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KMID : 0359319930330040623
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Korean Journal of Veterinary Research
1993 Volume.33 No. 4 p.623 ~ p.632
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Development of antigen for the microplate latex agglutination test on toxoplasmosis in animals
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Abstract
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This study was conducted to develop a sensitized latex-antigen for serodiagnosis of toxoplasmosis in animals.
Tachyzoites of T gandii (RH-strain) harvested from mouse peritoneal cavity were purified through the filtraton of polycarbonate membrane(pore size, 3.0μm, Costar Co.) and disrupted by ultrasonicator.
The tachyzoite suspension was ultracentrifuged for 30 min at 60,000×g(4℃ ) and the supernatant was used as a water-lysate antigen. Polystyrene latex particles of 0.8μm in diameter(Sigma) were used for the preparation of sensitized latex-antigen suspension.
The several parameters including the preparation conditions, incubation buffer. serum dilution buffer and stability of agglutination reactions were evaluated and the results obtained were summarized as follows :
1. The antigen consisting of a water-lysate of T gondii tachyzoites was adsorbed onto polystyrene latex particles of 0.8μm in diameter by adding a latex suspension to an equal volume of diluted antigen solution and by incubating the mixture at 37℃ under different conditions.
2. The optimum incubation buffer used for the antigen sensitization was 0.1M Tris-HCl buffer(pH 8.0).
3. The optimum serum dilution buffer used for the latex agglutination test was 0.1M Tris-HCl-NaCl buffer(pH 7.4) containing 300 mM NaCl. But 0.1M Tris-HCl-NaCl buffer(pH 7.4) containing 300-600 mM NaCl, 0.5% BSA and 0.01% Tween-20 improved the agglutination pattems and cleared the background of microplate well without the effects on I.A titer.
4. The time required for antigen sensitization was 40 and 60 min in incubation buffer(pH 8.0) at 37 C: . But the optimun time for antigen sensitization was min at 37℃.
5. The optimun quantity of antigen absorbed on latex particles for proper agglutination was the range of 20 to 32㎍ of latex particles.
6. The optimun concentration of the latex-antigen suspension for the proper agglutination reaction was determined as 0.2%(w/v).
7. The specificity, rapidity and simplicity of the latex-particle agglutination test suggested that it might be adaptable to large scale serum screening.
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