Immunoquantitation of human placental alkaline phosphatase using radial immunodiffusion.

A simple procedure is described for immunoquantitation of human placental alkaline phosphatase by radial immunodiffusion. Agarose gels in petri dishes were overlayed with diluted antiserum, and, after equilibration of the antibody with the gel, the overlay was poured off and the gel was used for quantitation of enzyme protein using a specific stain to amplify visualization. The method has a variation of 0–8.5% for triplicate determinations on a single plate, with a mean of 2.6%. Variations between plates were 0–13%, with a mean of 4.2%. By including Triton X-100 detergent in the overlay solution, it was possible to quantitate the amount of enzyme in membrane preparations from placentae and cancer fluids: A 1000-fold range of antiserum and enzyme dilutions was tested; over this range, only a 2–3 × deviation from the expected ring size was found. As little as 5 ng of enzyme protein could be measured by this technique.     

Alteration of human placental lactogen by mammary gland

Human placental lactogen was prepared in high purity and in good yield applying a minimum of purification steps. The isolated hormone was characterized with respect to isoelectric and electrophoretic properties, molecular size (estimated mol. wt. 23 000) and stability to heat, pH and organic solvents. Investigation of in vitro interaction between placental lactogen and mammary gland (mouse, rat) revealed a rapid alteration of hormone with loss of immunoreactivity resulting. The target organ as a selective alteration site of placental lactogen was suggested by a lack of similar action on the hormone by a number of other tissues tested, including liver, kidney and lung. The reaction involving hormone alteration by mammary gland was localized to a particulate-bound enzyme, sedimentable at 10 000 X g and undissociated by sonication in 0.5% Triton X-100. Examination of the reaction products revealed hormone degradation with formation of diffusible components and loss of original electrophoretic identity as well as immunoreactive properties. The reaction characteristics included: pH optimum between 7.5 and 8.0, an absolute salt requirement (NaCl, KCl, at concentration greater than 0.15 M for maximal activation), inhibition by Cleland's reagent and lack of reaction interference by pituitary prolactin.     

Two pathways of placental lactogen secretion by cultured human trophoblast

To assess the relative importance of regulated and of constitutive secretion of placental lactogen, a cell culture model of term human trophoblast was utilized. Time courses of secretion revealed a constant secretion rate over 9 days of culture, with relatively small constant intracellular hormone concentration. Potassium, 21 mM, produced a slight but significant increase in hormone secretion into the medium. Growth hormone-releasing hormone (5 X 10(-10)-5 X 10(-9)) stimulated a 27-48% increase in placental lactogen secretion. The data suggest a major process of constitutive secretion and a minor role for regulated secretion from a storage pool.     

Quantitation of apolipoprotein A-I in pooled human serum by single radial immunodiffusion and sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Apolipoprotein A-I was released from human HDL particles by treatment with 8 M urea, and the free apolipoprotein exhibited identical antigenicity and the same low mobility as purified apolipoprotein A-I in electrophoresis. Treatment of serum with 8 M urea enabled enabled quantitation of apolipoprotein A-I by single radial immunodiffusion assay, as judged by comparison with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Transcriptional products of the human placental lactogen gene

Poly(A+)RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to preplacental lactogen (pre-hPL) and a minor band co-migrating with mature hPL represent approximately 15% of the total radioactively labeled proteins. Analysis of the poly(A+)RNA by agarose gel electrophoresis showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with 32P-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460, and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A+)RNA was also used to construct a cDNA library. Approximately 5% of the clones were found to hybridize to hPL DNA sequences, indicating that hPL mRNA is indeed very abundant in term placental tissue. One recombinant plasmid containing an insert of approximately 815 base pairs was isolated and characterized by restriction enzyme mapping and electron microscopy. Heteroduplexes constructed between the cDNA and the DNA isolated from an hPL genomic clone revealed four small intervening sequences which can account for the lengths observed for the hnRNA molecules.     

Quantitative determination of human apolipoprotein D by electroimmunoassay and radial immunodiffusion.

1. An electroimmunoassay and a radial immunodiffusion procedure are described for the quantitative determination of human serum apolipoprotein D. Purified apolipoprotein D and antisera to both lipoprotein D and apolipoprotein D were used to standardize the assays. The assays are applicable to measurement of apolipoprotein D in serum and density classes. The electroimmunoassay is more sensitive (50 ng apolipoprotein D quantitatively detectable), rapid (time required for completion of assay is 5 h) and precise (the within- and between-assay coefficients of variation are 4 and 7%, respectively) than radial immunodiffusion. However, comparable results were obtained by both methods (r = 0.85). 2. Serum apolipoprotein D levels of normal subjects and hyperlipoproteinemic phenotypes IIa, IIb, III, IV and V were in the same range (10 to 12 mg/dl). In contrast, patients with hyperchylomicronemia (type I) had decreased apolipoprotein D levels (5 mg/dl; P less than 0.001). The apolipoprotein D in serum of normolipidemic subjects was detectable in all density classes but measurable only in HDL2 (21%), HDL3 (43%) and VHDL (36%). 3. Rocket electrophoresis is also a valuable tool for assessing the structural relationships among apolipoproteins or their constituent polypeptides. Interaction between serum and a mixture of antibodies to A-I, A-II and apolipoprotein D resulted in the formation of separate lipoprotein A and lipoprotein D rockets indicating that apolipoprotein D is not a constituent polypeptide of apolipoprotein A. This observation confirms the existence of lipoproteins A and D as separate lipoprotein families.     

Linkage arrangement of human placental lactogen and growth hormone genes

Human placental lactogen (hPL) and growth hormone (hGH) are two hormones thought to have evolved from a common ancestral gene (along with prolactin), yet they have quite different functions and specificities. The nucleic acid sequences of the respective cDNAs of the two genes share considerable homology, as well as the existence of multiple forms of each gene within the genome. In this study we report on the linkage arrangement of several genes from this group. Two hPL-like genes as well as an hGH gene are shown to be linked within a 38-kilobase pair region of DNA. Linkage between a variant hGH gene and an hPL gene is also shown. The orientation and structural organization of these genes was previously established using 5'- and 3'-specific probes from a placental lactogen cDNA clone and detailed restriction endonuclease mapping. Restriction fragments from the overlapping clones were verified by comparison to digests of high molecular weight genomic DNA. In addition, the location of a specific class of repetitive DNA sequences, the Alu family, was mapped on these clones using the recombinant clone BLUR 8. All members of this multigene family have Alu repeat sequences either immediately flanking their 3' or 5' untranslated regions or within their intervening sequences.     

The effects of insulin-like growth factor-I and insulin on placental lactogen production by human term placental explants

The effects of insulin-like growth factor (IGF)-I and insulin on placental lactogen production (hPL) by term human placental explants were studied. The hPL content in medium and explant decreased rapidly after first 24 hours of culture. The decrease thereafter was gradual and reached a plateau by day 4 of culture. The decrease of HPL content in placental culture has previously been suggested being due to the depletion of a rapidly secreting preformed pool of hPL. Addition of IGF-I (0.1-10 micrograms/ml) and insulin (1-20 micrograms/ml) stimulated the decreased level of hPL in tissue and medium after 24 hours in culture. IGF-I was 10 times more potent than insulin in stimulating hPL. These findings suggest that IGF-I and insulin effects the production of hPL by placenta. The lower potency of insulin may indicate that the effect of insulin on hPL production is via IGF-I receptor.     

Purification from human pregnancy serum of a low molecular weight mitogen similar to placental lactogen and growth hormone

Recently, we isolated from the serum of pregnant women a factor that induced rapid proliferation of a lactogen-dependent rat lymphoma cell line (Nb2). This mitogenic factor is reasonably specific to pregnancy, since it was present in serum samples from second trimester as well as term-pregnant women, but not in those of adult men or cycling females. It is unlikely that this mitogenic activity (referred to as pregnancy mitogen [PM]) is due to contamination by classical lactogens, since acetone fractionation of serum yielded a preparation devoid of placental lactogen and prolactin, as determined by radioimmunoassays. Further purification of acetone precipitates from term-pregnant serum by ion exchange chromatography and gel filtration yielded a mitogenic activity with a relative mol wt of approximately 10,000. PM activity in the NB2 cell bioassay was not affected by the presence of prolactin antiserum. However, its activity was immunoneutralized by coincubation with anti-placental lactogen serum and, to a lesser extent, anti-growth hormone serum. It appears that PM was not generated by our extraction procedure, since gel filtration of whole serum also yielded a bioactive fraction of approximately 10 kDa. PM was further purified to homogeneity by high-performance liquid chromatography. Examination of the preliminary amino acid composition of PM revealed differences from that of a bioactive fragment of growth hormone and a corresponding portion of placental lactogen, suggesting that PM could be either a molecular variant of these hormones or a novel protein.     

The identification of diphtheria, tetanus and pertussis vaccines by single radial and double immunodiffusion techniques

The identification tests for adsorbed diphtheria, tetanus and pertussis vaccines, which are required by the European Pharmacopoeia to be undertaken in animals, may be replaced by precipitation tests, for instance in agaros gels. Such in vitro tests eliminate the use of animals and are less expensive and time-consuming. The single radial immunodiffusion technique is a suitable semiquantitative test, while the double diffusion test is necessary for the investigation of complete or partial identity. The precipitates obtained in the single radial diffusion tests and in double diffusion tests with diphtheria toxoid were visible without staining; those obtained in the double diffusion tests with tetanus toxoid were weaker and staining was sometimes needed.