Development and characterization of monoclonal antibodies (mcabs) to trypanosoma (tryfanozoon) brucei evansi and their application as immunodiagnostic reagents
Parasitological techniques employed in the diagnosis of patent trypanosome infection often fail to detect animals with low or cryptic infection. Yet, serological tests lack precision and accuracy in detecting patent infection due to the presence of antibodies in circulation for a long time. So, there arises a need for a better test for the diagnosis of patent trypanosome infection in the field. In an effort to improve on the diagnosis of infection with T. (T) •b. evenei , murine t-1cAbs were developed against antigens of the bloodstream forms of T.(T}.b.evansi, KETRI1342 and used as antigen detecting probes for diagnosis of patent T.(T).b.evansi infection in experimental goats and field camels. The murine McAbs were produced by immunizing Balb/c mice with plasma antigen I and membrane antigen II preparation from bloodstream forms of T.(T).b. evansi KETRI1342. Later, spleen cells· from the immunized Balb/c mice were harvested and fused with NS-I myelomacells. A total of 69 hybridomas secreting McAbs to the above antigens were raised. The hybridomas were cloned and where applicable, recloned by limiting dilution. The classes and subclasses of McAbs secreted by hybridoma reclones were determined by the double immunodiffusion and immunoelectrophoresis techniques and their stability to freezing and thawing, and salt precipitation investigated. Twenty two hybrid reclones were selected and inoculated intraperitoneally into pristane-primed Balb/c mice so that antibody rich ascites could be produced from each hybrid reclone. Mouse IgM class McAbs were purified from the antibody rich ascites by gel filtration on Sepharose 6 column. Mouse IgGrich fractions were obtained by ammonium sulphate precipitation. In an indirect ELISA and/or IFAT, the McAbs showed a wide range of cross-reaction with antigens of other mammalian African trypanosome spp, but no cross reaction was observed with Theileria parva, Babesia bi gemi ne , Anaplasma mer g i ne l e , Plasmodium falciparum or Leishmania donovani lysate antigens. Two McAbs TE M5/17.4.6. and TE M3/12.3.6, showed high specificity for KETRI 1342 stock but no reaction with other trypanosome species. However, their reaction with thirteen T. (T).b.evansi Stocks of defined serodemes did not show that they were T(T).b.evansi specific. McAb TEAI/23.4.6. was selected for application in the sandwich antigen - ELISA (Ag-ELISA) studies for the detection of circulating trypanosome antigens in serum or plasma and cerebrospinal fluid of experimental goats infected with KETRI 1342. This McAb is a mouse IgM antibody which cross-reacts with all the 'species of mammalian African trypanosomes. In this experiment, group one goats in which each goat was inoculated intravenously by needle and syringe challenge with 2x106 trypanosomes of KETRI 1342 stock, the sandwich Ag-ELISA technique was able to detect circulating trypanosome antigens in plasma or serum 24 hours after the inoculation of the trypanosomes. In the group two goats, in which the trypanosomes were inoculated by intramuscular route, antigens could not be detected until day 6 after inoculation. In both groups, there was no parasitological or serological evidence of trypanosomes in cerebrospinal fluid. In these experiments, a high positive correlation was observed between plasma Ag-ELISA and serum Ag-ELISA values. A low positive correlation was observed between serum Ag-ELISA values and parasitaemia or rectal temperature. A negative correlation was observed between serum Ag-ELISA values and pev or total haemolytic complement. A positive correlation was observed between serum Ag-ELISA and antibody-ELISA (Ab-ELISA) values. After treatment with the trypanocidal drug, Berenil, antigen levels dropped more sharply in group one than in group two goats. By day 12 and day 41, antigen levels had fallen to preinfection levels in group one and two, respectively. However, by day 56 and 44, IgG antibody levels were still very high in group one and group two respectively. On the other hand, IgM antibodies had fallen almost to pre-infection levels by this period. Field studies were carried out in four camel herds to evaluate the usefulness of McAb TEA1/23.4.6.as an antigen detecting probe in the diagnosis of patent trypanosome infection in camels. In an Ag-ELISA sandwich assay designed as for the goat experiment, a very high significant difference (P<.OOOl) was observed between plasma or serum Ag-ELISA values of infected and non-infected camels. In the third camel herd, it was observed in 25% of camels, that by day 14 after treatment, there was no evidence of detectable trypanosome antigenaemia, while in only few 'camels ant.i.genaemi.apersisted beyond day 28. On the other hand, by day 48, anti-trypanosome antibody levels were still very high in about 95% of the previously infected camels. Thus McAb TEA1/23.4.6. was a very successful probe in detecting circulating T.(T).b evansi antigens in plasma or serum of both experimental goats and field camels. Since antigens were detected much earlier than the antibodies, it was concluded that Ag-ELISA technique employing McAb TEA1/23.4.6. was a superior technique to Ab-ELISA in detecting patent infection. There was a very high agreement between TEA1/23.4.6. Ag-ELISA and the parasitological test (HCT and MI) used. Since it is possible to modify the assay conditions so that Ag-ELISA technique can be used as a field test, McAb TEAl/23.4.6. could be employed in such a test as a diagnostic reagent in trypanosomiasis control programmes.