Production and characterization of monoclonal antibodies to a pilus colonization factor (colonization factor antigen III) of human enterotoxigenic Escherichia coli

Three monoclonal antibodies (MAbs) to a pilus colonization factor (colonization factor antigen III [CFA/III]) of human enterotoxigenic Escherichia coli (ETEC) were developed and characterized. All of the MAbs isolated belonged to the immunoglobulin G2a subclass. The specificity of these MAbs for CFA/III pili was demonstrated by the immunogold-labeling technique. The presence of more than one epitope in CFA/III pili was suggested. One of the three MAbs appears to recognize a polymeric conformational epitope(s) of CFA/III. CFA/III antigenicity distinct from that of other pilus colonization factors of ETEC was demonstrated by both a bacterial agglutination test and a sandwich enzyme-linked immunosorbent assay using the MAbs. Of the 100 strains of ETEC isolated from persons with traveler's diarrhea, 8% were found to carry CFA/III pili. Two enzyme-linked immunosorbent assay systems which could detect as little as several or 50 ng of CFA/III per ml were developed.     

A study to implement early diagnosis of HIV infection in infants born to infected mothers

A protocol for detecting HIV DNA from specimens collected on filter papers and the effect of storage temperatures on determination of HIV DNA from dried blood spots has been developed and optimized. Blood specimens collected from HIV-1 infected and normal persons were spotted onto blood collection cards (Whatman BFC 180). The HIV DNA was extracted by phenol-chloroform-isoamyl alcohol and was detected for C2V4 of HIV-1 env by nested polymerase chain reaction (nested PCR). One set was stored at -20 degrees C for 14 weeks, another at 37 degrees C for 1 week and then kept at -20 degrees C for 13 weeks and a third set at 25 degrees C for I week and then -20 degrees C for 13 weeks. The dried blood spots from each set were detected for the HIV DNA every 2 weeks for 14 weeks. The C2V4 region of HIV env DNA was determined from small amounts of the dried blood collected on the filter papers. The nested PCR procedure could detect as few as 5 copies of HIV proviral DNA, and HIV DNA could be detected from specimens with viral loads of 2x 10(4) copies/ml. HIV DNA could be detected from specimens collected at all temperatures tested for at least 14 weeks. Therefore, laboratory diagnosis of HIV infection can be done by PCR on dried blood spots. These techniques will be useful as a tool for studying the epidemiology of HIV infection among populations of interest such as mother to child infection using newborn screening specimens.     

Pilus colonization factors of enterotoxigenic Escherichia coli isolated from infantile diarrheal patients in Papua New Guinea

We examined the possibility of pilus colonization factor (or adhesin) production by 9 enterotoxigenic Escherichia coli isolated from infantile diarrheal patients in Papua New Guinea. By hydrophobicity, hemagglutination and bacterial agglutination with antisera against various known adhesins, none of the 9 strains produced known pilus adhesins. Three strains which produce heat-labile and heat-stable enterotoxins may produce a new type of pili which is not hydrophobic and differs from common (type 1) pili.     

Application of Streptomyces and Brevibacterium cholesterol oxidase for total serum cholesterol assay by the enzymatic kinetic method

BACKGROUND: Using non-esterified cholesterol standard, Brevibacterium and Streptomyces are found as suitable sources of cholesterol oxidase for kinetic cholesterol assay. For clinical use, we investigated the suitability of these enzymes for cholesterol determination in human serum. METHODS: We compared the performance of reagents containing 2 enzymes for the kinetic determination of total serum cholesterol with the standardized endpoint method. RESULTS: Reagent containing Streptomyces enzyme was more sensitive than that of Brevibacterium, with linearity up to 20.7 and 2.6 mmol/l, respectively. The analytical reaction for Streptomyces showed a shorter lag phase (148 s) and a steeper slope (absorbance vs. time) than that of Brevibacterium (246 s). The assay using Streptomyces reagent was precise and accurate and compared favorably with the endpoint method (y=1.06x-0.15, r=0.996, bias=0.21 mmol/l). Hemoglobin as high as 7.5 g/l did not interfere while turbidity greater than 2+ (absorbance >0.778 at 670 nm) and bilirubin concentrations >171.0 micromol/l did interfere (in a negative interference). Reagent was stable up to at least 8 weeks. CONCLUSIONS: The Streptomyces cholesterol oxidase, with 3,4-dichlorophenol, proved a suitable source for serum total cholesterol determination by the kinetic method.     

Bioactivities of Jc‐SCRIP,a Type 1 Ribosome‐Inactivating Protein from Jatropha curcas Seed Coat

In this study, a type 1 RIP, designated as Jc‐SCRIP, was first isolated from the seed coat of Jatropha curcas Linn. It was purified by ammonium sulfate precipitation and chromatography on DEAE‐Sephacel? and CM‐cellulose columns. Purification fold of Jc‐SCRIP increased 113.8 times, and the yield was 1.13% of the total protein in the final step. It was shown to be a monomeric glycoprotein with a molecular mass of 38 938 Da, as determined by MALDI‐TOF/MS. It exhibited hemagglutination activity and possessed strong N‐glycosidase activity. The antimicrobial activity of Jc‐SCRIP was tested against nine human pathogenic bacteria and one fungus; the most potent inhibitory activity was against Staphylococcus epidermidis ATCC 12228, with minimum inhibitory concentration value of 0.20 μm . Jc‐SCRIP demonstrated in vitro cytotoxicity against human breast adenocarcinoma cell line (MCF‐7), a colon adenocarcinoma (SW620), and a liver carcinoma cell line (HepG2), with IC₅₀ values of 0.15, 0.25, and 0.40 mm , respectively. The results suggested that Jc‐SCRIP may be a potential natural antimicrobial and anticancer agent in medical applications.     

Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method

BACKGROUND: Cholesterol oxidase is used for the determination of serum cholesterol. It can be derived from Streptomyces, Pseudomonas fluorescens, Cellulomonas, and Brevibacterium. This study compared the performance characteristics of four enzymes in the endpoint cholesterol determination. METHODS: Using the Mega analyzer, we studied assay optimization, linearity, precision, recovery, interference, stability, and compared 110 patient samples. RESULTS: The linearity for the four enzymes was up to 13.0 mmol/l at the optimal enzyme activity. The average within-run CVs ranged from 1.6% to 1.9% and between-day ranged from 2.8% to 3.0%, within the NCEP analytical criteria. The analytical recoveries obtained from four reagents ( approximately 96.5%) were excellent. The assays using these enzyme sources compared favorably with the commercial method and appeared accurate near the clinical decision cut-points. Hemoglobin concentration at 1.9 g/l interfered with the P. fluorescens cholesterol oxidase. Bilirubin caused a negative interference while lipemia generated a positive interference with all enzyme sources. Reagents were stable up to 6 weeks. CONCLUSIONS: Streptomyces, Cellulomonas, and Brevibacterium were essentially analytically equivalent. Streptomyces and Cellulomonas cholesterol oxidase are one-quarter as expensive Brevibacterium. Cellulomonas is a new source of cholesterol oxidase for determining serum cholesterol by the endpoint method.     

Jackfruit lectin: properties of mitogenicity and the inhibition of herpesvirus infection

Jackfruit lectin (JFL) from Artocarpus heterophyllus has been found to exhibit inhibitory activity in vitro with a cytopathic effect towards herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), and cytomegalovirus (CMV). The 50% inhibitory dose values from plaque reduction assay (inactivation) were 2.5, 5, and 10 Eg/ml of JFL for HSV-2, VZV, and CMV, respectively. Lymphocyte proliferation was significantly increased in the presence of the JFL in the concentration range of 2.5 to 50 Eg/ml, but was reduced at 500 Eg/ml. It was found that CD16(+)/CD56(+) cells (natural killer cells) were induced among the primary lymphocyte subpopulations. The activity of natural killer (NK) cells was not affected by JFL in the concentration range of 5 to 500 Eg/ml. These data suggest that JFL is mitogenic for NK lymphocyte (CD16(+)/CD56(+)) and also active against HSV-2, VZV, and CMV.