The use of monoclonal antibodies to differentiate isolates of herpes simplex types 1 and 2 by neutralisation and reverse passive haemagglutination tests

Monoclonal antibodies specific for herpes simplex type 1 or type 2 were used in reverse passive haemagglutination tests or infectivity neutralisation tests to serotype 100 isolates of herpes simplex virus (HSV). All isolates were independently serotyped by measuring their sensitivity to bromovinyl deoxyuridine. Reverse passive haemagglutination tests with type-specific antibodies directed against the HSV glycoprotein D and major DNA binding protein gave results in perfect agreement with the results of drug-sensitivity measurement. A single isolate behaved anomalously in the neutralisation test with a type 1-specific antibody directed against glycoprotein A/B. Restriction-enzyme analysis of virus DNA suggests that this isolate contains a variant glycoprotein A/B. The two methods used for serotyping proved very sensitive, giving adequate results with samples containing as little as 100 plaque forming units (pfu) of HSV. The reverse passive haemagglutination test has the additional advantages of speed and simplicity.     

A reverse passive haemagglutination test for the detection of respiratory syncytial virus in nasal secretions from infants

A reverse passive haemagglutination (RPH) test has been developed for the detection of respiratory syncytial (RS) virus in nasal secretions, taken from infants with acute respiratory illness. In the final form of the procedure, RS virus was detected in 24 of 25 samples positive for RS virus by tissue culture and/or fluorescence antibody staining and in two samples negative for RS virus by these techniques. The simplicity of the technique and the rapidity with which it may be performed together with its apparently high degree of sensitivity should make RPH useful in the rapid diagnosis of RS virus.     

A modified passive-haemagglutination technique for the detection of cytomegalovirus and herpes simplex virus antibodies: application in virus-specific IgM diagnosis

Cytomegalovirus (CMV) and herpes simplex virus (HSV) antibodies were detected by a modified passive haemagglutination (PHA) technique. The main features of this modification are the use of a simpler method for the removal of nonspecific sheep agglutinins in the sera, the deployment of commercially available CMV and HSV antigens, and a different sucrose density gradient (SDG) system for the separation of IgM from IgG. The modified procedure proved to be a reliable, specific, and sensitive technique in detecting antibodies to CMV and HSV in both whole serum and in the separated IgM and IgG fractions. It was as reliable as the complement-fixation (CF) test when applied in seroepidemiological studies and in the detection of antibodies in cord serum. Preliminary data are provided which suggest that the combination of SDG and PHA may prove to be a more reliable system for the detection of exclusion of CMV-specific IgM than an enzyme-linked immunosorbent assay (ELISA).     

A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus

A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus is described. It requires only a single tube culture showing viral cytopathology and can accommodate multiple specimens in a single assay. The monoclonal antibodies confer absolute specificity and the use of horseradish peroxidase-conjugated staphylococcal protein A or antiglobulin permits easy visual interpretation of the results following the 2-3 hour assay. Although it is possible that an occasional wild strain of HSV might escape recognition by monoclonal antibody, this potential problem has not been observed among the more than 500 clinical isolates tested to date, all of which have yielded an unequivocal result.     

A simplified procedure for measuring the class of anti-bacterial antibodies by mixed reverse passive antiglobulin haemagglutination (MRPAH).

A modified procedure is described for performing the MRPAH (mixed reverse passive antiglobulin haemagglutination) reaction as a simple micro-method to measure the classes of bacterial antibodies. This 'bacterial dilution procedure' gave results closely correlated with those obtained by the 'serum (sample) dilution procedure' previously reported and with great economy of materials, labour and time. The method was used to investigate human serum antibodies to Br. abortus and S. enteritidis and serum and secretory antibodies to Strep. mutans. The good reproducibility of the MRPAH reaction was demonstrated by re-examining brucellosis sera tested one year previously. MRPAH was sufficiently sensitive to demonstrate the small amounts of IgG and IgM antibodies to Strep. mutans in human colostrum and early milk. A rise of antibody levels in the different immunoglobulin classes G, A and M was readily demonstrated in sera from individuals with salmonellosis.     

Identification and typing of herpes simplex virus types 1 and 2 by monoclonal antibodies, sensitivity to the drug (E)-5-(2-bromovinyl)-2'-deoxyuridine, and restriction endonuclease analysis of viral DNA

With development of antiviral drugs, the need to identify a virus as to drug sensitivity becomes increasingly of importance. The compound (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) has been shown to be much more inhibitory to the replication of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus as opposed to herpes simplex virus type 2 (HSV-2). We have typed over 170 isolates, using an immunofluorescent technique and sensitivity to the drug BVDU. These results were then compared to the typing of isolates by analysis of viral DNA after restriction endonuclease digestion (EcoRI). Without exception the results were in agreement between the monoclonal antibody results and sensitivity to the drug BVDU. Furthermore, the typing with monoclonal antibodies was also in excellent agreement with the DNA analysis. Only those isolates inhibited with BVDU showed DNA characteristics of HSV-1 and reacted only with the S-200 antibody. On the other hand, those isolates which reacted with the monoclonal antibody S-141 were insensitive to BVDU, and again this was in agreement with the DNA analysis. These results could provide the basis for developing a diagnostic test using the two monoclonal antibodies to type either isolates or direct smears and to use the results as a basis for possible drug therapy.     

Detection and quantitation of rotavirus using monoclonal antibody coupled red blood cells: comparison with ELISA

A total of 125 faecal extracts from infants were tested by reverse passive haemagglutination (RPH) using red cells coated with a monoclonal antibody against the major group-specific rotavirus antigen (VP 6). Results were compared with those obtained using a rabbit anti-rotavirus capture, guinea pig anti-rotavirus detector-based ELISA. The specificity of the assay was confirmed by use of 'normal' immunoglobulin coupled red cells and by inhibition with rabbit antiserum. The antibody-coated red cells could be stabilised by treatment with glutaraldehyde and subsequent freeze-drying with no detectable loss of activity even after storage at 45 degrees C for 4 wk. Good correlation was obtained between RPH and ELISA. Purified bovine rotavirus could be detected by RPH down to approximately 10(5) particles in a 25 microliters vol. Similar results were obtained with polyclonal antibody coupled cells and an ELISA using monoclonal antibody. Experiments using subgroup-specific monoclonal antibodies indicated the feasibility of rapid subgroup determination.     

Glycoprotein gB of herpes simplex virus expresses type-common and type-specific antigenic determinants in vivo.

Four monoclonal antibodies directed against glycoprotein B of herpes simplex virus were evaluated for their ability to immunize mice passively against acute virus-induced neurological illness and death when administered intraperitoneally 2 hours prior to footpad challenge with type 1 or type 2 virus. Two monoclonal antibodies, H120 and H157, failed to reduce the severity of neurological disease in infected animals. In contrast, H233 and H368 antibodies provided significant protection in type-common and type-specific fashions, respectively. A direct correlation was observed between in vitro neutralization and in vivo protection. These results provide the first in vivo evidence that glycoprotein gB of herpes simplex virus expresses both type-common and type-specific determinants during the evolution of acute virus-induced neurological disease.     

The use of colloidal gold for screening monoclonal antibodies to cell surface antigens

An immunogold method in Terasaki plates is described which allows accurate and sensitive visualization of the binding of monoclonal antibodies to cell surface antigens and is suitable for large scale screening. Monolayers of fixed cells are prepared in the wells. The binding of monoclonal antibodies is detected by a protein A gold complex. The cell-bound gold can be visualized by either optical or transmission electron microscopy. The results obtained with various monoclonal antibodies are presented.     

Monoclonal antibodies to herpes simplex virus type 1 glycoproteins show that epitope location influences virus neutralization

Three monoclonal antibodies, G8D1 , C2D2 , and TI57 , reacting with herpes simplex virus type 1 glycoproteins have been characterised according to the location of their epitope and ability to neutralize infective virus. Immune electron microscopy and a blocking radioimmunoassay were used to locate the epitopes. The results indicate that the epitope recognised by G8D1 is located on the surface of the glycoprotein fringe, whereas those recognized by C2D2 and TI57 are interior with respect to this. Only G8D1 has neutralizing activity alone, whereas C2D2 can neutralize when antiglobulin is added. Thus, epitope location and density determine the neutralizing capacity of individual antibody molecules.     

Red cell-labelled monoclonal antibodies for assay of human chorionic gonadotropin and luteinising hormone by reverse passive haemagglutination

The assay of human chorionic gonadotropin and luteinising hormone by reverse passive haemagglutination reaction, using monoclonal antibodies coupled to red cells, is described. Quantitation is achieved by end-point determination for serial dilutions of standard or sample, the haemagglutination reaction being observed after settling under gravity for 90 min. Red cell-labelled antibodies were stabilised with glutaraldehyde without loss of sensitivity and allowing long term storage. Various antibody combinations were assessed, and the best combination under optimum conditions gave a positive haemagglutination reaction down to 0.2 ng/ml with HCG.     

Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies

A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens.     

Production of monoclonal antibodies to gluten proteins and their use in developing tests for gluten quality

There is a need for rapid, simple tests to give added assurance in gluten quality evaluation which address the needs of plant breeders and the milling and baking industry. As part of fundamental research into gluten protein structure‐function relationships, a library of monoclonal antibodies (MAbs) has been developed to various gluten protein fractions, including both high and low molecular weight subunits of glutenin. The difficulties presented by working with insoluble, heterogeneous gluten proteins in raising MAbs of the requisite specificity are discussed. Several of the MAbs have been used to develop rapid immunoassays which are capable of analyzing a single sample in 10 min, and these have been applied to the quantification of total glutenin proteins, and high and low molecular weight subunits of glutenin in flour samples, illustrating the potential of immunotechnology for monitoring flour quality.     

Evaluation of Rh monoclonal antibodies for flow cytometry tests

As participants of the “III International Workshop on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens”, we tested 43 RH monoclonal antibodies by the flow cytometry technique. Besides the anti-D antibodies (not included in this paper), we tested the following antibodies: three (3) anti-C; one (1) anti-C^w; six (6) anti-c; eight (8) anti-E; three (3) anti-e; two (2) anti-G; one (1) anti-CcEe (total = 24 antibodies). These antibodies (from different lab sources) were tested against antigen positive cells (homozygous or heterozygous) and antigen negative cells. When available, some of them were tested against “rare” phenotypes like r_yr, r″^Gr, r^Gr, R₂R_z. All the three anti-C tested, showed poor discrimination between positive and negative cells; from the six anti-c tested, only three had good results (Workshop n° 18, 20, 21) with a superior performance of one of them (Workshop n° 18). From the eight anti-E tested, we found two (Workshop n° 139 and 153) with good performance; all the three anti-e were non reactive; the two anti-G failed to react with r′r red cells; the anti-C^w reacted better with R₁^wr cells than R₁^wR₁; and the anti-CcEe antibody showed good results with all the phenotypes tested. From the 24 antibodies tested, we found six (25%) antibodies with a good performance.     

Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA)

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.     

An enzyme-linked double antibody immunoassay to measure murine immunoglobulins--its application to determine the specific activity of radiolabeled monoclonal antibodies

A sensitive, simple and reproducible biotin-avidin amplified double antibody immunoassay to quantitate low concentrations of mouse immunoglobulins is described. The assay is a useful technique to measure trace levels of murine monoclonal antibodies in culture supernatants of hybridoma cells metabolically labeled with radioactive isotopes. A combination of radioactive counting and measurement of the absorbance of a peroxidase catalyzed reaction permits accurate determination of the specific radioactivity of labeled monoclonal antibodies.     

Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-beta-adrenergic ligand antibody

A new method has been developed to raise monoclonal anti-idiotypic antibodies. Monoclonal anti-idiotypic antibodies were obtained by fusion of NS-1 myeloma cells with splenocytes of mice immunised by intravenous injections of fixed hybridoma cells bearing a monoclonal antibody specific for beta-adrenergic ligands. New screening tests were developed to analyse the resulting hybridoma supernatants for different anti-idiotypic properties. Among 23 hybridoma supernatants recognising the idiotype, 6 were found to inhibit hapten binding and 3 of these recognised beta-adrenergic receptors.     

The development of monoclonal antibodies to respiratory syncytial virus and their use in diagnosis by indirect immunofluorescence

Twelve clones of murine hybridoma cells secreting antibody specific for respiratory syncytial (RS) virus were classified into four groups on the basis of their pattern of staining of unfixed RS virus-infected HEp-2 cells in an indirect immunofluorescence test. Three of the groups reacted with virus antigens present on the membrane of the cells, whilst the fourth group failed to stain most live cells, suggesting specificity for an antigen expressed internally. Representative monoclonals from the membrane antigen staining groups immunoprecipitated the 86K glycoprotein (G), 50K plus 19K glycoprotein (F1,2) and a 23K non-glycosylated protein (VP23). A representative monoclonal from the fourth group that appeared to stain an internally expressed protein immunoprecipitated the virion 34K phospho-protein (P). All four monoclonals stained acetone-fixed tissue culture cells infected with either the Long strain of RS virus or with strains isolated in Newcastle during the 1965, 1972, and 1983 winter epidemics. The anti-fusion protein antibody stained acetone-fixed cells from all of 26 nasopharyngeal secretions from infants with RS virus infection. The anti-G glycoprotein antibody and the anti-VP23 antibody stained cells from secretions poorly or not at all, whilst the anti-P protein antibody stained cells in half the secretions tested but reacted with only a small proportion of cells in comparison with the anti-F or polyclonal antibodies. A pool of all four monoclonals produced more intense staining than the anti-F monoclonal alone and gave a more clearly defined staining reaction than the polyclonal antiserum used for routine diagnosis in over half the secretions. These results indicate that monoclonal antibodies will be of value in the diagnosis of RS virus by indirect immunofluorescence if care is taken in the selection of a suitable pool.     

Detection of herpes simplex virus type-specific antibodies by an enzyme-linked immunosorbent assay based on glycoprotein G

In order to develop a simple and quantitative method to detect herpes simplex virus (HSV) type-specific antibodies, the usefulness of an enzyme-linked immunosorbent assay (ELISA) using HSV glycoprotein G (gG) captured on a plate by monoclonal antibodies as antigen was studied. The gG1- and gG2-specific IgG antibody activities were measured by the ELISA for 54 sera which had been collected from culture-proven genital herpes patients and pre-characterized by an immunodot assay using purified gG antigens. Thirty control sera without antibodies against the HSV whole antigens were also included. In comparison with the immunodot assay as standard, the sensitivities of the ELISA were 88.9% (32/36) for HSV-1 antibody and 89.2% (33/37) for HSV-2 antibody and the specificities were both 100%. Sera taken within a few months after primary infection tended to give false negative results. The HSV type-specific ELISA based on easy-to-prepare gG antigens might be useful to help improve the serological assessment of HSV infections. J. Med. Virol. 53:319–323, 1997. © 1997 Wiley-Liss, Inc.     

Antibody assay for measles virus: comparisons of immune-adherence haemagglutination, single radial haemolysis, enzyme immunoassay and haemagglutination inhibition

Immune-adherence haemagglutination (IAHA); single radial haemolysis and enzyme immunoassay (ELISA) versus haemagglutination inhibition (HAI) for detecting antibodies to measles virus were evaluated. The rank correlation computed according to Spearman for the sera of healthy individuals gave values of 0.66, 0.73, 0.72, respectively, for HAI-IAHA, HAI-single radial haemolysis (SRH) and HAI-ELISA. High percentages of accordance were observed as regards sera from both healthy and vaccinated individuals. In the case of vaccinated individuals the discordance analysis showed significant differences between HAI and IAHA, HAI and SRH, SRH and ELISA. The discordances indicate higher sensitivity of HAI and ELISA for the detection of seroconversion.