Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens

Infection with Helicobacter pylori is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of H. pylori gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against H. pylori cell lysate and the H. pylori urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an H. pylori-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the H. pylori Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the H. pylori urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related H. pylori binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following H. pylori infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of H. pylori antigens to be determined, which might be of use for vaccine development.     

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.     

Multiple reactivity of monoclonal antibodies

The reactivity of 31 purified monoclonal antibodies (MAbs) with 10 unrelated protein antigens was determined by an enzyme-linked immunosorbent assay. Twenty-two of the monoclonal antibodies (71%) reacted with at least one of these nonhomologous antigens. Overall, there was positive reactivity in 27% of the tests. A larger incidence of multiple reactivities was observed with increasing concentration of the MAbs. Certain of these antibodies (29%) did not react with any nonhomologous tested antigens, whereas others had reactivity with multiple antigens. Multiple reactivity was observed with 6 of 14 of the hybridomas in a test that measured binding to a cell line. Multiple reactivity was more frequently observed when the MAbs were tested with complex antigens, such as keyhole limpet hemocyanin or a cell surface. These experiments demonstrate that MAbs frequently react with multiple antigens.     

Analysis of Campylobacter jejuni antigens with monoclonal antibodies

To develop monoclonal reagents for antigenic analysis and serotyping of Campylobacter spp., hybridoma cell lines were produced by fusion of mouse myeloma cells and spleen cells from mice immunized with Formalin-treated Campylobacter jejuni organisms. An enzyme immunoassay was used for preliminary screening of the cell culture supernatants and ascites. Twenty-nine clones which reacted with the immunogen were obtained. Seven of these clones were positive in passive hemagglutination tests with sheep erythrocytes coated with boiled saline extract of whole bacteria; four of these reacted with the purified polysaccharide preparation and with the autoclaved saline extract, but not with lipopolysaccharide prepared from the immunogen strain. Two of the antipolysaccharide clones agglutinated live bacteria in slide tests. Four additional clones gave positive slide agglutination tests with live bacteria, but in tube testing no clones agglutinated Formalin-treated bacteria. No cross-reactions with unrelated bacteria were seen, but several clones reacted in the enzyme immunoassay with many of the 24 Campylobacter strains studied. The clone which gave the highest mean enzyme immunoassay values with Campylobacter coli and C. jejuni strains also reacted with Campylobacter fetus subsp. veneralis and C. fetus subsp. fetus strains. This clone also gave the highest enzyme immunoassay value with an acid glycine extract of the immunogen, which indicates the presence of common antigens in the extract. The results suggest that monoclonal antibodies may be used to devise serotyping schemes for Campylobacter spp.     

Cell-mediated immunity to chemically xenogenized tumors--III. Generation of monoclonal antibodies interfering with reactivity to novel antigens

To develop monoclonal antibodies (MAbs) recognizing drug-mediated tumor antigens on a chemically xenogenized murine lymphoma, hybridomas were constructed with splenocytes from histocompatible mice hyperimmunized with L5178Y cells antigenically altered by triazene treatment in vivo (clone D, derived from a polyclonal L5178Y/DTIC subline). Screening of supernatants with parental and xenogenized cells showed that nine MAbs displayed exclusive or preferential reactivity with clone D cells as detected by immunofluorescence, and failed, as a rule, to bind normal or unrelated malignant cells of the same or different haplotype. Moreover, no reactivity was displayed to the triazene-xenogenized variants of antigenically unrelated tumors. All nine MAbs, however, were capable of binding a panel of L5178Y/DTIC clones in addition to clone D. When the ability of these antibodies to interfere with the development of cell-mediated immunity to clone D cells in vitro was tested, it was found that the proliferative reaction and generation of cytolytic activity by syngeneic lymphocytes were inhibited by addition of several MAbs to the tumor--lymphocyte co-cultures.     

Rat monoclonal antibodies. VII. Enhancement of ascites production and yield of monoclonal antibodies in rats following pretreatment with pristane and Freund's adjuvant

The effect of intraperitoneal injections of pristane, incomplete Freund's adjuvant (IFA) and a v/v mixture of pristane and IFA (called PIFA) on ascites production and the yield of monoclonal antibodies has been studied in Louvain rats. The best results were obtained following injection of 2 ml PIFA at the moment of i.p. transfer of hybridoma or immunocytoma cells. Ascites production was increased by as much as 4.7 times and monoclonal antibody production by more than six times compared with untreated control rats.     

Lectin binding patterns and monoclonal antibodies to epidermal antigens in tumours of the skin

Monoclonal antibodies to epidermal antigens and cell surface carbohydrate markers, as defined by lectin binding, were used to analyze the cells in squamous and basal cell carcinomas of the skin (SCC and BCC). The cells in BCC failed to stain with the lectin peanut agglutinin (PNA), which stains surface carbohydrates of cells in the stratum spinosum and stratum granulosum layers of normal epidermis, confirming histological observations that the cells in BCC are incapable of differentiation beyond the basal cell stage. Conversely, the central cells in SCC did react with PNA, suggesting that they can differentiate to a stage equivalent to the stratum spinosum of epidermis. The zone immediately surrounding BCC differed from that around SCC in lectin binding and staining with antisera to laminin and fibronectin, an observation which could be connected with the failure to metastasize. It was of interest that histologically normal skin immediately adjacent to and overlying these tumours showed marked changes in reaction with markers of normal epidermis. The outer layers of this epidermis showed aberrant retention of the lower molecular weight cytokeratins marked by the monoclonal antibodies LMM2 and LMM3, and occasional strong staining of individual cells by the stratum granulosum-reactive LMM1. These changes appear to be indicative of a 'premalignant' state in these cells and the monoclonal antibodies are thus potentially useful reagents for early detection of skin malignancies.     

Muscle and species reactivity of mouse monoclonal antibodies to human eye muscle membrane antigens.

We studied the tissue and species reactivity of mouse monoclonal antibodies (MCAB) produced by immunizing mice with a 100,000g ultracentrifuged preparation of human eye muscle (HEM) membranes. Twenty-three MCABs, 20 of which reacted in an enzyme-linked immunosorbent assay (ELISA) with HEM membrane, 2 with human thyroid membrane, and 1 nonreactive negative control, were selected for the study. The muscle and species specificity of 6 of the most reactive and more restrictively reactive MCAB were studied in more detail. All reacted in ELISA with human skeletal muscle membrane and, to a lesser extent, with human cardiac muscle membrane, but not with human brain membrane. The 6 MCAB cross-reacted with eye muscle membrane prepared from pig but not rat, although reactivity with human tissue was greatest for all MCAB tested. When tested in immunoblotting with HEM and thyroid membranes, 3 of 6 MCAB reacted with a 64-kDa protein in HEM, 2 of which also reacted with an antigen of the same molecular weight in thyroid membrane. In a complement-mediated antibody-dependent cytotoxicity assay, 5 of 19 MCAB lysed HEM cells, 6 of 21 lysed human skeletal muscle cells, and 10 of 22 lysed human thyroid cells. These findings support results from earlier clinical studies which showed that eye muscle membrane reactive autoantibodies in the serum of patients with thyroid-associated ophthalmopathy cross-react with membrane prepared from other striated muscle. The significance of eye muscle, skeletal muscle, and thyroid cross-reactivity of MCAB is discussed in the context of autoimmune thyroid disease and ophthalmopathy.     

Detection of urothelial Lewis antigens with monoclonal antibodies.

The detectability of Lewis a and b antigens (Lea, Leb) was examined in the normal urothelium of 28 human subjects whose red blood cells (RBCs) were also tested for Lea and Leb. Mouse monoclonal antibodies as well as goat and human antisera were used on paraffin-processed and fresh-frozen tissues. Multiple biopsy specimens from the same individual were studied in order to evaluate temporal as well as topographic consistency of the results. In addition, the expression of the Lewis antigens in the appendiceal mucosa was investigated in 9 of these patients. The antibodies against Lea reacted with the urothelium of all patients with either Lea+b- or Lea-b+ RBC phenotype. None of the 5 patients with Lea-b- RBCs had Lea urothelial reactivity. The antibodies against Leb reacted with the urothelium of all patients with Lea+b- or Lea-b+ RBCs and with 2 of the 5 patients with Lea-b- RBCs. The mucin in the goblet cells of the appendiceal mucosa was positive only for the Lewis antigen that was also detectable of the individual's RBCs. These findings indicate that the expression of Lewis antigens in nonsecretory epithelia may not follow the same principles as in the blood and secretions.     

Characterization of Borrelia coriaceae antigens with monoclonal antibodies.

Three monoclonal antibodies (F6F3, F6B11, and F6B3) were developed against Borrelia coriaceae antigens. All three antibodies appeared to be specific for this species and did not cross-react with Borrelia burgdorferi (strains B31 and IRS), Borrelia hermsii, Borrelia anserina, Leptospira interrogans serovar hardjo, or Treponema hyodysenteriae, as determined by indirect fluorescent antibody staining, enzyme-linked immunosorbent assay, and Western immunoblot analysis. Only one of these antibodies, F6B3, bound to spirochetes present in organ smears from the argasid tick, Ornithodoros coriaceus. The antigens recognized by F6F3, F6B11, and F6B3 have apparent molecular weights of ca. 37,000, 35,000, and 16,000, respectively, as determined by Western blot analysis. Antigens were analyzed by immune electron microscopy as well as Western blot and indirect fluorescent antibody staining analysis of spirochetes after enzyme (trypsin and protease K) and detergent (Triton X-100) treatments. These studies suggest that all three antigens are integral membrane proteins. The characteristics of the 37K and 35K proteins are consistent with the outer surface proteins of B. burgdorferi (OSP A and OSP B) described by Barbour et al. (A. G. Barbour, S. L. Tessier, and S. F. Hayes, Infect. Immun. 45:94-100, 1984), while data regarding the 16K protein are less conclusive but may suggest a cytoplasmic membrane location. We suggest that the 37K, 35K, and 16K antigens be designated integral membrane proteins A, B, and C, respectively, as a result of these studies.     

Glutaraldehyde fixation of target cells to plastic for ELISA assays of monoclonal anti-HLA antibodies produces artefacts

We found that an ELISA method for screening mouse monoclonal antibodies raised against antigens of the HLA system, that involves glutaraldehyde fixation of the target cells, produces both false negatives and false positives. The false negative results are due to destruction and/or modification of the antigenicity of some HLA-D region coded molecules; the false positives are mostly caused by non-specific adhesion of IgM, even at 1.625 μg/ml, to glutaraldehyde fixed cells. To a lesser extent there is nonspecific binding of IgG2a and IgG2b monoclonal antibodies particularly at concentrations above 12.5 μg/ml. These findings are unfortunate because the fixation of cells to the plates appeared to be a major technical advance as it removed the need for multiple centrifugation steps in the ELISA assay.     

Simultaneous enzyme-immunocytochemical detection of antigens in monocellular specimens with monoclonal antibodies

An immunoperoxidase and immunoalkaline phosphatase technique is described specially devised for separate cell specimens such as smears, cytospins, and tissue imprints. The reaction may be used alone or in combination for simultaneous staining with 2 different monoclonal antibodies. Monoclonal antibodies Leu3a, Leu2a, T4, T8, anti-kappa, and anti-lambda light chain were used in combination to visualize subsets of human lymphocytes selectively. Six cases of chronic lymphocytic leukemia were classified by this method.     

Characterization of smooth lipopolysaccharides and O polysaccharides of Brucella species by competition binding assays with monoclonal antibodies.

Previously, four epitope specificities on the O chain of Brucella species were reported: M, A, C, and C/Y. In this work, according to monoclonal antibody binding to smooth lipopolysaccharides of Yersinia enterocolitica 0:9, Brucella abortus W99 (A-dominant strain), and B. melitensis Rev1 (M-dominant strain), seven O-chain epitope specificities were defined: M, A, C (M > A), C (M = A), C/Y (M > A), C/Y (M = A) and C/Y (A > M). Competitive binding assays between these monoclonal antibodies suggested that these different epitopes are probably overlapping structures.     

Discrimination of epitopes identified by monoclonal antibodies by competitive binding to nitrocellulose bound antigens

A new method is described for determining the distribution of epitopes identified by monoclonal antibodies. The method utilizes nitrocellulose membranes as a solid support for antigens which are rapidly adsorbed to nitrocellulose by vacuum-blotting and then used in competitive antibody binding assays. The distribution of epitopes is established by the reciprocal cross-blocking of radiolabeled antibody by increasing concentrations of unlabeled antibody. When unlabeled antibody does not block the binding of labeled antibody to antigen, the 2 antibodies recognize distinct epitopes. When unlabeled antibody blocks the binding of labeled antibody to antigen, the 2 antibodies recognize the same epitope. The method is rapid, sensitive and should be applicable to screening monoclonal antibodies to any epitope.     

Studies with monoclonal antibodies against brush border antigens in Heymann nephritis

The authors have prepared and studied three murine monoclonal antibodies that are reactive with antigens in brush border regions of proximal renal tubules of rats. Two of the antibodies, 14C1 and AG3, were derived from mice immunized with Fx1A and the third, 4H6, from a mouse immunized with isolated glomeruli obtained from rats with Heymann nephritis. Immunohistochemical, immunoelectron microscopic, and immunochemical studies showed that the three antibodies recognized different antigens. The antibody 14C1 recognized the previously described nephritogenic glycoprotein, gp 330, in microvillar brush border preparations, and reacted with material present on podocyte cell surfaces of normal rat kidneys, especially in coated pits, as well as with material in the glomerular deposits of rats with Heymann nephritis. The antibody 4H6 recognized a 110-kilodalton microvillar antigen and reacted with material in the glycocalyx of podocytes of normal glomeruli, but showed only equivocal reactivity with material in the deposits in Heymann nephritis. AG3 failed to immunoprecipitate a distinctive antigen in microvillar preparations and did not react with the glomeruli of normal rats or of rats with Heymann nephritis. All three antibodies reacted with epithelial cells in the intestine, epididymis, and placenta; 4H6 also reacted with thin loops of Henle as well as with endothelial cells or other cells in lung, lymphoid tissue, liver, and spleen. The results demonstrate that not all brush border antigens participate in Heymann nephritis and confirm that an antigen (gp 330) involved in the formation of glomerular deposits in Heymann nephritis is normally present on podocyte surfaces, especially in coated pits, but is not present in extracellular sites.     

A rapid solid-phase enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens

A solid-phase enzyme-linked binding assay is described for screening monoclonal antibodies to cell surface antigens. E. coli β-galactosidase was coupled to rabbit anti-rat Ig and used to detect the binding of rat monoclonal antibodies to cells which had been fixed to the wells of microtitre plates using a combination of poly-l-lysine and glutaraldehyde. This method was found to be advantageous for the large scale screening of monoclonal antibodies with a panel of cell types, and has been useful in the selection of antibodies which would be candidates for differentiation markers within the human and mouse haemopoietic systems.     

DR antigens on melanoma cells: analysis with monoclonal antibodies

Two monoclonal antibodies (691-13-17 and 37-7) can precipitate DR antigens from radioiodinated, detergent solubilized melanoma cells. However, after complete depletion of lysates with one antibody (691-13-17) alpha-like chains can be still be precipitated from some melanoma cells by the other antibody (37-7). Antibody 37-7 also precipitates an additional distinct antigen from SK-MEL 37 but not from any five other melanoma cell lines.     

Recognition of binding sites on Candida albicans by monoclonal antibodies to human leukocyte antigens.

Candida albicans exhibits examples of human molecular mimicry, including receptors resembling human steroid receptors and human complement receptor (CR)-like receptors that bind the complement fragments C3d and iC3b. To further characterize the epitopes of the Candida human CR-like molecules, a panel of monoclonal antibodies (MAbs) against epitopes within the human CR3 was used. MAbs Mo1, M1/70HL, and 7C3 bound to the germ tube, as assessed by immunofluorescence. MAbs Leu15, 60.1, and 95G8 did not bind. Miscellaneous MAbs against other antigens on human leukocytes (B2, 3D9, and OKT4) did not bind. However, MY9, which recognizes a monocyte antigen, bound extensively to the germ tube. The binding of certain anti-CR3 MAbs suggested limited identity between the Candida CR3-like receptor and the human CR3. The binding of MY9 identified an antigen recognized by a MAb to a human cell antigen not previously known to exist on C. albicans.