A sensitive and rapid immunochemical method for quantitation of proteins.

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.

Novel application of quantitative immunoassays for screening seed globulins of cowpea varieties.

Using antibodies raised in rabbits, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are standardized for cowpea (var. Pusa Barsati) seed globulins. The RIA, when used to screen three stages of seed development, reveals that maximum globulins are detected at 28 days after flowering. When three different varieties of cowpea are assayed for their globulin content by RIA and ELISA, it is observed that the Bold Grain cowpea has the highest amount of related globulin as compared to two other varieties, namely Pusa Phalgun and Asparagus Bean.

Oligonucleotide fingerprint analysis of a wild strain & its attenuated variant of Japanese encephalitis virus.

A strain of Japanese encephalitis (JE) virus was passaged serially through primary chick kidney cell cultures (45 times) and primary baby hamster kidney cell cultures (21 times). The resultant virus lost its lethal effect to 3 wk old mice by the ic route and 10 day old mice by the ip route. The oligonucleotide fingerprint analysis of the parent and the passaged strains showed 64 spots in common; 17 spots were present in the parent strain which were absent in the passaged virus, while the latter had acquired 9 spots which were not present in the parent virus.

Cloning & expression of chikungunya virus genes coding structural proteins in Escherichia coli.

DNA complementary to the single stranded RNA genome of Chikungunya (CHIK) virus with poly A tract was cloned into the plasmid pGEM-3Zf(-) and 5Zf(+) by blunt end ligation strategy. Clones containing the cDNA inserts were selected by X-gal, IPTG system. They were tested for the expression of structural protein(s) of CHIK virus by in situ enzyme immunoassay and Western blot. The former assay system showed the presence of expressed viral proteins. Analysis of Western blot shows that three structural proteins, E1, E2 and capsid (C) are expressed in Esch. coli. The molecular weights of envelope proteins E1 and E2 were 44-46 Kd and 42-44 Kd respectively, which are lesser than the actual molecular weights of virional proteins (50-52 Kd). This may be due to the absence of glycosylation of these proteins in Esch. coli. In clone no. 382, a high molecular weight protein (56-58 Kd) was observed, which was probably the unglycosylated form of P62 polyprotein coded by the virus during its multiplication. A small protein of MW 6-8 Kd was also expressed in clone nos. 382 and 504, and this appeared to be the unglycosylated form of E3 protein of CHIK virus.

Oligonucleotide fingerprint analysis of Japanese encephalitis virus strains of different geographical origin.

RNA fingerprint analysis was carried out with different strains of Japanese encephalitis virus which were isolated from Japan, China, India and Sri Lanka. From the similarity ratios, a similarity matrix was worked out which yielded a dendrogram. Geographical proximity of the place of isolation did not contribute much to the similarity of the fingerprint of the strains, nor did temporal proximity. The Japanese Nakayama strain had greater similarity with the Asansol strain from West Bengal. However, another strain from West Bengal, the Bankura strain, showed marked difference. Similarly the Bhopal and Beijing (China) strains were relatively close to each other while the Japanese JaGAr15460 strain was nearer to the strain from Gorakhpur. Serial mouse passage of the Asansol strain did not change the fingerprint pattern drastically.

Isolation of the new variant influenza A (H3N2) strains during an outbreak in Pune, February, 1996.

During January-February, 1996, an outbreak of influenza-like illness occurred in Pune. The throat and nasal swabs collected from the patients during this outbreak were processed in MDCK and LLC-MK2 cell cultures and influenza A(H3N2) viruses were isolated. They were identified as being similar to the recent circulating global strains A/Johannesburg/33/94 and A/Wuhan/359/95.

Putative chikungunya virus-specific receptor proteins on the midgut brush border membrane of Aedes aegypti mosquito.

Two proteins (putative receptors) of 60 and 38 kDa, for chikungunya (CHIK) virus were detected in the brush border membrane fraction (BBMF) of the normal population of Aedes aegypti mosquitoes. Mosquitoes were infected orally with CHIK virus and infectivity checked by testing the head squashes. BBMF was prepared from proved positive and negative mosquitoes. The receptor proteins were found to be present in both the proved genotypes. However, dot-b'ot assays showed that the CHIK virus binding activity of BBMF/mg protein was noticeably low in the proved negative mosquitoes as compared to the positives. BBMF from the larvae of the normal populations also showed the presence of the receptor proteins, binding to CHIK virus. Receptor proteins from larvae as well as the adults were found glycosylated. CHIK virus receptor proteins of 24, 45, 58, 60 and 62 kDa were also seen in the membrane fraction of the C6/36 cells.

Antibody-based enzyme-linked immunosorbent assay for determination of immune complexes in clinical tuberculosis.

Immune complexes were isolated from sera of tuberculosis patients by precipitation with 2.5% polyethylene glycol. The precipitates were characterized by quantitative determination of different immunoglobulin classes by single radioimmunodiffusion, sodium dodecyl sulfate polyacrylamide gel electrophoresis for presence of serum components, Ouchterlony's double diffusion method for detection of complement components C3 and C4, and immuno-dot assay for detection of Mycobacterium tuberculosis antigens. The results showed that polyethylene glycol precipitates of patients' sera were indeed immune complexes, as they contained immunoglobulins, albumin, complement components, and mycobacterial antigens, whereas precipitates from control sera contained mainly albumin. The antibodies present in immune complexes were specific to M. tuberculosis antigen and showed no binding to Escherichia coli antigens. Immune complex levels, as determined by the ability to bind M. tuberculosis antigens in an enzyme-linked immunosorbent assay, were significantly higher in tuberculosis patients (n = 22) than in healthy control subjects (n = 18). Thus, immune complex level could be a useful parameter in the diagnosis of active tuberculosis.

Humoral immune response in tuberculosis: initial characterization by immunoprecipitation of 125iodine labelled antigens and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

125Iodine-labelled Mycobacterium tuberculosis antigens were immunoprecipitated with tuberculosis patients' sera and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A group of four polypeptide antigens of 55, 38, 28 and 18 kD were thus identified. The 38 and 28 kD polypeptides were the major antigens. Antibody response differed from one patient to another, both with respect to the number and quantity of antigens precipitated. Untreated patients and those undergoing treatment with antimycobacterial drugs also showed marked differences in their antibody response. Generally, immunoprecipitates from treated patients showed a larger number of antigen bands and the relative intensities of the bands was also greater. No correlation was observed between the immunoprecipitation profile and antibody titres determined by enzyme linked immunosorbent assay.

Betel quid chewing and oral cancer: experimental studies on hamsters.

Betel quid ingredients--betel nut, betel leaf, lime, catechu and tobacco--were tested separately and in various combinations for carcinogenicity, using hamster cheek pouch as the experimental site. The four modes of administration used were (1) tri-weekly painting of the cheek pouch with aqueous extracts of test materials, (2) deposition of replaceable wax pellets containing the test material, (3) gelatin capsules containing the powdered material and (4) insertion of natural material into the pouch for trauma and direct exposure. Untreated controls and standard carcinogen DMBA-treated controls were also maintained. A total of 317 young adult golden Syrian hamsters (Mesocricetus auratus) used for the experiments were killed in two age groups: 6-12 months and 13-24 months, only when signs of general debility were observed. In the untreated controls, animals were free of any malignancy. In the experimental series, various betel quid ingredient combinations under test induced both oral and gastric lesions ranging from massive atypia and precancerous lesions to frank carcinomas. Maximum lesions were observed in the groups receiving betel nut, lime and tobacco combinations and in the polyphenol fraction of betel nut containing major tannins. The mode of administration of test material resulted in distinct differences; tri-weekly paintings giving oral lesions in the range of 22-23% and gastric lesions 39-48%; the same material given either through the replaceable gelatin capsule or in natural form induced 69% oral lesions and 63 to 82% gastric lesions. Overall evaluation of the data of all the four series confirms the potent carcinogenicity of betel nut, particularly its tannin-containing polyphenolic fraction and its combination with lime and tobacco. Maximum oral lesions induced in the hamsters by continuous exposure to capsules and natural material, highlight the direct relationship of frequency of chewing in habitual chewers with oral carcinogenesis. The high incidence of gastric (forestomach) lesions invites special attention.