Studies On The Cloing And Expression Of Lactate Dehydrogenase(LDH)gene From Plasmodium Falciparum,preparation Of LDH Based McAbs And The Development Of New Malaria Diagnosis Techniques

Supervisor Prof. LI Yingjie and Prof. LI Ming Malaria remains one of the leading causes of morbidity and mortality in the developing countries in tropical and subtropical regions. It was estimated that malaria killed 2.7 million people worldwide annually. Fast and accurate diagnosis was one of the key measures for malaria control. Diagnosis of malaria using microscopy examination of stained thick and thin smears remains the gold standard and also a most cost-effective method. However, not only it is labor-intensive, requiring a well- functioning, high-quality microscope, but also the interpretation of data demands considerable expertise, particularly at low levels of parasitaemia. Although two new recently developed diagnosis methods, i.e. the QBC and Kawamoto test, are shown to be rapid and accurate, they do need highly trained specialists to operate. Moreover, these two methods also require specialized centrifugation and viewing instruments, and electricity supply, thus making their uses in the field rather difficult. Though PCR-based tests are extremely sensitive, they are limited by the needs for considerable expertise, specialized equipment, and by the high cost of reagents. These limitations, therefore, have prevented the widespread applications of PCR-based methods in the field. The new-generation antigen-capture tests, including ParaSight-F (Becton Dickinson, Cockeysoville, Maryland, USA), ICT Malaria Pf and ICT Malaria Rf/P.v (ICT Diagnostics, Sydney, Australia) and OptiMAL (Flow mc, Portland Oreg), are capablq of detecting fewer parasites and of producing a result more rapidly. They are commercially available as kits, which include all the necessary reagents, and do not require extensive training or equipment to perform or to interpret the results. Moreover, these kits are reported to perform well in the field. Unfortunately, these kits are still too expensive for widespread applications in the developing countries. Patients who suffer from malaria normally live in remote areas and lack the necessary resources to go through the current diagnosis procedure. Hence, it is very obvious that a cheaper, but accurate and rapid diagnosis kit is urgently required. 8 Y~4~: I L~Th~扟3V~ ~ ~ Previous studies have shown that Plasmodium lactate dehydrogenasc (LDHp) is significantly different from the host LDH in biochemical, immunological and enzymatic properties. One of the biochemical characteristics that distinguishes LDHp from human LDH is the ability of LDHp to rapidly utilize 3-acetylpyridine NAD(APAD) as a coenzyme in the reaction leading to the formation of pyruvate from lactate. Human erythrocyte LDH could not make use of APAD. This unique feature makes it feasible to differentiate these two types of LDHs either by spectrophotmetry or by electrophoresis. In addition, it has been suggested that Plasmodium LDH possess species- and genus-specific antigens, which are considered to be promising candidates for the diagnosis purpose. As LDHp is only produced by live parasites, and a correlation between the level of parasitemia and the activity of parasitic LUll was demonstrated in previous studies, consequently, LDHp activity assay could be developed to specifically detect a particular plasmodium clone, and to monitor the effectiveness of drug therapies. In this study, we successfully amplified the gene encoding lactate d...