Isolation of a Vaccinia-specific Hapten from Vaccinia-infected Sheep Dermis

Abstract

SUMMARYVirus-free buffer extracts of vaccinia-infected sheep dermis were fractionated by elution from DEAE-cellulose with increasing concentrations of NaC1. The fraction eluted with 4-o M-NaC1 was serologically homogeneous when examined in precipitation-in-gel tests. Further fractionation on Sephadex G-2oo gave a material which was also physically homogeneous when examined by both ultracentrifugal and electrophoretic techniques. A sedimentation coefficient of 3"47 S and a molecular weight of 3 I,OOO were calculated from ultracentrifugal data. The isolated material contained 74"3 % DNA, 9"4 % protein estimated as bovine serum albumin and 14"9 % carbohydrate estimated as glucose. The DNA contained the bases adenine, thymine, guanine and cytosine in the ratios I.O: I. t : o.8: o.8. Paper chromatography of formic-acid hydrolysates of the material resolved seven substances reacting with ninhydrin. Qualitative colorimetric tests indicated, apart from deoxyribose, the presence of a hexose and a hexuronic acid.The serological activity of the isolated material was heat-stable and identical with that of a previously described heat-stable extract of vacciniainfected rabbit dermis. Treatment with enzymes indicated the presence of two different, serologically active sites. Failure to elicit an antibody response following injection into rabbits suggested that the isolated material was a hapten. Serum absorption studies showed serological identity with an antigen present on the surface of the vaccinia virus particle.

Studies on the LS antigen of vaccinia virus

“…centrifuge. As stated by Williamson & Rondle (1964) it gave a single sharp peak and from the data a molecular weight of approximately 31,000 was calculated.…”

“…This fractionation yielded two chemically and physically different substances, one possessing the serological specificity of L, the other that of S. No serological relationship could be demonstrated between the two materials; indeed, experimental results indicated that they were serologically distinct. The isolation from vaccinia-infected tissue of a material exhibiting L activity only has not been reported previously, although Smadel, Hoagland & Shedlovsky (1943) (Williamson & Rondle, 1964). When tested against suitable antisera it gave an S reaction up to a dilution of 1 in 750.…”

“…Calculated from a standard curve of commercial calf thymus nucleic acid, S(PR) contains less than 6 % and LS and L less than 2 % nucleic acid, while S contains approximately 70 % of it. Chemical studies have shown that the nucleic acid of sheep S is DNA (Williamson & Rondle, 1964).…”

“…With antisera prepared as described below the eluate at 0-137 m-NaCl reacted serologically as a specific L substance and the eluate at 4 M-NaCl reacted serologically as a specific S substance. Further work on the latter material gave a physically and serologically homogeneous S substance (Williamson & Rondle, 1964).…”

The inhibition of growth of vaccinia and cowpox viruses in RK 13 cells

“…lpc f in Rondle & Dumbell, 1962). Lpc 3 is heat labile and lpc 5 and 7 heat stable; they are serologically related to the 'L' and 'S' antigens of vaccinia (Smadel & Shedlovsky, 1942;Wiliamson, 1963;Williamson & Rondle, 1964). Lpc d was the material normally present in cowpox soluble antigens, but absent from antigens prepared from vaccinia and the white pock variant of cowpox (Rondle & Dumbell, 1962).…”