Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Abstract

Background: The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.

Methods: Six bovine foetuses were chronic cannulated on the aorta via the medial tarsal artery. Infusion of rabbit anti-bPL IgG was performed during late gestation. Pooled rabbit anti-bPL antisera had a maximal neutralization capacity of 25 microg bPL/mL of immunoglobulin. Interference of rabbit anti-bPL immunoglobulin with radioimmunoassay measurement using guinea pig anti-bPL as primary antibody was first evaluated in vitro. Polyclonal anti-bPL antibodies raised in rabbit were added in foetal sera to produce 100 samples with known antibodies titers (dilutions ranging from 1:2,500 till 1:1,280,000).

Result(s): Assessment of the interference of rabbit anti-bPL antibody showed that bPL concentrations were significantly lower (P < 0.05) in samples added with dilutions of rabbit antiserum lower than 1:80,000 (one foetus) or 1:10,000 (four foetuses). It was also shown that the recovery of added bPL (12 ng/mL) was markedly reduced in those samples in which exogenous rabbit anti-bPL were added at dilutions lower than 1:20,000. Concentrations of foetal bPL were determined in samples from cannulated foetuses. In foetuses 1 and 6, bPL concentrations remained almost unchanged (<5 ng/mL) during the whole experimental period. In Foetus 3, bPL concentrations decreased immediately after IgG infusion and thereafter, they increased until parturition.

Conclusion(s): The use of a bPL RIA using a guinea pig anti-bPL as primary antiserum allowed for the measurement of bPL concentrations in foetal plasma in presence of rabbit anti-bPL IgG into the foetal circulation. Long-term foetal catheterization allowed for the study of the influence of direct infusion of anti-bPL IgG on peripheral bPL concentrations in bovine foetuses.

Figure 1

Arterial cannulation in bovine foetuses. Bovine fetal hind limb was identified by intra-abdominal palpation and moved so that the foot lay in the maternal abdominal incision (A). After an incision through the uterine wall and after opening of the fetal membranes, they were progressively fixed together by Collins forceps. The fetal limb was withdrawn from the uterus until the anterior surface of the hock joint was easily accessible (B). The fetal medial tarsal artery was exteriorized and catheterized with a polyvinyl catheter (C).

Figure 2

Plasmatic profiles of bPL concentrations and rabbit anti-bPL titers in peripheral circulation of four bovine foetuses. Concentrations of fetal bPL (ng/mL) are represented by black dots. Rabbit anti-bPL titers measured as B/T (bound activity (B) regarding total tracer (T) added) are represented by white circles. Plasma samples from cannulated foetuses (Foetuses 1 to 4) were collected from Days 232 (Foetus 1) to 249 (Foetus 4) of pregnancy until term. Concentrations of bPL were measured by RIA with guinea pig anti-bPL antiserum (AS#276) as primary antibody. Solid line arrows indicate day of infusion of a pool of rabbit anti-bPL IgG into the fetal catheter. Broken line arrow indicates the day of calving.

Figure 3

Plasmatic profiles of bPL concentrations and rabbit anti-bPL titers in peripheral circulation of two bovine foetuses. Concentrations of bPL in fetal plasma (ng/mL) are represented by black dots. Anti-bPL titers measured as B/T (bound activity (B) regarding total tracer (T) added) are represented by white circles. Plasma samples from cannulated foetuses (Foetus 5 and 6) were obtained during late pregnancy. Concentrations of bPL were measured by RIA with guinea pig anti-bPL antiserum as the primary antibody. Solid line arrows indicate day of infusion of a pool of rabbit anti-bPL IgG into the fetal catheter. Broken line arrow indicates the day of calving.