Radioimmunoassay of bovine placental lactogen using recombinant and native preparations: determination of fetal concentrations across gestation

Abstract

Concentrations of bovine placental lactogen (bPL) were determined in fetal plasma samples by twelve double-antibody competitive radioimmunoassay systems (RIA I-XII) based on either recombinant bPL (non-glycosylated) or native bPL (glycosylated). Both preparations were used as standard and tracer, and for primary antisera production. The minimum detection limit measured by these RIA varied from 0.02 to 0.6 ng bPL mL(-1). The coefficients of correlation of different bPL RIA systems were up to 90% (P < 0.0001) when each RIA was tested against the average values of all twelve RIA systems. All developed RIA were used to investigate the incidence of different bPL isoforms in bovine fetal serum samples (n = 71). Fetal concentrations ranged from 11.8 to 35.7 ng mL(-1) at the third month and from 1.1 to 13.5 ng mL(-1) at the ninth month of gestation. They tended to decrease with advancing gestation. In general, those RIA systems that used recombinant bPL as the standard measured higher values than those using the native bPL preparation. These differences decreased toward the end of gestation (P < 0.05), suggesting a lower rate of glycosylation. Our results provide evidence of different glycosylated isoforms of bPL in fetal serum at different gestation periods.

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

“…The infusion of anti-bPL IgG immediately immunoneutralized the circulating bPL, as reported by Waters et al [8]. Before the infusion of anti-bPL, basal concentrations of bPL are in accordance with those reported by different authors [17,22]. After bPL immunoneutralization, a rapid decline of anti-bPL was observed, alleviating the neutralization effect.…”

“…Optimal dilution titers (20 to 30% binding ratio of the radiolabeled rbPL ( 125 I-rbPL) to the antiserum in the zero standard (B 0 ) assay tube) were 1:130,000 for guinea pig AS#276 and 1:400,000 for rabbit AS#295 [22]. …”

“…In brief, 100 μL of guinea pig AS#276 (1:130,000), rabbit AS#295 anti-bPL (1:400,000), or a mixture of both primary antisera (0.05:0.05 mL; vol:vol) were incubated with 100 μL of 125 I-rbPL (25,000 cpm) [22]. The volume was adjusted to 500 μL by adding 300 μL of assay buffer (phosphate 0.05 M, pH 7.3 containing 0.1% BSA).…”

“…Samples were incubated overnight at room temperature. The next day, bound and free fractions were separated after addition of 1 mL of second-antibody PEG solution containing 0.87% v:v sheep anti-rabbit Ig, as described elsewhere [22]. …”

“…The effects of antisera dilutions on bPL concentration measurements ( in vitro study) were analyzed using a general linear model (Proc GLM, SAS) according to the following model: Yij = ai + bj + eij, where Yij = difference in bPL concentration measured in control sample and sample that received antisera, in animal i (i = 1 to 5) and at dilution j (1:1,280,000 to 1:2,500 step 2 dilution), ai = the effect of animal i, bj = effect of the dilution j, and eij is the random residual effect (N [22]). The animal effect was considered as random and the dilution one as fixed.…”