Antigenic determinants of pregnancy-associated alpha 2-glycoprotein and alpha 2-macroglobulin defined by poly- and monoclonal antibodies.

Human alpha 2-macroglobulin and pregnancy-associated alpha 2-glycoprotein (PA alpha 2G) share several physicochemical characteristics. By the use of unabsorbed or absorbed polyclonal antibodies to these antigens, the existence of common epitopes in these molecules were demonstrated in crossed immunoelectrophoresis. Two monoclonal antibodies out of 9 raised against purified PA alpha 2G were demonstrated to react with both antigens, indicating close immunochemical relatedness between these macroglobulins. The findings might have functional implications.

Characterization of tryptic fragments of human complement factor C3.

C3c and C3d fragments were prepared in pure form from trypsin-digested human C3, and the individual chains of tryptic C3c were isolated by gel filtration on Sepharose 4B in 6M guanidinium hydrochloride. No low mol. wt (Mr) fragments were identified. The polypeptide chains were characterized with regard to Mr, amino acid composition and N-terminal amino acid sequence. Tryptic C3c consisted of one fragment from the beta-chain (Mr 64,000) and two from the alpha'-chain (Mr 40,000 and 23,000). The beta-chain fragment was derived from the C-terminal part of the chain, and the 23,000-Mr component constituted the amino terminal end of the alpha-chain. The 40,000-Mr fragment emanated from the C-terminal end of the alpha-chain. Tryptic C3d displayed microheterogeneity on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, but possessed a homogeneous N-terminal, identical to that described by Tack et al. (1980) (Proc. natn. Acad. Sci. U.S.A. 77, 5764-5768). By utilization of antisera against subunits of C3 and C3c in immunoblotting a degradation scheme for C3 by trypsin was proposed and the positions of the fragments in the intact molecule indicated.

Hypersensitivity to intravenous iron: classification, terminology, mechanisms and management.

Intravenous (IV) iron therapy is widely used in iron deficiency anaemias when oral iron is not tolerated or ineffective. Administration of IV-iron is considered a safe procedure, but severe hypersensitivity reactions (HSRs) can occur at a very low frequency. Recently, new guidelines have been published by the European Medicines Agency with the intention of making IV-iron therapy safer; however, the current protocols are still non-specific, non-evidence-based empirical measures which neglect the fact that the majority of IV-iron reactions are not IgE-mediated anaphylactic reactions. The field would benefit from new specific and effective methods for the prevention and treatment of these HSRs, and the main goal of this review was to highlight a possible new approach based on the assumption that IV-iron reactions represent complement activation-related pseudo-allergy (CARPA), at least in part. The review compares the features of IV-iron reactions to those of immune and non-immune HSRs caused by a variety of other infused drugs and thus make indirect inferences on IV-iron reactions. The process of comparison highlights many unresolved issues in allergy research, such as the unsettled terminology, multiple redundant classifications and a lack of validated animal models and lege artis clinical studies. Facts and arguments are listed in support of the involvement of CARPA in IV-iron reactions, and the review addresses the mechanism of low reactogenic administration protocols (LRPs) based on slow infusion. It is suggested that consideration of CARPA and the use of LRPs might lead to useful new additions to the management of high-risk IV-iron patients.

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.

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.

Production of antibody against C3c epitopes which are inaccessible on native C3.

Anti-C3c antiserum was induced by immunizing rabbits with autologous erythrocytes coated with human C3b/C3bi. By absorption with human plasma this antiserum was rendered specific for C3c epitopes which are not expressed on native C3. This specificity may be analogous to that of immunoconglutinin.

An automatic multi-programmed affinity chromatographic system.

An automatic immunospecific affinity-chromatographic system for continuous operation is described. The system comprises time-controlled sample application, washing and elution steps and automatic dialysis of eluted fractions. The applicability of the system is illustrated by the purification of pregnancy zone protein on immunosorbent gel.

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.

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.

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.

Investigations into the molecular heterogeneity of pregnancy-specific beta 1-glycoprotein (SP1).

The molecular heterogeneity of pregnancy-specific beta 1-glycoprotein (SP1) was examined by analytical immunoelectrophoresis and a radioimmunostaining technique of immunoelectrophoretic plates. Analytical crossed immunoelectrophoretic analysis and radioimmunostaining of these plates demonstrated the presence of normal human serum components in the alpha-mobile precipitate previously considered to be exclusively the high molecular pregnancy-specific protein, SP1 alpha. This observation suggested that two molecular populations were contributing to the alpha-mobile precipitate. Following fractionation of late-pregnancy serum by size chromatography, the radioimmunostaining technique further demonstrated the presence of normal serum components in the intermediate fraction but not in authentic SP1 (i.e., SP1 beta) or the high molecular weight form (SP1 alpha). We suggest that SP1 antigenic determinants are distributed in three different fractions of pregnancy serum, one of which (intermediate fraction) is a complex of authentic SP1 (SP1 beta) and a normal serum protein, whereas non-pregnancy serum components were not demonstrable in the remaining two (i.e., SP1 beta or SP1 alpha).

Specific assay for pregnancy-specific-beta 1-glycoprotein (Sp1-beta). Preparation of antiserum specific for SP1-beta by absorption with a crossreacting high molecular weight serum protein.

Two pregnancy-associated proteins reacting with antibody against pregnancy-specific beta 1-glycoprotein (PS beta G or SP1) were separated by means of ammonium sulphate precipitation, size chromatography and preparative zone electrophoresis. The antibody preparation was made monospecific to the beta-mobile protein by liquid phase cross-absorption of anti-SP1 immunoglobulin preparation with the isolated alpha2-mobile high molecular weight protein. The specificity of the absorbed antibody was tested in line immunoelectrophoresis, rocket immunoelectrophoresis and crossed immunoelectrophoresis.

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.

Immunochemical and biological studies on two molecular variants of pregnancy specific beta 1 glycoprotein (SP1) SP1 alpha and SP1 beta.

Analytical crossed immunoelectrophoresis was performed to examine the relationship of a high molecular mass alpha 2 mobile glycoprotein (SP1 alpha) which is immunochemically identifiable with pregnancy specific beta 1 glycoprotein (SP1 beta). There was no evidence for any reaction of SP1 alpha with antisera to normal human serum protein, nor for complex formation following incubation with normal serum proteins. The half-life of SP1 alpha was apparently shorter than that of SP1 beta after delivery of the placenta.

Trophoblastic tumour, oral contraceptive therapy and pregnancy associated alpha2-glycoprotein (PAAG), case report.

Pregnancy associated alpha2-glycoprotein (PAAG) and human chorionic gonadotrophin (HCG) levels were studied in a patient with an invasive mole who was being treated with cytotoxic drugs and oral contraceptives. Both the PAAG and HCG levels fell during cytotoxic drug treatment while oral contraceptives caused a rise in PAAG concentrations. A relation between oestrogen-induced PAAG and prolonged survival of trophoblastic tumour is proposed.

Arthritis in ducks. II. Condemnation rate, economic significance and possible preventive measures.

During the period 1959 to 1968 0.4% of ducks slaughtered in Denmark were condemned because they had arthritis. The condemnation rate due to arthritis decreased 2 years after compulsory measures to control salmonella infections were introduced and in the 10 years from 1969 to 1978 this condemnation rate was 0.12%. The condemnation rate due to arthritis was relatively high during the period 1961 to 1967. A positive correlation between the condemnation rate due to arthritis and the number of ducks slaughtered and the overall condemnation rate existed at this time. Seasonal variations in the condemnation rate due to arthritis were observed. Mallards were found to be less susceptible to arthritis than White Pekin and Muscovy ducks reared under the same conditions. The hock was most frequently affected (43%) with arthritis. In 37% of arthritic birds the knee was affected, hip and toe joints being affected in 18% and 1% of cases respectively. Wing joints were affected in less than 1% of cases. The national annual cost of arthritis from 1959-1968 was estimated at 207, 579 Danish crowns. In the subsequent 10 years the cost was only one third of this sum.

Application of electroblotting technique to studies of the intestinal antibody response to extractable fecal antigens.

Water-soluble and sodium dodecyl sulfate (SDS)-extractable fractions were prepared from feces of four patients with inflammatory and six with non-inflammatory bowel disease. Both types of fractions were run on polyacrylamide gel electrophoresis, followed by electroblotting on nitrocellulose sheets of the separated components. The antibody reactivities in serum and in the intestinal wall against the extracted and separated fecal components were investigated. A striking lack of reactivity was observed when serum was used as antibody source against the fecal extracts both in patients with inflamed and in those with non-inflamed intestinal mucosa. The locally produced gut-associated IgG reacted more intensely with SDS-extractable fecal components than with water-soluble components. A strong reaction between intestinal-wall IgG extracts and a water-soluble antigen of 46-48 kD in the feces of two colitis patients was, however, observed. No clear correlation between the single bands and the occurrence of inflammatory bowel disease could be established because of the small number of patients.

Charge and size heterogeneity of C3d following in vivo and in vitro activation of the complement system.

Split products of the third complement factor (C3) expressing D but not C epitopes (C3d) were analyzed by crossed immunoelectrophoresis and size chromatography. Four molecular forms (termed 1, 2, 3, and 4 from the anodic side) were identified. The precipitation pattern of C3d in serum following acute in vivo activation was similar to the patterns observed using in vitro activation by MgCl2, zymosan, Escherichia coli, and delta IgG. These patterns were different from those observed in normal human serum and serum from a patient suffering from systemic lupus erythematosus. The molecular weights of forms 1 and 4 were approximately 170,000 daltons and those of forms 2 and 3 approximately 40,000 daltons.