K39 strip test--easy, reliable and cost-effective field diagnosis for visceral leishmaniasis in India.

OBJECTIVE: A firm diagnosis of visceral leishmaniasis (VL) requires demonstration of the parasite in splenic or bone marrow aspirate. The aim of this prospective study was to assess the usefulness of K39 strip test as a noninvasive method of diagnosing visceral leishmaniasis under field conditions by testing serum antibody to the leishmanial antigen K39. MATERIAL AND METHODS: One drop of serum/blood was applied to the sample application pad on the test strip, which was diluted with 2 drops of chase buffer solution. The development of two visible red lines indicates the presence of IgG anti-K39. In the first phase of the study (2001), a total of 200 patients (Active VL-70, ex-VL-30, healthy endemic control-20 and patients with other tropical diseases-80) were tested with the K39 strip test at the School of Tropical Medicine, Kolkata. In the second phase of the study (2002), the test was applied in a remote tribal area of West Bengal where an epidemic of VL had occurred. Thirty-two patients were identified in 207 villagers of the affected area; all of them were tested with the K39 strip test. RESULTS: In the first phase, all VL and ex-VL cases gave positive results (100%). Ten percent of the healthy endemic controls were positive. The test results were negative in all other prevalent tropical diseases (100%). The estimated sensitivity of the test was 100% and the specificity was 98.18%. In the second phase of the study, all 32 patients of the epidemic were shown to be positive. All patients were treated with sodium stibogluconate injections and they recovered uneventfully. CONCLUSIONS: K39 strip test is ideal for rapid reliable field diagnosis of visceral leishmaniasis. The test has high sensitivity and specificity but it remains positive long after treatment (up to 3 years).

Effect of addition of proton carriers in culture medium on growth and secretion of hybridoma cell line OKT3.

Monoclonal antibodies (MAb) constitute the centre of all in-vitro diagnostic measures and almost all in-vivo therapeutic manoeuvres now. Production emphasis for these antibodies is having a current shift from animal-based large-scale culture to in-vitro bioreactor-based high-density culture. One of the major difficulties in high-density culture is end-metabolite accumulation in batch and fed-batch cultures in the forms of H+, NH4+ etc.. thereby reducing cellular growth and secretions. In the present study, effects of added proton carries--NAD and NADP--over and above the metabolic pools of the molecules, were examined on the cellular growth and secretion kinetics. Although NADP fortification showed a remarkable improvement in cellular growth (time dependent 200-300% improvements compared to controls) and size, cumulative MAb titre was better with NAD fortification. Combined additional loads of the proton carriers would be interesting to study in high density culture conditions.

Getting higher yields of monoclonal antibody in culture.

Getting higher yields of monoclonal antibody (MAb) is a problem in Hybridoma Technology which has two major bottlenecks: a) poor yield of hybridized cells, b) low cellular productivity of MAb in culture. There are three ways of obtaining high MAb yield in vitro a) Large scale culture, b) high density culture and c) enhancing individual cellular productivity in culture. Currently, the focus is on the correct synergistic combination of fortified nutrient media, bioreactor design and mode of operation. Maximization of cell culture longevity, maintenance of high specific antibody secretion rates, nutrient supplementation, waste product minimization and control of environmental conditions are important parameters for improvement of large scale production of MAb. Though, MAb yields have improved rapidly over the decade, there is a growing concern for the decrease in quality of MAb secreted. Further research is therefore necessary to take full advantage of MAb as a potential diagnostic agent for in vivo therapy.

Monoclonal antibody: high density culture of hybridoma cells and downstream processing for IgG recovery.

Getting higher yields of monoclonal antibody (MAb) is a problem in Hybridoma Technology which has two major bottlenecks--(a) poor yield of hybridized cells; and (b) low cellular productivity of MAb in culture. There are three ways of obtaining high MAb yield in vitro--(a) large scale culture of hybrid cells; (b) high density culture; and (c) enhancing individual cellular productivity in culture. Currently, focus is on correct synergistic combination of fortified nutrient media, bioreactor design and mode of operation. Maximisation of cell culture longevity, maintenance of high specific antibody secretion rates, nutrient supplementation, waste product minimization and control of environmental conditions are important parameters for improvement of large scale production of MAb. Though, MAb yield has enhanced rapidly over the decade, there is a growing concern for decrease in quality of MAb secreted. Further research is therefore necessary to take full advantage of MAb as a potential diagnostic agent for in vivo therapy.