Development of an ELISA to detect antibodies to a protective lipopolysaccharide fraction of Leptospira borgpetersenii serovar hardjo in cattle.

Monoclonal antibodies (Mabs) were produced against Leptospira borgpetersenii serovar hardjo-type Bovis antigens. A panel of 28 Mabs were characterised. Only the nine Mabs toward a lipopolysaccharide (LPS) fraction of 18, 24 kDa bands and a 26-28 kDa smear showed agglutinating, leptospiricidal and growth-inhibition activities, and passively protected hamsters against renal infection with hardjo. They also reacted strongly in the CH-ELISA, captured killed whole hardjo leptospires, gave good fluorescence in indirect FAT against smears of hardjo culture and exhibited no cross reactivity with strains in heterologous serogroups. On the basis of optimal activity in a range of tests, one IgG class Mab (designated 25) was selected for use in an antibody-capture ELISA system for the detection of bovine anti-hardjo antibodies. The system gave a wide separation of absorbance values between positive and negative sera at a 1:10 dilution. The antibodies detected by this assay are believed to be protective anti-LPS IgG.

Expression of grass carp growth hormone by baculovirus in silkworm larvae.

A total of five recombinant Bombyx mori nuclear polyhedrosis viruses (BMNPV) carrying the grass carp (Ctenopharyngodon idellus) growth hormone (GH) cDNA were constructed in this study. Two of them were able to express the hormone up to a level of 12 microgram/ml medium when cultured B. mori cells were infected for 4 days. Inoculation of the viruses into silkworm (B. mori) host significantly increased the level of GH achievable. The amount of hormone produced per larva was estimated to be around 1 mg. The recombinant grass carp GH had immunological and biological activities similar to the native hormone. The N-terminal sequence of the recombinant hormone was the same as the native one, indicating that the fish signal peptide was correctly processed by the insect cells. Silkworm powder prepared from larvae infected with the recombinant virus was used as food supplement for fish. Compared with the control, this dietary supplement was effective in increasing the growth rate of juvenile carp.

Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine.

A magnetic bead antigen capture system which combined the use of two evolving techniques - immunomagnetic separation (IMS) and time-resolved fluoroimmunoassay (TR-FIA) - was developed to detect Leptospira borgpetersenii serovar hardjo in bovine urine. The assay utilised monoclonal antibody coated magnetic beads to capture leptospiral antigen which was in turn detected using another monoclonal antibody (Indicator) labelled with biotin. Signal was generated by the binding of europium labelled streptavidin to indicator antibody. The sensitivity of the assay was improved from 10(3) to 10(2) leptospires per ml by using an ethanol precipitation procedure to treat each sample. The assay detected only 31 of 56 (55 per cent) urine specimens culture-positive for hardjo, but seven of 24 urine samples culture-negative for hardjo were identified as positive by the assay. These seven samples were from animals which were culture positive on at least one other occasion. These results suggest that this system should be further investigated as a complementary test to culture for the identification of hardjo carrier animals.

Magnetic immuno capture PCR assay (MIPA): detection of Leptospira borgpetersenii serovar hardjo.

Magnetic immuno PCR assay (MIPA) was developed for the rapid detection of leptospires excreted in urine samples (n = 59) collected from 35 experimentally infected cattle. The immunomagnetic separation of leptospires from inhibitors in frozen formalin fixed bovine urine prior to PCR detection resulted in a marked improvement on previous detection methods. MIPA is a rapid 5 step protocol requiring 70 mins preparation time prior to amplification, which consistently detects 10(1) organisms. MIPA detected 76% (38/50) of culture positive urines and in addition three urines that were culture negative were shown to be positive by this method of detection. Consequently we conclude that whilst MIPA is an improvement on previously published PCR detection methods, the culture of the organism is still the standard against which other detection methods have to be compared.

Selective killing of choriocarcinoma cells in vitro by trichosanthin, a plant protein purified from root tubers of the Chinese medicinal herb Trichosanthes kirilowii.

Trichosanthin is an abortifacient plant protein purified from the Chinese medicinal herb Tian-hua-fen, obtained from root tubers of Trichosanthes kirilowii (Cucurbitaceae). Recently, trichosanthin has also been used in the treatment of trophoblastic tumours, including hydatidiform mole, invasive mole and choriocarcinoma. We have examined the effects of trichosanthin on cultured cells and observed selective cytotoxic effects on choriocarcinoma cells. Mouse melanoma cells were also sensitive to its cytotoxic action. Cytotoxic effects on cultured cells were usually not prominent after 24 hr treatment. Continued incubation for 48 hr in control culture medium after treatment with trichosanthin enhances the cytotoxicity on cells. Our findings suggest that trichosanthin exerts specific cytotoxic activity towards trophoblastic cells.