Purification of tulip breaking virus and production of anti-sera for use in ELISA

Abstract

After comparison of various homogenization buffers and clarification methods, tulip breaking virus (TBV) was purified on a large scale by homogenization in 0.1 M tris buffer (pH 9.0), clarification with Triton X-100, and differential centrifugation. The TBV suspensions were absorbed with an anti-host protein antiserum. The purity of the product was verified by mechanical inoculation of test plants, serology, electron microscopy, spectrophotometry, and analytical ultracentrifugation.The TBV antisera obtained from rabbits after intramuscular injections had titres of 1/1280 to 1/10240 in the microprecipitin test and were mainly used in conjugate dilutions of 1/1000 in ELISA. The absorbance values for healthy tulips and lilies were (very) low. The antisera prepared against TBV from tulip cv. Jack Laan reacted best with TBV in lilies, whereas the antisera prepared against TBV from tulip cv. Texas Flame reacted best with TBV in tulips.

Characterization of Potyviruses from Tulip and Lily which Cause Flower-Breaking

“…Rembrandt tulip-breaking virus (ReTBV) originated t'rom a stock of Rembrandt tulip cv. Jack Laan with a deep purple and white flower break and light green bands in the leaves (Derks et al, 1982).…”

“…The antisera against tulip-breaking viruses from tulip cvs. Jack Laan (RD60) and Texas Flame (REI02) were described previously (Derks et al, 1982); the LiMV antiserum (UT478) was prepared against LiMV propagated in N. benthamiana.…”

“…These differences in opinion about the systematic classification of tulip-breaking viruses were mainly based on host plant reactions. Later on, these viruses were classified as potyviruses (Brandes & Wetter, 1959) with small serological differences between them (Derks et al, 1982;Maeda et al, 1984). Differences in transmissibility to dicotyledonous plants have been reported as well (Alper et al, 1982;Maeda et aI., 1984).…”

Detection of the tulip breaking virus (TBV) in tulips using optical sensors

“…To have sufficient infected bulbs for testing, sets of bulbs were selected that had high rates of TBV infection. These bulbs had been assessed in the former breeding season by ELISA, an enzyme-linked immunosorbent assay (Derks et al 1982). The established infection rates for the bulb sets were 14% in Yokohama, 16% in Barcelona and 31% in Monte Carlo.…”

“…The ELISA analysis, using TBV-specific antisera in a validated protocol (Derks et al 1982), was carried out on the same leaves as those measured with the optical sensors. These ELISA measurements were used as the reference by which to compare the human visual assessment and the optical sensor techniques.…”