J. Folkersen

Circulating levels of pregnancy zone protein: normal range and the influence of age and gender.

Serum levels of pregnancy zone protein (PZP) were measured in 506 apparently normal males and 329 normal non-pregnant females, the age range being 18 to 70 years. The estimations of PZP were performed by sensitive radio-rocket-line immunoelectrophoresis. The distribution of the data had a marked positive skew in both sexes which was reduced following logarithmic transformation. Serum concentrations in both men and women were found to increase significantly with advancing age. This increase and the mean concentrations were significantly higher in females.

Immunohistochemical demonstration of pregnancy-associated plasma protein A (PAPP-A) in the syncytiotrophoblast of the normal placenta at different gestational ages

The immunoperoxidase technique was used to study the localization of pregnancy-associated plasma protein A (PAPP-A) in formaldehyde-fixed paraffin-embedded tissue from normal human placentae at the gestational age of 8, 15 and 40 weeks. Sections of formaldehyde-fixed tissue treated with a proteolytic enzyme and incubated in antiserum against PAPP-A either raised in goats or rabbits showed that PAPP-A was distributed in the cytoplasm of the syncytiotrophoblast. The protein was not found in the cytotrophoblast. Sections without pretreatment with trypsin and incubation in goat anti-PAPP-A showed no staining reaction, whereas incubation in rabbit anti-PAPP-A revealed a staining of the syncytiotrophoblast surface. The results indicate that PAPP-A is probably synthesized in the syncytiotrophoblast.

Comparison of different antibody preparations against pregnancy‐associated plasma protein‐A (PAPP‐A) for use in localization and immunoassay studies

Summary. Four antibody preparations against pregnancy‐associated plasma protein (PAPP‐A) were compared in order to find an explanation for the contradictory results published on tissue localization, clinical usefulness and biological function of PAPP‐A. One of the preparations studied was a rabbit anti‐PAPP‐A antiserum which has been offered for general scientific use (Bischof et al. 1979). Only the IgG fraction of anti‐PAPP‐A antisera which appeared to be monospecific and had been further absorbed with fetal connective tissue gave specific uniform staining of the cytoplasm of the syncytiotrophoblast exclusively. Circulating PAPP‐A could not be detected by RIA employing this IgG preparation in the non‐pregnant state, or before 18 days after conception. Circulating PAPP‐A could be detected in all seven pregnant women studied within 4 weeks after conception. Identical results were obtained with a commercially available IgG fraction against PAPP‐A.

Purification of pregnancy-associated plasma protein-A by a two step affinity chromatographic procedure

A high molecular weight pregnancy specific protein (PAPP-A) was purified by immunospecific affinity chromatography. The purification procedure was based on a new method for affinity chromatography using direct coupling of precipitable immune complexes to the gel matrix. The procedure was performed in two steps: a positive immunospecific affinity chromatography followed by a negative affinity chromatography in which balanced amounts of anti-contaminant antibodies were used as ligant. The purified material at a concentration of 300 microgram/ml was tested by immunoelectrophoretic methods and no contaminants were detectable. WHO beta-1 SP1 reference material 78610 contained 45 microgram PAPP-A/ml.

A method for preparation of immunosorbents by direct coupling of dissociated immune complexes

A simple and rapid method of preparing immunosorbents by use of isolated immune complexes is described. Dissociated immune complexes may be directly coupled to activated Sepharose and an immunosorbent gel with complementary molecular populations of immunospecifically purified ligands is obtained. The antigen binding capacity of insolubilized antibodies was about 50% of the antigen bound at equivalence using liquid phase precipitation. The batch technique described may be scaled up and the only limitation is the availability of the ligands. The usefulness of the technique is illustrated in two model systems.

Affinity chromatographic purification of the pregnancy zone protein

An immunospecific affinity chromatographic method for purification of human pregnancy zone protein (PZP) directly from serum is described. Highly purified goat-anti-human PZP-immunoglobulin was applied as a ligand. Recovery of PZP varied from 56--75%. The impurities constituted maximally 5--10% of the total protein in the eluate. The purification factor was approximately 100.

An immunoprecipitation-dissociation technique for large scale antibody purification and an antigen consumption electroimmunoassay for antibody quantitation. A model study with antibodies to pregnancy zone protein

A simple immunoprecipitation--dissociation technique for large scale purification of antibodies is described, which comprises selective denaturation of the antigen and recovery of the antibody fraction by exclusion chromatography at low pH. Its use is illustrated by the purification of antibodies to pregnancy zone protein. A purification factor of about 60 was achieved. An antigen consumption electroimmunoassay was also developed which permits quantitative determination of the antigen binding activity of antibodies with a given specificity. The methods have general application.

Specific assay for pregnancy-specific-β1-glycoprotein (SP1-β). Preparation of antiserum specific for SP1-β by absorption with a crossreacting high molecular weight serum protein

Abstract Two pregnancy-associated proteins reacting with antibody against pregnancy-specific β 1 -glycoprotein (PSβG or SP1) were separated by means of ammonium sulphate precipitation, size chromatography and preparative zone electrophoresis. The antibody preparation was made monospecific to the β-mobile protein by liquid phase cross-absorption of anti-SP1 immunoglobulin preparation with the isolated α 2 -mobile high molecular weight protein. The specificity of the absorbed antibody was tested in line immunoelectrophoresis, rocket immunoelectrophoresis and crossed immunoelectrophoresis.

Complement activation, circulating protease inhibitors and pregnancy-associated proteins in severe preeclampsia

Summary. Circulating protease inhibitors, pregnancy‐associated proteins and the split product of complement factor 3 (C3d) were measured in 14 women with severe pre‐eclampsia and their matched controls. Only the mean levels of antithrombin III were observed to be significantly lower in pre‐eclampsia (P<0.02).

The selection of antibodies with defined desorption properties from precipitated immune complexes for use in immunoadsorption procedures

A general method for preparing immunosorbents with preselected antibody avidity is described. The method, which is a modification of a method described previously, also includes immunospecific purification of the ligand prior to coupling on the gel matrix. Polyclonal anti-alpha-1-fetoprotein antibodies in precipitated immune complexes were separated according to their avidity (low, intermediate and high) by dissociation with agents of increasing efficiency. After solid-phase coupling the antigen binding activity of the separated antibody preparations was examined according to recovery, capacity and binding strength. Antibodies of intermediate avidity derived from the immune complexes demonstrated optimal properties for preparative affinity chromatography.