Dennis B. Rylatt

Protein determination on an automatic spectrophotometer.

Abstract Rapid and sensitive estimations of protein can be carried out in 96-well microplates in an automated spectrophotometer of the type developed for microenzyme linked immunosorbent assay determinations. The procedure is based on the Coomassie blue method of J. J. Sedmark and S. E. Grossberg (1977, Anal. Biochem. 79 , 544–552) and can detect less than 200 ng of bovine plasma albumin and results in up to a 50-fold reduction in processing time over that required with a conventional manual spectrophotometer.

Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.

A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596). The assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human red blood cells). This anti-red blood cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579-613). Addition of this reagent to 10 microliters of whole blood results in the Fab-peptide conjugate coating the red blood cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, red cell agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

Physicochemical, immunochemical and functional comparison of human histidine-rich glycoprotein and autorosette inhibition factor

Abstract The present study shows that the histidine-rich glycoprotein previously isolated from haman plasma (Heimburger, N., Haupt, H., Kranz, T. and Baudner, S. (1972) Hoppe-Seylers Z. Physiol. Chem. 353, 1133–1140; Lijnen, H.R., Hoylaerts, M. and Collen, D. (1980) J. Biol. Chem. 255, 10214–10222) is a partially degraded molecule with a molecular weight of 60000. The native molecule has a molecular weight of 81000 and is shown to be identical with the autorosette inhibition factor of human plasma (Rylatt, D.B., Sia, D.Y., Mundy, J.P. and Parish, C.R. (1981) Eur. J. Biochem. 119, 641–646). The native molecule can be converted to the proteolytic derivative by limited digestion with plasmin. Both the native and the degraded molecule have a similar affinity for the lysine-binding site(s) of plasmin(ogen), but the latter is devoid of autorosette inhibition activity.

A competitive enzyme immunoassay for the detection of bovine antibodies to Brucella abortus using monoclonal antibodies

A competitive enzyme immunoassay (EIA) for the detection of circulating bovine antibodies to Brucella abortus has been developed using horseradish peroxidase conjugated monoclonal antibodies (MAb) raised against B. abortus cell surface antigens. Antibodies present in the serum of either vaccinated or infected cattle can apparently displace the conjugated MAb from the lipopolysaccharide antigen (LPS) in a quantitatively different manner allowing an assessment of immune status of the animal. The results from a panel of sera from animals with a known status of vaccination or infection indicated that the test was more selective in the detection and discrimination of infected from uninfected or immunized animals, than conventional complement fixation, agglutination or indirect enzyme immunoassay procedures.

Monoclonal antibodies directed against Brucella abortus cell surface antigens

A panel of monoclonal antibodies (MAb) has been raised against Brucella abortus cell surface antigens from mice immunized with either heat/phenol treated or UV killed bacterial suspensions of B. abortus. The hybridomas were screened by either a microagglutination procedure or by an indirect enzyme immunoassay (EIA) on sonicated bacterial preparations. From a large number of MAb generated by various procedures, two distinct types of MAb emerged. The most numerous type was capable of agglutinating B. abortus and reacting with a soluble preparation of lipopolysaccharide (LPS). A second type was not capable of agglutinating the bacterial suspensions or of binding to the soluble LPS preparation but reacted with an antigen present in bacterial sonicates. Two MAb of this type react differentially with sonicates prepared from virulent and avirulent strains of B. abortus. There appeared to be sufficient evidence from our analysis of the relative degree of cross reaction with antigens present on a range of B. abortus strains and Brucella and xenogenic bacterial species to conclude that each of the seven MAb was recognising a separate antigenic site on the B. abortus cell surface.

Presence of receptors for self MHC antigens on non-T cells

It is generally accepted that the major hislocompatibility complex (MHC) plays a key role in the recognition of foreign antigens by T lymphocytes. However, recent studies indicate that a range of non-T cells can also recognize sell MHCstructures. This review discusses these recent findings and suggests that the MHC plays a fundamental role in cell-cell communication and differentialion of both lymphoid and non-lymphoid cells.

Rapid screening of monoclonal antibodies by spin adherence double immunosorbent test (SADIST)

The spin adherence double immunosorbent test (SADIST) is a simple, rapid immunoassay with sensitivity similar to the enzyme-linked immunosorbent assay (ELISA). A 1-step SADIST has been found suitable for rapid screening of hybridomas for antigen-specific monoclonal antibodies (MAb). In this procedure hybridoma supernatants are added to antigen coated microplates followed by commercially available antiglobulin beads. The microplate is immediately centrifuged. Wells containing antigen-specific MAb produce a mat of beads whilst wells without antigen-specific MAb produce a button of beads. No washing or incubation steps are necessary and results are read within minutes of adding beads to test supernatants. By comparison, ELISA tests require several hours to perform with multiple wash steps and further reagent additions. A 2-step SADIST was also assessed. Supernatants are incubated in the microplate as for an ELISA and a wash step precedes the addition of antiglobulin beads. A panel of 117 hybridoma supernatants was selected to assess the suitability of the SADIST techniques for hybridoma screening. The supernatants were added to antigen-coated microplates and SADIST and ELISA tests performed. The SADIST correctly discriminated most hybridoma supernatants that were clearly positive or negative by ELISA. It was also found possible to perform SADIST followed by ELISA tests on the same microplate well without significantly affecting ELISA values.

Immunocytochemical studies on the localization of 5-aminolevulinate synthase in rat liver

The localization of 5-aminolevulinate synthase (ALAS) in hepatocytes of untreated and porphyrinogenic drug-treated rats has been examined by an immunocytochemical approach using a monoclonal antibody and protein A-gold labeling. Gold particles representing antigenic sites for ALAS were observed in the mitochondria and cytoplasm of untreated and drug-treated cells. Quantitative analysis of the labeling density showed that levels of ALAS increased significantly in both of these cellular compartments following drug treatment. Evidence that the detected cytoplasmic form of ALAS represents the precursor of the enzyme was obtained from immunoblotting experiments. The direct detection of cytosolic ALAS in vivo rules out the possibility that enzyme activity previously detected in the cytosol fraction resulted from mitochondrial leakage during cell fractionation. The results indicate that the cytosolic accumulation of ALAS is not a consequence of the inability of mitochondria to accommodate more enzyme. However, the molecular basis for this cytosolic accumulation is not known. The studies also established that the mitochondrial enzyme is predominantly, if not exclusively, associated with the matrix side of the inner mitochondrial membrane.