Development of Antiserum against the major basic protein isolated from the medium of peri-attachment porcine conceptus cultures

Studies were performed to isolate the major secretory protein produced by the peri-attachment porcine conceptus and produce antiserum against it. Gilts were surgically hysterectomized on day 16 +/- 1 post-breeding and, conceptuses flushed from uteri and cultured 3 for 24 hours in a minimum essential medium with either ³H-leucine or ³⁵S-methionine. Proteins released into the incubation medium were subjected to carboxymethylcellulose ion exchange and Sephacryl-200 gel filtration chromatography to isolate the protein. Isolation of the protein (Mr 43000; pI 7.5-8.0) from other components of conceptus secretion was verified by non-equilibrium pH gradient electrophoresis and fluorography. A rabbit received an initial injection of 720 ug of the isolated protein in Freund's complete adjuvant and was boosted with 160 ug protein in Freund's incomplete adjuvant four weeks later. Using Ouchterlony immunodiffusion, antibody against the isolated conceptus protein was identified in the serum of the treated rabbit. Antiserum was characterized by Ouchterlony immunodiffusion, antigen-antibody immunoprecipitation, one dimensional polyacrylamide gel electrophoresis and fluorography. The molecular weights of the labelled Ag-Ab complex and the peri-attachment conceptus protein in major production are approximately 43000. The antiserum did not crossreact with 1) uterine flushings from pseudopregnant gilts, 2) peripheral plasma from nonpregnant gilts, or 3) amnionic and allantoic fluid and media from cultures of endometrial, chorionic or amnionic tissue from late pregnant gilts. This antiserum will be used in future studies designed to examine the site of action and function of the major protein product of the peri-attachment porcine conceptus.