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.     

Identification of woodchuck class I MHC antigens using monoclonal antibodies

Abstract: Two class I major histocompatibility complex (MHC) proteins with molecular masses of 43- and 39-kDa were identified in the cell surface membranes of normal woodchucks using a newly developed anti-woodchuck class I monoclonal antibody (mAb) B1b.B9 and immuno-blotting. B1b.B9 was generated by immunizing mice with viable wood-chuck peripheral blood mononuclear cells and was selected for anti-class I MHC reactivity using a cellular enzyme–linked immunoassay, indirect immunofluorescence on tissue sections and flow cytofluorimetry. The distribution pattern of class I MHC antigen on woodchuck lymphoid cells was found to be similar to that reported in other species. Also, the antigen expression on normal woodchuck hepatocytes was comparable to that observed on normal human liver parenchymal cells; thus, the antigen was not detected on hepatocytes by staining of liver tissue sections, but was found by indirect immunofluorescence staining of isolated liver cells. Western blot analysis of the plasma membranes from normal woodchuck hepatocytes revealed the presence of a single species of class I MHC heavy chain protein with a molecular mass of 43-kDa, whereas splenocyte plasma membranes showed intense expression of a 43-kDa species, as well as the presence of a 39-kDa protein. The 39- and 43-kDa proteins were extracted with Triton X-114 to the hydrophobic protein phase, suggesting that they both contain a hydrophobic transmembrane domain. The data obtained indicate that the B1b.B9 identifies a nonpolymorphic epitope of woodchuck class I MHC heavy chains, providing an important reagent for the study of the pathogenesis of hepatitis B virus infection in a woodchuck model.     

Immunotherapy with monoclonal antibodies directed against the immunosuppressive domain of p15E inhibits tumour growth.

Immunosuppressive retrovirus-related proteins, like p15E, are involved in tumour-associated immunosuppression. In the present study we investigated whether such proteins could be used as targets in tumour immunotherapy using MoAbs. Immunotherapy was performed in mice inoculated with the Rauscher virus-transformed myeloid cell line RMB-1. RMB-1 cells express retroviral antigens at their cell surface. In order to obtain constant serum titres of MoAbs over a prolonged period of time during therapy, anti-p15E antibody-producing hybridoma cells were encapsulated in alginate and injected intraperitoneally in tumour-bearing mice. Using this technique, serum antibody titres of 50-100 micrograms/ml were obtained, which remained constant over a period of at least 3 weeks. Therapy experiments were performed using anti-p15E antibodies 19F8, which recognizes both cell surface-associated as well as circulating p15E, and ER-IS5, which did not react with surface-bound p15E beyond background, but which neutralizes circulating p15E. Inoculation of alginates containing anti-p15E hybridoma cell lines in RMB-1 tumour-bearing mice showed inhibition of tumour cell growth. In survival experiments, 19F8 cured eight of 23 tumour-bearing mice. The p15E neutralizing antibody ER-IS5 caused a significant longer survival, but therapy with this MoAb alone was not sufficient to cure the animals of the RMB-1 tumour.     

Study of antibodies directed against antigens of the gut epithelium of Schistosoma mansoni using an immunofluorescence reaction. IV. Blocking action on immunity of an IgM monoclonal antibody

The inhibitory effect on immunity is demonstrated by passive transfer experiments in mice. It can be shown in a secondary infection with Schistosoma mansoni and also in primary infection. The results indicate that blocking activity probably interfers with non specific immunity mechanisms.     

Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I

The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide-linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty-one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope-mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I.
Two monoclonal antibodies directed against reduced/alkylated chain I bound to the overlapping peptides 53–66 and 60–70 of chain 1. The monoclonal antibody directed against reduced/alkylated chain 2 bound to the overlapping peptides 36–49 and 43–56 of chain 2. Binding specificity was demonstrated by inhibition by reduced/alkylated Fel d I for all three monoclonal antibodies.
Another monoclonal antibody against reduced/alkylated Fel d I had been found to bind predominantly to reduced/alkylated chain 2 on immunoblot in previous studies (27). It bound to peptides 1–16 and 60–70 of chain 1 and peptides 1–14 and 50–63 of chain 2; it is therefore probably directed against a conformational epitope formed by these four regions. Possibly because of low affinity of this monoclonal antibody, specificity of its binding could not be verified by inhibition studies.
A panel of monoclonal antibodies directed against native Fel d I bound to peptides 1-16 and 60–70 of chain 1 and peptides 1–14 and 43–56 of chain 2. For two monoclonal antibodies, binding to each peptide was investigated and shown to be inhibitable by native Fel d I. These antibodies are therefore probably directed against a conformational epitope formed by these four regions.
These studies give us substantial information about the quaternary structure of Fel d I.     

Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry

Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-μm polystyrene beads through the Fc portion by the use of protein A. The antibody-coupled beads showed a high capacity to bind HLA-A and -B proteins as well as their chains by the intracytoplasmic domain, keeping the extracellular domains solvent exposed. The density of HLA class I proteins bound on the beads was approximately the same as that on cultured B cells. The antibody beads made it possible to quantitate peptide-HLA class I binding, i.e., in vitro HLA class I assembly by flow cytometry. The assembly rate determined by the provisionally called flow cytometric HLA class I assay was 15%–19% for the reassembly of dissociated HLA class I proteins with the released selfpeptides. With single synthetic peptides, the highest rate so far obtained was 6.5%. The assay specificity and reproducibility were satisfactory.     

Interaction of blood sera from patients with autoimmune diseases with expressed cDNA fragment of topoisomerase I and with monoclonal antibodies to the enzyme

V. A. Éngel'gardt Institute of Molecular Biology, Academy of Sciences of the USSR. Institute of Rheumatology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR I. B. Zbarskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 12, pp. 598–600, December, 1990.     

Efficient production of mouse and rat monoclonal antibodies against the O antigens of Salmonella serogroup C1, using LPS-coated bacteria as immunogen.

The - -galactopyranosyl binding lectin from the seeds of Bandeiraea simplicifolia (a.k.a. Griffonia simplicifolia) termed BS-I, strongly reacts with murine IgD and with no other protein in ascites including all other classes of immunoglobulins as determined by immunoprecipitation, hemagglutination inhibition and affinity binding. Based on this finding, murine IgD could be rapidly purified directly from whole ascitic fluid by passage over affinity beads of BS-I linked to Sepharose 4B and subsequent elution by a buffer containing 0.1 M -galactose. The sugar eluted product is 95–99% pure as determined by SDS-PAGE and represents 90–95% of the total IgD in the initial ascites by ELISA assay. Both monomeric and dimeric murine IgD may be purified by this procedure. Human IgD is unreactive with this lectin. Treatment of purified IgD with endoglycosidases that remove either O- or N-linked glycosides indicates that BS-I binds to IgD only via N-linked carbohydrate chains.     

Accuracy of the determination of S100 protein expression in malignant melanoma using a polyclonal antibody directed against S100 and monoclonal antibodies specific for S100 alpha and beta

Abstract

S100 proteins are present in a variety of tissues and perform regulatory functions in numerous metabolic processes. They have an important role in many human cancers, including malignant melanoma. Both polyclonal and monoclonal antibodies have been used to investigate S100 expression in melanoma tissue sections. This study aimed to determine the accuracy and sensitivity of these two types of antibodies in detecting S100 proteins in paraffin processed tissue cases of malignant melanoma. The study compared routinely used rabbit polyclonal anti-S100 antibody raised against both anti-S100A and B isoforms (Dako, Glostrup, Denmark), as per studies by Timar, and compared and contrasted findings with mouse monoclonal anti-S100A and anti-S100B antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The study involved the assessment of formalin-fixed paraffin-embedded tissue blocks from 56 cases of malignant melanoma, consisting of 23 superficial spreading, nine nodular, eight lentigo maligna, five acral lentigenous forms, five metastatic melanomas (two sentinel lymph node positive cases and three cases of nodal involvement from cases of elective nodal groin dissections), and six cases of desmoplastic malignant melanoma (DMM). The slides were stained by immunohistochemical methods on an automated platform (BenchMark XT; Roche, USA) and employing the iView detection system. All slides were examined by routine light microscopy by two independent assessors. The best results for both intensity of staining and percentage of positive tumor cells were achieved with polyclonal anti-S100 antibody and monoclonal anti-S100B antibody. Anti-S100A antibody yielded weaker staining intensity (with mean intensity of 1·8, compared to 2·8 for both anti-S100B antibody and polyclonal anti-S100 antibody), and a lower percentage of positive melanoma cells (an average of 74% for anti-S100A, compared to 95% for both anti-S100B antibody and polyclonal anti-S100 antibody). This result was statistically significant (P<0·01). Staining in cases of DMM gave the same results (P<0·01). The conclusion from this study is that polyclonal anti-S100 antibody and monoclonal anti-S100B antibody are more suitable than monoclonal anti-S100A antibody for diagnostic investigations of malignant melanoma, irrespective of the histological type of melanoma.