A radioimmunoassay of the pregnancy-specific beta1-glycoprotein (SP1).

A radioimmunological method for determination of the pregnancy specific beta1-glycoprotein (SP1) is described. Antigen specificity of rabbit anti-human SP1 serum (Behringwerke) was investigated against human placental and pituitary hormones. The sensitivity was about 10 microgram/l. The concentration ranges of SP1 in umbilical cord blood and amniotic fluid were 0.10--0.60 mg/l and 0.3--3.8 mg/l, respectively. A good correlation (r = +0.95) was found between the radioimmunological and electroimmunological methods for determination of SP1 in maternal blood.     

Oligomerization of pregnancy-specific beta 1-glycoprotein (SP1) at physiologic pH and ionic strength

Highly purified pregnancy-specific beta 1-glycoprotein (SP1) migrated in gel electrophoresis as a homogeneous species and behaved as a single species in 6 mol/l guanidinium chloride (GdmCl), both in the ultracentrifuge and HPLC. At physiologic pH and ionic strength, in the absence of GdmCl, SP1 existed in the form of oligomers of apparent molecular weights of 40 000 to greater than 300 000. The specific activity of these oligomers varied over a 5-fold range. Electrophoretic mobility also varied among SP1 oligomers, with increasing (alpha-like) mobility shown by oligomers of increasing molecular size. Oligomerization may explain some or all of the reports of SP1 heterogeneity.     

Immunoturbidimetric determination of pregnancy-specific beta 1-glycoprotein (SP-1)

We describe a simple and rapid immunoturbidimetric end-point assay for measuring pregnancy-specific beta 1-glycoprotein (SP-1) in human serum with the Kone Progress clinical chemistry analyser, using a commercially available antiserum. The absorbance was measured at 340 nm 5 min after addition of antiserum. The assay range was 3-250 mg/l. Over this range the CV for between-day precision was below 6%. Bilirubin up to 160 mumol/l, haemoglobin up to 2 g/l and lipaemia up to 320 mg/l did not interfere. The reference interval in maternal serum at the 15th week of gestation, was 13 to 46 mg/l with a median of 25 mg/l (n = 78) and that of the 16th week was 6 to 60 mg/l with a median of 28 mg/l (n = 78). Comparison of results obtained by two commercially available radio-immunoassays and by the present method shows acceptable correlation. Rapid and reproducible, the immunoturbidimetric method is suitable for use in routine determination of SP-1.     

Problems encountered in the measurement of pregnancy-specific beta1-glycoprotein.

Evidence is presented here that variants of the human trophoblastic plasma protein, pregnancy-specific beta1-glycoprotein (PSbetaG) are detectable in plasma samples from a small percentage of subjects and may be recognised by their capacity to form indistinct immunoprecipitates and give artificially low levels when measured by radioimmunoassay. Urinary PSbetaG shows a marked degree of heterogeneity and is difficult to measure using conventional immunoprecipitation assay techniques. By radioimmunoassay, late pregnancy urinary PSbetaG concentrations have been found to be in the range 0.35 to 6.9 microgram/ml, approximately 3% of the term plasma concentrations.     

Incidence of pregnancy-specific beta-1-glycoprotein in sera of malignant tumor patients.

Pregnancy-specific beta-1-glycoprotein (SP1) is said to be found only in sera of pregnant females. In this paper, SP1 in the sera of malignant tumor patients was investigated by means of Ouchterlony's method and counter immunoelectrophoresis (CIEP). On the examination by Ouchterlony's method, all samples were negative, but by CIEP, 34 out of 90 samples (38 percent) showed positive reaction; 25 out of 61 (41 percent) in males and 9 out of 29 (31 percent) in females. There was no indication that the patients with a certain type of cancer tend to have a high incidence of SP1, but the examination of SP1 in the sera of patients with cancer or suspicious cancer had some clinical significance.     

Particle-counting immunoassay (PACIA) of pregnancy-specific beta 1-glycoprotein, a possible marker of various malignancies and Crohn's ileitis

Pregnancy-specific beta 1-glycoprotein (SP1) was assayed by particle-counting immunoassay (PACIA) with a sensitivity of 1 microgram/L. In serum from 50 men, the SP1 concentration was less than 1 microgram/L, whereas three of the specimens from 46 nonpregnant women had values exceeding 1 microgram/L. In 29% of 950 consecutive patients' sera, SP1 concentrations exceeded 1 microgram/L--in sarcoma (six of six), in malignant hemopathies (101/127, 80%) such as myeloma (20/26, 92%) and acute myeloblastic leukemia (23/27, 90%), and in various other types of cancer (11/19, 58%) except for bronchial epithelioma, which did not lead to any significant increase of SP1 in the five patients examined. The concentration of SP1 was also frequently increased in patients with Crohn's ileitis (28/43, 65%) but not in patients with other inflammatory disorders.     

Pregnancy-specific beta 1-glycoprotein (SP1) determined by means of electroimmunoassay, radial immunodiffusion and nephelometry.

A comparison has been made between rocket-immunoelectrophoresis (RIE), radial immunodiffusion (RID) and automated immunonephelometry (AIP) in the assay of pregnancy-specific beta 1-glycoprotein (SP1) in serum from pregnant women. Using RIE an interaction was demonstrated between the various SP1-reactive molecular populations causing a bias of up to 10%. An interaction corresponding to this phenomenon cannot be demonstrated when using RID and AIP. When correlating the serum-SP1 concentration of samples containing various ratios of SP1-reactive molecules by means of RIE, RID and AIP, it was demonstrated that there was no correlation between the results achieved using one method compared to the results achieved by either of the other methods. The results achieved using one method can therefore exclusively be judged from reference values determined using the same method. The analysis time is essentially shorter with AIP than with RIE and RID.     

Differences in the slopes of dose-response curves measuring pregnancy specific beta 1-glycoprotein (SP1) by means of radioimmunoassay.

Variants of pregnancy specific beta 1-glycoprotein have been described previously. These variants seem to cause artificially low levels when measured by radioimmunoassay. We demonstrate that sera with indistinct precipitates in electroimmunoassay give less steep dose-response curves in radioimmunoassay than do sera with well defined precipitates. Until parallel dose-response curves are demonstrated for all variants, previously published data must be treated with reserve.     

Distribution of SP1, immunoreactivity among different plasma proteins: real molecular heterogeneity or adsorption of SP1-beta to other plasma proteins?

Three SP1-containing factors from pooled term pregnancy sera were subjected to crossed immunoelectrophoresis. New patterns as far as electrophoretic mobilities and shapes of the immunoprecipitates were revealed. The appearance of an additional anodic radioimmunoassayable activity in agarose electrophoresis of mixed SP1-alpha and SP1-beta suggested a binding capacity of SP1-alpha for SP1-beta determinants. In the serum of a single patient at the third trimester of pregnancy we also found two SP1 variants, possessing little radioimmunological reactivity and with crossed immunoelectrophoretic characteristics quite different from those of the 'usual' alpha and beta SP1 forms. These results suggest that, in this particular case, the overall SP1 production cannot be evaluated by competitive binding assay and, that in general, SP1 is a complex antigen the heterogeneity of which can be determined following adsorption of some beta epitopes to another serum protein.     

Measurement of SP1 in samples with varying SP1 alpha: SP1 beta ratios

Mixtures containing a fixed amount of SP1 alpha, increasing amounts of SP1 beta and vice versa have been measured by rocket immunoelectrophoresis, by enzyme immunoassay and by radioimmunoassay. Both proteins have immunological determinants which react with standard antisera to SP1 and the value obtained for any particular mixture is dependent on the ratio of the proteins. The effect of the ratio on measurement by immunoelectrophoresis is different from that on measurement by enzyme immunoassay or radioimmunoassay. As these two proteins exist together in the blood of pregnant women, Results obtained by the two types of assay cannot be compared. Even with the same method the results will depend on the ratio which varies from subject to subject. It is concluded that until specific measurements for SP1 alpha and SP1 beta are designed, clinical application studies must be viewed with reserve.     

Pregnancy-specific beta1-glycoprotein (sp-1): clinical aspects (author's transl)]

Clinical aspects of pregnancy-specific beta1-glycoprotein (SP-1) were evaluated. Serum levels of SP-1 were determined by the method of single radial immunodiffusion (Partigenplatten, Behring-Werke). This method provides a simple and highly reproducible technique for routine clinical assay. SP-1 levels in maternal serum correlated directly with placental weight and birth weight of the infant (p less than 0.01).     

Heterogeneity of the pregnancy-specific β1-glycoprotein (SP1)

The pregnancy-specific β₁-glycoprotein (SP₁) in pregnant serum fractionated by ammonium sulfate precipitation and anion exchange chromatography was investigated by various electrophoretic methods. From these investigations it appears that in addition to the major component SP₁(β₁) three other components are present, termed SP₁(γ), SP₁(α₂² and SP₁α₂¹) on the basis of their electrophoretic mobilities.The isoelectric point (pI), estimated by isoelectric focusing, and the relative molecular mass (M_r), estimated by Sephadex G-200 gel filtration chromatography, are for SP₁(γ): pI 2.9–4.9, M_r ～ 80000; SP₁(β): pI 2.5–4.5, M_r ～ 80000; SP₁(α₂²): pI ? 6, M_r ? 200000; SP₁(α₂¹): pI < 3, M_r ～ 30000. Based on the interaction with phenyl-Sepharose and concanavalin A all four species of SP₁ are amphiphilic glycoproteins.