Production of monoclonal antibody to deltorphin-I and its immunocytochemical application to adult rat brain and cultured rat brain neurons

A monoclonal anti-deltorphin-I antibody specifically recognizing its NH2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Characterization and immunocytochemical application of monoclonal antibodies against enkephalins

Monoclonal antibodies were produced following immunization of mice with either [Leu5]enkephalin-bovine serum albumin or [Met5]enkephalin-keyhold limpet hemocyanin conjugates. Two monoclonal antibodies coded NOC1 and NOC2, respectively, were derived. These monoclonal antibodies did not discriminate between Leu- and Met-enkephalin in either radioimmunoassay or immunocytochemistry. NOC1 was characterized in detail. In radioimmunoassay NOC1 displayed about 40% crossreactivity with C-terminal extended Met-enkephalin hexapeptides and 7% with the extended heptapeptide (-Arg-Phe-OH), but did not recognize other endogenous peptides. In immunocytochemistry the NOC1 and NOC2 recognized all well-established "enkephalin immunoreactive sites," but they did not bind to areas known to contain beta-endorphin or high levels of pro-enkephalin. NOC1 was shown to be a suitable tool to demonstrate enkephalin immunoreactive sites by radioimmunocytochemistry utilizing both internally and externally labeled monoclonal antibodies.     

Production of a monoclonal antibody against Aeromonas hydrophila and its application to bacterial identification

AIMS: to develop a monoclonal antibody (MAb) for the rapid detection of Aeromonas hydrophila in human faeces. METHODS AND RESULTS: A monoclonal antibody with strong specificity against Aer. hydrophila was obtained by the fusion of myeloma cells and splenocytes of a mouse immunized with vegetative cells of Aer. hydrophila ATCC 7966, followed by a two-step selection against other species of the genera. ELISA analyses revealed that MAb 5F3 strongly reacts with all the Aer. hydrophila strains evaluated, showing a just basal reactivity against other species of the genera, especially Aer. sobria and Aer. caviae. CONCLUSIONS: MAb 5F3 was characterized as an IgG that recognized a polypeptide of approximately 110 kDa. SIGNIFICANCE AND IMPACT OF THE STUDY: This MAb could be used to detect Aer. hydrophila in human stool samples.     

Production of a monoclonal antibody specific for Salmonella spp.

A monoclonal antibody (MAb) specific for heat-treated Salmonella spp. which shows no cross-reactivity with other enteric bacteria when tested by an ELISA system, has been developed. The MAb isotype is IgG2a and has been successfully purified using protein A. Preliminary work suggests that the MAb recognizes a protein present in the Boivin lipopolysaccharide extracts of Salmonella spp.     

Production and characterization of a mouse monoclonal antibody specific for lentil lectin

A murine IgA monoclonal antibody (MoAb) was produced against the widely used glucose/mannose-specific two-chain mitogen from Lens culinaris (lentil) belonging to the Vicieae tribe of the Leguminosae family. The MoAb designated, 98, F-10, was found to be specific for lentil lectin when tested in dot blotting against 22 different native lectins. The antigenic specificity was also tested against subunits of 13 completely sequenced legume lectins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. The MoAb showed a strong reaction only against the lentil heavy subunit. Comparison of the amino-acid sequences revealed 13 amino-acid residues which might be involved in the epitope reactive with this antibody. The MoAb did not react with synthetic peptides from the heavy subunit of lentil.     

Production and characterization of monoclonal antibodies against the polyamine,spermine: immunocytochemical localization in rat tissues

Two monoclonal antibodies of types IgG_2b and IgG_2a, anti-spermine-(Spm)-1 (ASPM-1) and anti-Spm-2 (ASPM-2) respectively were found among five clones of murine monoclonal antibodies, which were raised against Spm conjugated with bovine serum albumin via the cross-linker N-(-maleimidobutyryloxy) succinimide (GMBS). Antibody specificity was evaluated by a recently developed ELISA binding test, and led to the study of tissue sections by immunocytochemistry (ICC). ASPM-1 showed exclusive immunoreactivity with Spm, with the exception of a negligible cross-reactivity (2.0%) with spermidine (Spd). ASPM-2, on the other hand, reacted almost equally with acetylspermine (Ac-Spm) and N ¹-acetylspermidine (N¹-Ac-Spd) but with none of the other polyamine-related compounds tested. Complete agreement was obtained with the results of immunoblot analysis. Furthermore, results for antibody specificity obtained with the ELISA inhibition test and ICC model experiments using Sepharose gel beads strongly suggested that ASPM-1 recognizes the Spm molecule possessing at least a free terminal primary amino group, while ASPM-2 recognizes the Spm molecule acylated at both the terminal primary amino groups. An ICC method using ASPM-2 produced strong staining for polyamines (PAs) in the cytoplasm (but very few in the nuclei) of two different tumor cell lines and protein- or peptide-secreting cell systems, including exocrine and endocrine cell types; ASPM-1 showed immunoreactivity only with the tumor cell lines. These results strongly suggest that ASPM-2 may be useful for studies on actively proliferating and neoplastic cells, supporting our previously proposed idea that in immunocytochemistry PAs were converted to a variety of PA derivatives during the fixation process.     

Production of monoclonal antibody against Listeria monocytogenes and its application to immunochromatography strip test

An immunochromatography (ICG) strip test based on a monoclonal antibody for the rapid detection of L. monocytogenes in meat and processed-meat samples was developed in this study. A monoclonal antibody (MAb) specific to L. monocytogenes was produced from cloned hybridoma cells (FKLM-3B12-37) and used to develop an ICG strip test. The antibody showed a stronger binding to L. monocytogenes than other Listeria species, and a weak cross-reaction to S. aureus based on an ELISA. The detection limit of the ICG strip test was 10(5) cell/ml. In total, 116 meat and processed-meat samples were collected and analyzed using both the ICG strip test and a PCR. The ICG strip test and PCR indicated L. monocytogenes contamination in 34 and 27 meat samples, respectively. The 7 meat samples not identified as L. monocytogenes positive by the PCR were also tested using an API kit and found to be contaminated by Listeria species. In conclusion, the ICG strip test results agreed well with those obtained using the PCR and API kit. Thus, the developed ICG has potential use as a primary screening tool for L. monocytogenes in various foods and agricultural products, generating results within 20 min without complicated steps.     

Development and application of a monoclonal antibody against Thiothrix spp.

Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml.     

A monoclonal antibody specific for rat intestinal lymphocytes

A monoclonal antibody, RGL-1, was produced by fusion of NSI myeloma cells with spleen cells of a mouse immunized with isolated rat intraepithelial lymphocytes (IEL). SDS-PAGE analysis revealed that RGL-1 precipitated two major noncovalently bound chains of about m.w. 100,000 and 125,000, and a minor component of m.w. 200,000. Examination of both tissue sections and isolated cells indicated that RGL-1 stained the majority of the lamina propria lymphocytes and IEL but only very few cells (less than 2%) in the lymphoid organs and small numbers of lymphocytes in other mucosae. In the small intestine, RGL-1 stained lymphocytes with the helper (W3/25) as well as the cytotoxic/suppressor (OX8) phenotype. The antibody reacted with 95% of the granular IEL but with less than 0.1% of the blood large granular lymphocytes. Although mature IgA plasma cells in the lamina propria were RGL-1-, some large IgA-containing cells were weakly positive. In the gut-associated lymphoid tissues (GALT), studies combining immunofluorescence and autoradiography indicated that 56 and 73% of rapidly dividing cells of mesenteric lymph nodes and of thoracic duct lymph (TDL) stained with RGL-1, respectively. In addition, 90 to 100% of the IgA-containing blasts of MLN and 75% of those of TDL were labeled by RGL-1. In contrast, rapidly dividing cells of spleen and of peripheral lymph nodes did not stain with RGL-1. Because RGL-1 can be demonstrated on both intestinal lymphocytes and their immediate precursors in the GALT, its expression may be related to the homing of lymphocytes into the gut mucosa.     

Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

Production and characterization of a monoclonal antibody specific for Salmonella O5-antigen

The present report described the production and characterization of a monoclonal antibody, TMY1, specific for an O-antigen of Salmonella bacilli. The following results were obtained: The slide agglutination test against strains of Salmonella serovars indicated the responsiveness of TMY1. TMY1 was reactive only to strains that also possessed O5-antigen in group O4. The agglutinating ability of TMY1 was absorbed completely with bacilli possessing O5-antigen in group O4. When treated with 1 N HCl, bacilli possessing O5-antigen in group O4 showed no agglutinability in the presence of TMY1. The data indicates that TMY1 was specific for O5-antigen. Strains of Salmonella group O4 were classified by the agglutination test with TMY1 into two subgroups, O5-antigen positive and negative, suggesting the usefulness of TMY1 as an epidemiologic tool for the serotyping of Salmonella.     

Production of a monoclonal antibody specific for a Flavobacterium species isolated from soil

Abstract Hybridomas secreting monoclonal antibodies (MABs) specific for a soil Flavobacterium species (P25) were isolated. The MAB (D10) was used to target P25 using an enzyme-linked immunosorbant assay (ELISA) and indirect immunofluorescence. Cross-reactivity of the MAB with other Gram-negative bacteria (including Flavobacterium spp.) and a number of Gram-positive bacteria was investigated but none were found. Cross-reactivity with other orange/yellow pigmented Gram-negative rods ( Pseudomonas/Flavobacterium type) isolated from the soil into which P25 has been introduced in field experiments was also assessed using a modified colony blotting procedure. None of the indigenous species tested were recognised by the monoclonal antibody, thereby allowing unambiguous identification of P25 in soil. The MAB D10 was shown to recognise P25 growth under low-nutrient or stored under starvation conditions, suggesting that the antigen is a constitutive component of the cell and that the microorganism should be detected in oligotrophic environments such as soil. The pattern of fluorescence of P25 gave a clear indication of the localisation of the antigen in the outer membrane/cell wall region, and this was confirmed by immunogold labelling. Preliminary studies on the limits of detection of P25 using immunofluorescence suggest that densities as low as 20 bacteria g⁻¹ soil can be enumerated.     

Development and application of a monoclonal rat peroxidase antiperoxidase (PAP) immunocytochemical reagent

Summary Monoclonal antibodies are being increasingly used in immunocytochemistry but their localisation by the peroxidase antiperoxidase (PAP) procedure requires the use of rat or mouse PAP. In this paper we describe the development and application of a monoclonal rat PAP. This reagent has been used successfully for immunocytochemistry at light and electron microscopy level in combination with rat monoclonal antibodies against serotonin (5-HT), substance P and somatostatin. The monoclonal rat PAP has several advantages over conventional polyclonal rat PAP and is likely to be a valuable developing reagent in immunocytochemistry using rat monoclonal antibodies.     

A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly.

Abbreviations: B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-l-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II     

Production of monoclonal antibody against histamine and its application to immunohistochemical study in the stomach

A monoclonal antibody against histamine has been produced. A histamine–haemocyanin conjugate prepared using 1-ethyl-3-(3-dimethylami nopropyl) carbodiimide as a coupling agent was used for immunizing mice. Immunized mice were sacrificed to prepare monoclonal antibody using a hybridoma technique. On immunospot assay, the hybridoma culture supernatant containing a monoclonal antibody was capable of detecting 50 pmol of histamine. Using this antibody, we examined the cellular localization of histamine-like immunoreactivity in the stomach of normal or -fluoromethylhistidine-treated rats and mice. Immunoreactive cells were abundant in the gastric mucosal layer. These positive cells were often located in the basal half of the fundic gland but were rare in the pyloric gland. The cells, small or medium in size, spindle or cone in shape, were intermingled with immunonegative epithelial cells. In the cytoplasm of the positive cells, granular reaction products were densely deposited. In addition, a few positive cells, identified as mast cells by Toluidine Blue staining, were distributed mainly in the submucosal and muscular layer. The antibody preabsorbed with 10 mm histamine gave no positive immunostaining. For pharmacological study, some rats were injected six times with -fluoromethylhistidine every 8 h. In these rats, positive cells except mast cells were no longer detected. In conclusion, the monoclonal antibody produced appears to be highly specific for histamine. Its application in immunohistochemistry should provide a powerful tool for analysing the roles of histamine in enterochromaffin-like or mast cells in the stomach. © 1998 Chapman & Hall     

Binding site of the rat liver specific monoclonal antibody

A rat liver-specific antigen (RLSA) lost its binding ability to the corresponding monoclonal antibody after treatment with N-glycanase or sialidase, which suggested that the specific binding site might be in a portion of the sugar chain containing sialic acid. The specific antigen reacted with wheat germ agglutinin, lentil lectin, erythroagglutinating phytohemagglutinin and Ricinus communis agglutinin, but not with concanavalin A or peanut agglutinin. These results suggest that the specific antigen has asparagine-linked complex-type sugar chains which might be the binding sites of the monoclonal antibody.     

Production of monoclonal antibody against protopectinase from Kluyveromyces wickerhamii

Four monoclonal antibodies (MCA 3–6, 4-2, 9-2, and 10-2) against protopectinase (an endo-polygalacturonase) from Kluyveromyces wickerhamii were prepared. MCAs 3-6 and 4-2 reacted to protopectinases produced by K. fragilis and K. marxianus as well as to the protopectinase produced by K. wickerhamii. However, 9-2 and 10-2 were specific for the protopectinase from K. wickerhamii. These MCAs did not inhibit the protopectinase reaction or the polygalacturonase reaction of protopectinases produced by these species. We used these MCAs to find protopectinases in the culture filtrates of Kluyveromyces yeasts.     

Functional properties of a rat monoclonal IgE antibody specific for Schistosoma mansoni

A rat monoclonal antibody of IgE isotype (B48-14) raised against Schistosoma mansoni has been generated by the fusion of mesenteric lymph node cells from LOU/M rats immunized with a preparation of adult schistosome worms and IR973F nonsecreting rat myeloma cells. Investigation of the in vitro effector functions of this IgE antibody showed a high level of cytotoxicity against S. mansoni schistosomula in the presence of eosinophils, macrophages, and platelets. A significant level of protection (40 to 60%) against a challenge infection with S. mansoni cercariae was achieved by passive transfer experiment of B48-14 IgE to naive recipient rats. By immunoprecipitation, B48-14 IgE antibodies were shown to react with an antigen of 26 kDa present in excretion-secretion products of schistosomula, previously described as a potential immunogen eliciting a protective IgE response against schistosomiasis.