DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR DETECTION OF MELOIDOGYNE SPP. (ROOT-KNOT NEMATODE) ANTIGENS

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Date
2010
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PAU
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Plant parasitic nematodes are destructive pests worldwide that cause severe losses in agriculture. Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary plant endoparasites that are extremely polyphagous in nature with a wide host range. Keeping in view the huge economic losses by this parasite, it is essential to control the disease at an early stage. For any control strategy to be effective, an early and accurate diagnosis is of paramount importance. Immunoassays have the inherent advantages of sensitivity and specificity; have the potential to identify and quantify these plant-parasitic nematodes. Hence, in the present studies, ELISA has been developed for the detection of Meloidogyne spp. antigens. This is based on detection of egg antigens, for which anti-Meloidogyne antibodies were produced by immunization of rabbits with soluble proteins of the eggs. The production of antibodies was confirmed by the appearance of precipitin lines in double immunodiffusion method. An indirect enzyme-linked immunosorbent assay (ELISA) was developed for detection and titration of these antibodies. Results indicated as high as 320 K titre of the antisera. Finally competitive inhibition ELISA was developed employing these anti-Meloidogyne antibodies for detection of Meloidogyne antigens. This is based on competition between solid phase bound antigens and free antigens for limited antibodies. Sensitivity of ELISA was 10 femtograms. This sensitivity was further enhanced to 1 femtogram with an additional step of pre-incubation, in which antibodies and free antigens are allowed to react before the competition. Competitive inhibition ELISA developed in the present studies has the potential of being used as an easy, rapid, specific and sensitive diagnostic tool for the detection of Meloidogyne infection.
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