Eiji Okuhara

Anti-poly(I)·poly(C) antibody bound to cellulose and its use in the specific separation of double-stranded RNAs

Abstract Antibodies specific to double-stranded RNAs were purified from complexes of poly(A)·poly(U) and anti-poly(I)·poly(C) antibodies by a method of dissociation with 5% dimethylsulfoxide-alkali solution and DEAE-cellulose column chromatography. The purified antibodies were coupled to CNBr-activated cellulose. Synthetic and naturally occurring double-stranded RNAs were bound specifically by the antibody-linked cellulose and were eluted with alkali solution (pH 10.5) or 10% dimethylsulfoxide. The antibody-linked cellulose column appeared to be of practical value for the separation of rice dwarf virus ribonucleic acids from rice plant nucleic acids.

IMMUNOLOGICALLY ACTIVE LESIONS INDUCED ON DOUBLE-STRANDED DNA WITH ULTRAVIOLET

Abstract— Some immunochemical properties of double‐stranded DNA irradiated with UV were studied, using a radioimmunoassay with irradiated [3H]‐DNA and experimentally produced antibodies to DNA. Reactivity of antibodies revealed that irradiated DNA contained an immunologically active structure other than the irradiated DNA specific structure, resembling that of thermally denatured DNA. inhibition assay demonstrated that while DNA‐antibody binding was effectively inhibited by mixed purine and pyrimidine oligonucleotides, thymine dimer containing pyrimidine oligonucleotides derived from the irradiated DNA showed no appreciable inhibition. The antigenic structure specific for irradiated DNA was found to be thermally labile in low salt medium. Cupric and ferrous ions and cysteine added to the DNA solution inhibited antigenicity formation during irradiation, but these substances exhibited no effect on dimer formation in irradiated frozen thymine solution. Calcium ions and histidine were inert for the former reaction but inhibited the latter effectively. This suggests that different mechanisms are involved in the 2 processes. The immunologically active UV‐induced lesions appeared to depend mainly on a conformational structure change of the DNA strands rather than on a single modified base moiety.

Reduction of the non-specific binding of DNA to gamma-globulin in Farr radioimmunoassay by addition of dextran sulfate and calcium chloride.

The effect of non-specific binding caused by the interaction between gamma-globulin and denatured DNA was markedly reduced by addition of dextran sulfate or CaCl2 at alkaline pH. This method was shown to be applicable in the detection of anti-DNA antibodies in sera from cases of human systemic lupus erythematosus.

Demonstration of extrinsic DNA from immune complexes in plasma of a patient with systemic lupus erythematosus

Antigen DNA isolated from immune complexes present in plasma of three patients with active systemic lupus erythematosus using an affinity column was cloned and sequenced. One clone, designated pKS7, was found to have a region homologous with that of the E. coli metK gene, and another, designated pKS8, had a region homologous with a sequence including the replication origin of bacteriophage f1. Gel retardation assay revealed that pKS7 and pKS8 interacted with the patients IgG fraction to form immune complexes, respectively. The affinity-purified antigen DNA was proved to be originated from bacteria or bacteriophage.

An adjuvant effect of poly A - poly U on production of antibodies to DNA in rabbits.

The effect of polyadenylate-polyuridylate copolymer (poly A-poly U) on the formation of anti-DNA antibodies in rabbits was investigated. 1) Poly A-poly U proved to be an effective adjuvant for production of antibodies to DNA. These were elicited in rabbits at high titer and were formed at an early stage of immunizations. 2) The antiserum obtained reached potently with denatured DNA, but with neither methylated bovine serum albumin (MBSA) used as carrier protein nor poly A-poly U used as adjuvant. This was demonstrable both by C-fixation and double diffusion tests. 3) The antibodies occurred exclusively in the IgG fraction separated by Sephadex G-200 column chromatography.

A method for producing anti-DNA antibodies in ascitic fluid of mice

Immunological methods for the production of antibodies to DNA in ascitic fluid of DDY, A/He, and C57B1/6 mice were investigated. Relatively large volumes of ascitic fluid containing antibodies for DNA in high concentration were obtainable after seven injections of denatured ssDNA-MBSA complexes emulsified with CFA into peritoneal cavities of DDY mice. The quantity of anti-ascitic fluid from 4 to 5 DDY mice was equivalent to that of antiserum from one rabbit. DDY mice accumulation of ascites is much greater than in other strains, and the ascitic fluid contains antibodies of higher titer regardless of sex. The antibody titers in ascitic fluid are not affected by alteration of the ascites volume, and are proportional to those of the corresponding sera. Antibodies to DNA in ascitic fluid of these mice occurred exclusively in IgG as in DNA antisera.

The specific binding of parotin on duct cells of human parotid gland.

ZusammenfassungDie Beziehungen zwischen Parotin (Speicheldrüsenhormon) und der menschlichen Parotis wurden mit der indirekten Technik der Enzym-Antikörper-Methode untersucht. Die Ergebnisse zeigen, daß Parotin selektiv an die Zellen des Gangsystems der menschlichen Parotis gebunden ist.SummaryThe relationship between parotin and human parotid gland was investigated using indirect technique of enzyme antibody method. Normal rabbit IgG has non-specific affinity to duct cells of human parotid gland, but it was suppressed by adding bovine serum albumin. Anti-parotin rabbit IgG has no reaction products on the sections of adult human parotid gland. When the sections, initially incubated with parotin solution in PBS, were treated with anti-parotin rabbit IgG containing bovine serum albumin, and then HRPO-labeled anti-rabbit gamma globulin antibodies (swine IgG), intercalated and striated ducts excepting excretory ducts were stained with diaminobenzidine reaction, but also acinar cells did not. These results indicate that parotin is bound specifically on duct cells of human parotid gland.

Production of antibodies specific for double stranded antigen DNA cloned from immune complexes in plasma of a SLE patient

The antigenicity of antigen DNA isolated from immune complexes in plasma of a SLE patient was examined. DDY mice were immunized with the cloned antigen DNA carrying a sequence homologous with a part of bacteriophage f1 (KS8 DNA) by the coupling method, and the antibody response was estimated by radioimmunoassay. Antibodies specific to double stranded DNA were elicited. Moreover, the antibodies showed preferential binding to KS8 DNA than other DNA derived from Escherichia coli. These results suggest that KS8 DNA has a significant antigenicity in mice.