Neutralization of toxic shock syndrome toxin-1 by monoclonal antibodies in vitro and in vivo.

Sixteen monoclonal antibodies (MAbs) directed against toxic shock syndrome toxin-1 (TSST-1) were generated by immunization of mice with purified TSST-1 and subsequent fusion of spleen cells with myeloma cells. Antibody-producing clones, identified by an enzyme-linked immunosorbent assay, were maintained as ascites tumors, and MAbs were purified by protein A chromatography. High-titered clones were further characterized and tested for the ability to neutralize several biological activities of TSST-1. The MAbs, which are of several immunoglobulin subtypes, reacted specifically with purified TSST-1 and TSST-1 present in Staphylococcus aureus culture supernatants. Three MAbs neutralized TSST-1-induced mitogenesis in a dose-dependent manner. Three of eight MAbs tested were able to neutralize induction by TSST-1 of interleukin-1 production by human monocytes. One neutralizing MAb, 8-5-7, was tested for the ability to protect rabbits from a constant infusion of TSST-1. Rabbits given the MAb had an attenuated clinical illness and were protected from the hypocalcemia, lipemia, and hepatic and renal insufficiency seen in control rabbits. Six of seven control rabbits died, compared with only one of seven rabbits treated with MAb 8-5-7. These experiments suggest that MAb 8-5-7 is directed against an antigenic determinant critical to the toxicity of TSST-1 and that the MAbs should be useful as probes in structure-function analyses of the TSST-1 molecule.     

Efficient laboratory-scale production of monoclonal antibodies using membrane-based high-density cell culture technology

Monoclonal antibodies (MAbs) are important tools used in basic research as well as in the imaging and therapy of cancer. Many countries have limited the use of animals for large-scale production of MAbs, obliging laboratories to find efficient in vitro alternatives to ascites production. In this report we describe a protocol for laboratory-scale production of MAbs by culturing hybridoma cells in the two-chamber cell culture device CELLine 1000. This culture flask supports high cell densities (10⁷–10⁸ cells/ml) and generates high concentrations of MAbs (0.7–2.5 mg/ml). Two hybridomas producing MAbs directed against the gastrointestinal antigen GA733-2, GA733 MAb and CO17-1A MAb, were evaluated over culture periods of up to two months using several alternative conditions. Two different sets of conditions are reported; the first using serum-supplemented medium (20% v/v) and the second using serum-free medium (SFM). Average weekly yields of the purified MAbs in serum-supplemented medium were 24 mg and 33 mg, and in SFM were 21 mg and 17 mg for GA733 MAb and CO17-1A MAb, respectively. Experimental variables that can affect antibody production and economy include: nutrient medium and cell compartment medium compositions (cell line dependent), the proportion of the cell compartment medium harvested every 3 days (50% to 80% with 80% optimal) and the frequency of nutrient medium changes (3 to 9 days with 6 days as most cost effective). Protein-A Sepharose purification followed by antigen-specific affinity purification showed that MAbs obtained from serum-supplemented cultures contain less than 0.6% of bovine IgG contamination, while MAbs obtained from serum-free cultures contained no extraneous IgG. In addition, MAbs from both culture media were fully active (essentially 100%) as measured by their ability to bind to an antigen column. In contrast, the same MAbs purified from ascites using Protein-A-Sepharose typically contained a major portion of inactive IgG. This in vitro method for laboratory-scale production of MAbs (10 to 500 mg) proved to be simple, reproducible and cost effective. It represents a useful alternative to the in vivo production of MAbs in mice.     

Isolation and characteristics of anti-adenovirus monoclonal antibodies in immunoenzyme and immunofluorescent reactions

New monoclonal antibodies (MAbs) to adenovirus hexon, highly active in ELISA and immunofluorescent analysis, were prepared. According to competitive ELISA, new MAbs differed in their blocking activity and were directed to 2 different hexon epitopes. MAb 3H8 did not modify antigen binding of the rest MAbs labeled with peroxidase (PAb x Pox), and none of unlabeled MAbs suppressed the reaction of MAb x Pox 3H8. MAbs 1E8 7F1, 1E11, and 3B1 reacted with each other but differed by the spectrum and level of competitive inhibition, which indicated that they were directed to different epitopes of adenovirus hexon. Comparison of the specific activity of MAbs 7F1 and 1E8 in direct immunofluorescent detection of adenovirus antigens in infected cell cultures and clinical materials from patients showed a good coincidence (90-97%) of the results with the IMAGEN Adenovirus test (Dako) and with polyclonal FITC conjugates to adenovirus hexon.     

Modulation of polymorphonuclear cell interleukin-8 secretion by human monoclonal antibodies to type 8 pneumococcal capsular polysaccharide

Pneumococcal capsular polysaccharide (PS) vaccines induce type-specific immunoglobulin M (IgM), IgG, and IgA. Type-specific IgG to the PS is sufficient to confer protection against the homologous serotype of the pneumococcus, but the efficacies of type-specific IgM and IgA are less well understood. We examined the in vitro activities and efficacies in mice of two human monoclonal antibodies (MAbs) to type 8 PS, NAD (IgA) and D11 (IgM). MAb-mediated opsonophagocytic killing was evaluated after coculture of type 8 pneumococci with human polymorphonuclear cells (PMNs), type-specific or control MAbs, and human complement sources. The effects of the MAbs on PMN interleukin-8 (IL-8) and IL-6 secretion were determined in supernatants from cocultures containing pneumococci and PMNs by enzyme-linked immunosorbent assay. MAb efficacy was determined in an intratracheal model of type 8 infection in mice with classical complement pathway deficiency. Both MAbs were protective in 100% of infected mice. Neither MAb promoted a significant amount of killing of type 8 pneumococci compared to its isotype control MAb. Both type-specific MAbs mediated complement-dependent modulation of PMN IL-8 secretion, with increased secretion at effector/target (E:T) ratios of 500:1 and 50:1 and reduced secretion at 1:5. Trypan blue staining revealed that PMNs cocultured with D11 were less viable at an E:T ratio of 1:5 than PMNs cocultured with the control MAb. PMN IL-6 secretion was increased by both type-specific and control MAbs. These results suggest that certain type-specific IgM and IgAs might contribute to host defense by modulation of the inflammatory response to pneumococci.