The production of antiserum against Myrobalan latent ringspot virus for detection of the virus using ELISA

Abstract

<I>Myrobalan latent ringspot virus</I> (MLRSV) was purified from the extract of infected <I>Chenopodiumquinoa</I> Willd., using <I>n</I>-octanol, differential centrifugation, and saccharose gradient centrifugation. Electron microscopy showed 28 nm large isometric particles in a sample of purified virus. Antiserum to MLRSV was prepared by immunisation of rabbits with intravenous injections of antigen in combination with intramuscular injections of antigen with Freund’s adjuvant. The antibody titer of the obtained antiserum was determined by drop-precipitation method to be 1:1024. Immunoglobulins against MLRSV were isolated from antiserum with ammonium acetate, caprylic acid, and precipitation with ammonium sulphate. The isolated immunoglobulins (IgG) were conjugated with alkaline phosphatase. The optimal dilution of IgG for detection of MLRSV using ELISA was 1×10<sup>–3</sup>μg/ml which was also the optimal dilution of conjugated IgG. Using this dilution of antibodies, the absorbance of samples from MLRSV-infected plants of <I> C. quinoa</I> varied between 0.71 and 1.45, while absorbance of samples from healthy plants (control) was 0.01 to 0.07.