Intrauterine Infusion of High Doses of Pig Trophoblast: MATERIALS AND METHODS(2)

Sources and Assay of IFNs

Pure recombinant porcine IFN-7, produced in Escherichia coli, was kindly provided by Ciba-Geigy (Basel, Switzerland). Its specific antiviral activity was 1.2-1.5 X 105 U/mg protein. A specific sandwich-type ELISA was set up in our laboratory using monoclonal antibody G47 for the first step, and rabbit serum 652 for the second step. This test has a maximal sensitivity of 0.2-0.4 ng/ml for IFN–y and is very specific since it does not detect IFN-8 (unpublished data). Recombinant IFN-8 was produced in insect SF9 cells using a baculovirus vector and purified as previously described. Its specific antiviral activity exceeded 5 X 107 IU/mg protein.

The current antiviral activity in flushings and blood plasma was assayed using the bovine MDBK cell line, after challenge with the Vesicular Stomatitis Virus (VSV), and titers were expressed in human international units per milliliter, as described elsewhere. A particular antiviral assay was used for specific dosage of IFN-8 in flushings, since no specific ELISA exists for this IFN. The swine testicular cell line ST was used in the same microassay as with MDBK cells, with challenge infection by VSV, as we repeatedly found that ST cells are completely refractory to the IFN-7-induced antiviral effect (unpublished data). birth control pills

Preparation of IFN Mixture

A mixture of the two IFN types with nearly the same proportions as the native mixture was prepared as follows: a pool of uterine flushings from Day 14 pregnant Meishan gilts was prepared as described and separately assayed for each IFN species. IFN-7 was assayed by ELISA, and its concentration was directly estimated in micrograms per milliliter with reference to pure IFN-7 provided by Ciba-Geigy. To estimate the amount of IFN-8, the same pooled flushing was assayed in international units per milliliter by an antiviral activity test on ST cells.

This entry was posted in Trophoblast and tagged Interferons, Intrauterine Infusion, Pig, Trophoblast.