The Development of a Semi‐automated Latex Agglutination Test for the Detection of Antibodies toAnaplasma marginaleUsing a Cell Culture‐derived Antigen

Abstract

Serologic diagnosis of anaplasmosis is currently done by the complement-fixation, ELISA, and card agglutination tests. These tests have utilized A. marginale harvested from bovine erythrocytes as antigen which is often contaminated with erythrocyte stroma. We are currently testing A. marginale propagated in a Ixodes scapularis cell line as antigen for serologic tests. In this study, we report the use of the cell culture-derived A. marginale as antigen for development of a rapid, semi-automated latex agglutination test. Diluted serum and latex (polystyrene microspheres), sensitized with cell culture-derived A. marginale proteins, were dispensed into 96-well microtiter plates. An initial reading of light transmission was recorded by a computer-interfaced scanning autoreader. After 30 minutes, the plates were mixed and read a second time, recording the delta % light transmittance. The sensitized latex microspheres (latex) agglutinated in the presence of A. marginale antibodies, thus producing an increase in light transmittance. In preliminary tests, 724/977 of the sera were positive for A. marginale antibodies with an apparent agreement of 83.3% when compared with the complement-fixation test. Sensitization and sera dilution buffers were shown to have a marked effect on the sensitivity and specificity of this assay. Results will be presented on the optimization of buffers and the testing of sera from experimentally and field-infected cattle.

Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against Anaplasma marginale in cattle

“…Agglutination tests to detect antibody against A. marginale have been used for decades in capillary tubes, cards or plates (Welter, 1964; Amerault and Roby, 1968; Howarth and Hokama, 1976; Rodgers et al , 1998). However, in all cases the A. marginale antigen used is derived from initial body semi-purified from bovine erythrocytes (Wilson et al , 19778) or in vitro propagated in tick cell line (Rodgers et al , 1998).…”

“…However, in all cases the A. marginale antigen used is derived from initial body semi-purified from bovine erythrocytes (Wilson et al , 19778) or in vitro propagated in tick cell line (Rodgers et al , 1998). These antigens are formed by a complex protein mixture, making it difficult to standardize different batches of antigen, resulting on low reproducibility.…”

Manutenção in vitro de células IDE8 em dois tipos de soro bovino

“…Alguns sistemas de cultivos de células de carrapatos possibilitam o cultivo de antígenos, livres de membranas animais ou patógenos, para serem utilizados em testes de diagnóstico Rodgers et al, 1998) e no desenvolvimento de vacinas Kocan et al, 2001;de la Fuente et al, 2002).…”

Use of Refrigeration as a Practical means to Preserve Viability of in Vitro-Cultured IDE8 Tick Cells

“…Some tick cell culture systems allow the production of such antigens free from host or pathogen contaminants for use in diagnostic tests Rodgers et al 1998) and for the development of vaccines Kocan et al 2001;de la Fuente et al 2002).…”