Yuichi Oku

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin‐producing Escherichia coli (VTEC), a sensitive bead‐enzyme‐linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo‐ and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.

Purification and some properties of a Vero toxin from a human strain of Escherichia coli that is immunologically related to Shiga-like toxin II (VT2)☆

A cytotoxin to Vero cells (Vero toxin), which was immunologically related to Shiga-like toxin II (SLT-II) (or VT2), was purified from a stain of Escherichia coli isolated from a patient with hemolytic uremic syndrome. The toxin was active on Vero cells but much less active on HeLa cells, a property similar to that of the recently identified SLT-II variant from E. coli strains that caused edema disease of swine. Thus the toxin purified in this report was tentatively named Shiga-like toxin II variant (Vero toxin 2 variant). The purification procedures consisted of ammonium sulfate fractionation, DEAE-Sepharose CL-6B column chromatography, chromatofocusing column chromatography, and repeated high performance liquid chromatography (HPLC) on TSK-gel G-2000SW column and on TSK-gel DEAE-5PW columns. About 90 micrograms of purified toxin was obtained from 451 of the culture supernatant with a yield of about 16%. The purified toxin consisted of A and B subunits of molecular sizes similar to those of SLT-II (VT2). The isoelectric point of the purified toxin was 6.1, which was different from that of SLT-II (VT2) (pI = 4.1). In an Ouchterlony double gel diffusion test, purified toxin and SLT-II (VT2) formed precipitin lines with spur formation against anti-purified toxin and anti-SLT-II (anti-VT2), respectively. The purified toxin was cytotoxic to Vero cells, about 6 pg of the toxin killing 50% of the Vero cells, and showed lethal toxicity to mice when injected intraperitoneally, the LD50 being about 2.7 ng per mouse.

Development of oligonucleotide lateral-flow immunoassay for multi-parameter detection

We have developed a highly sensitive and rapid oligonucleotide lateral-flow immunoassay (OLFIA), using a colloidal gold as an indicator, for the simultaneous detection of antigens and/or antibodies in specimen. This system can detect more than two types of antigens and/or antibodies in a single assay device at the same time. The device is basically composed of colloidal gold-labeled antibodies and oligonucleotide-labeled antibodies fixed in a conjugate pad, and the complementary oligonucleotide-labeled proteins are immobilized on a nitrocellulose membrane. If the target antigen is present in a specimen, the colloidal gold-labeled antibody and oligonucleotide-labeled antibody will make a complex with the antigen. Subsequently, the formed complex migrates to the place where complementary oligonucleotide is immobilized and is bound to the solid phase via the DNA-DNA interaction. As a result, more than two types of reactions can be detected on a single assay device by the combination of colloidal gold-labeled antibodies, different oligonucleotide-labeled antibodies and complementary oligonucleotide-labeled proteins immobilized at different places on a nitrocellulose membrane.

Detection of Cholera Toxin by a Highly Sensitive Bead-Enzyme Linked Immunosorbent Assay

A bead‐enzyme linked immunosorbent assay (bead‐ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1 has been developed. Under optimal buffer and pH conditions the bead‐ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the bead‐ELISA. Evaluation of the sensitivity and specificity of the bead‐ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the bead‐ELISA was more sensitive than the RPLA. Quantification of CT by the bead‐ELISA revealed that the concentration of CT produced by the strains of V. cholerae O1 which were negative by the RPLA was lower than 1 ng/ml and therefore below the minimum detection ability of the RPLA. The bead‐ELISA is a simple, specific and highly sensitive assay for routine detection of CT and is recommended for routine use in clinical microbiology laboratories.

Synthesis and Deposition of Spore Coat Proteins during Sporulation of Bacillus megaterium

Rabbit (anti‐spore coat protein) IgG was prepared by immunization with coat proteins extracted with sodium dodecyl sulfate and dithiothreitol from isolated spore coats of Bacillus megaterium ATCC 12872. Coat proteins were detected from 3 hr after the end of exponential growth (t3) in the mother cell cytoplasmic fiaction by sandwich enzyme immunoassay using this antibody. The proteins in the forespore coat protein fraction increased from t3 and reached a plateau at t10. Immunoblot analysis for the coat proteins in sporulating cells revealed the sequential synthesis of various proteins in the mother cell cytoplasmic fraction and simultaneous deposition of the same proteins as in the forespore coat fraction. These results suggest that turnover of precursor proteins of the spore coat is very rapid if precursor proteins are produced and they are proteolytically processed to produce mature proteins. Specific antibody to the 48,000‐dalton protein, which is a major protein, did not cross‐react with any other major (36,000, 22,000, 19,500, and 17,500‐dalton) proteins. Specific antibody to the 22,000‐dalton protein did not cross‐react with the 48,000, 36,000, 19,500, 17,500, and 16,000‐dalton proteins, but did cross‐react with the 44,000, 25,000, and 12,000‐dalton proteins.

Immunoelectron Microscopic Studies on Spore Coat Proteins of Bacillus megaterium

An immunochemical staining technique for the spore coat proteins of Bacillus megaterium ATCC 12872 was developed using colloidal gold as a second antibody. For reducing the non‐specific immunogold binding and increasing the specific binding, the affinity‐purified IgG was used as a first antibody. In sporulating cells at t10, gold particles were found not only in the spore coat but also in the mother cell cytoplasm, suggesting that some coat proteins were synthesized in the cytoplasm. Use of the specific affinity‐purified antibody to 48K‐protein demonstrated that this protein was one of the components of the outer coat.

Comparative Antigenicity of Spore Coat Proteins from Bacillus Species Using Antibody to Spore Coat Proteins of Bacillus megaterium

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross‐reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross‐reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross‐reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross‐reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.