Katsumi Tachibana

Demonstration of a hepatitis C virus-specific antigen predicted from the putative core gene in the circulation of infected hosts

An ELISA was used to detect a protein derived from the core gene of the hepatitis C virus (HCV) in human plasma. The solid phase antibody in the assay was a murine monoclonal antibody against a synthetic peptide deduced from the putative core gene of HCV (residues 39 to 74). An enzyme-labelled affinity-purified human antibody directed at another region within the HCV core (residues 5 to 23) was the second antibody tracer. The ELISA had a sensitivity capable of detecting a few ng/ml of the HCV core polypeptide expressed in Escherichia coli. Core antigen activity in plasma of infected hosts was detected after treatment of HCV RNA-rich fractions from buoyant density centrifugation with the detergent Tween 80. There was a direct correlation between core antigen ELISA values of a plasma fraction and intensities of polymerase chain reaction signals for HCV RNA. These observations are consistent with the proposal that the N-terminal sequence of the predicted polyprotein of HCV is a nucleocapsid protein, and that improved core antigen assays may correlate with viraemia.

Application of monoclonal antibodies to the isolation and characterization of a killer toxin secreted by Hansenula mrakii.

A strain of yeast, Hansenula mrakii, secretes a toxin that kills sensitive yeasts, such as Saccharomyces cerevisiae. Monoclonal antibodies raised against the toxin had both binding and neutralizing activities. The toxin in culture media was isolated by an affinity column of monoclonal antibody. The toxin is a basic polypeptide with an isoelectric point at pH 9.1, and devoid of mannosides. It is composed of 88 amino acid residues with a molecular size of 10721 Da. The monoclonal antibodies could be applicable to the analysis of biologically active sites on the toxin, in an attempt to synthesize chemically a small peptide with killer activity and little immunogenicity.

A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies

Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype.

Immunoglobulin a antibody against hepatitis B core antigen in the acute and persistent infection with hepatitis B virus

Antibody to hepatitis B core antigen of immunoglobulin A class was determined in the serum of patients infected with hepatitis B virus by a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies. The antibody, as defined by a sample to normal ratio greater than 2.1, was detected in all of 39 patients with acute hepatitis, with titers varying widely depending on the time of blood sampling. In persons with persistent infection, the antibody was detected in only 2 (4%) of 46 asymptomatic carriers of the virus, contrasting with the positivity in as many as 15 (41%) of 37 patients with chronic persistent hepatitis, in 45 (94%) of 48 patients with chronic active hepatitis, and in 40 (87%) of 46 patients with liver cirrhosis with or without hepatocellular carcinoma. The mean +/- SE titer of antibody in chronic persistent hepatitis (3.8 +/- 0.9) was significantly lower than those in chronic active hepatitis (13.8 +/- 3.2) and cirrhosis with or without carcinoma (25.6 +/- 6.1) (p less than 0.001). Based on the results obtained, the antibody may reflect hepatic injury in the persistent hepatitis B virus infection.

Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a.

Beta‐Propiolactone for the Inactivation of Non‐A/Non‐B Type 1 Hepatitis Virus Capable of Inducing Cytoplasmic Tubular Ultrastructures in Chimpanzees

Non‐A/Non‐B type 1 hepatitis virus may be recognized because it induces characteristic tubular ultrastructures in the hepatocyte cytoplasm of chimpanzees. 3 chimps received 0.1 ml of a chimp serum containing more than 100 chimp infecting units of non‐A/non‐B type 1 hepatitis virus after it had been treated with β‐propiolactone with or without combined ultraviolet irradiation. All of the chimps escaped infection throughout the observation period of 23 weeks. The treatment of the serum with β‐propiolactone at the mildest condition employed (0.05%, 4°C, 20 min) was still effective in inactivating the virus. The susceptibility of the chimps was ascertained by the subsequent challenge with 0.1 ml of the untreated serum which invariably induced non‐A/non‐B type 1 hepatitis in them. On the basis of these results, β‐propiolactone was extremely efficacious for the cold sterilization of non‐A/non‐B type 1 hepatitis virus.

A two-site sandwich radioimmunoassay of β2-microglobulin with monoclonal antibodies

Among 21 batches of monoclonal antibodies raised against beta 2-microglobulin (anti-beta 2m), 6 reacted with soluble beta 2m, as well as with beta 2m present in association with HLA heavy chains on the surface of leukocytes. The remaining 15 anti-beta 2m antibodies bound with soluble beta 2m, but failed to react with beta 2m on the cell surface. No monoclonal anti-beta 2m antibodies revealed precipitin lines when they were tested against beta 2m in immunodiffusion. When 2 anti-beta 2m antibodies with different specificities were mixed together, however, they developed a precipitin line against beta 2m. Based on these observations, there was only 1 epitope on beta 2m that was available for the binding with a monoclonal anti-beta 2m antibody with either specificity. This allowed the development of a solid-phase radioimmunoassay in which beta 2m in test specimens was sandwiched between immobilized anti-beta 2m of 1 specificity and radiolabeled anti-beta 2m of a heterologous specificity in a single step. Monoclonal antibodies with at least 2 different specificities would be required for developing a sandwich-type immunoassay of polypeptides, such as beta 2m, that do not display 2 or more epitopes of the same specificity.

A radioimmunoassay that sandwiches human interleukin-2 between radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line

Two monoclonal antibodies were raised against human interleukin-2 (IL-2) produced by E. coli harboring recombinant complemental DNA. Both antibodies did not neutralize its activity, nor did they inhibit the binding of IL-2 to the receptor on target cells. Taking advantage of the ability of monoclonal antibodies to detect IL-2 that had bound to the receptor, a radioimmunoassay was developed that sandwiched IL-2 between the radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line infected with human T cell leukemia virus Type I. The assay had the advantage of detecting only IL-2 with the ability to bind to the receptor, and displayed a linear dose-response relationship over concentrations ranging from 5 to 100 ng/ml.