A rapid and inexpensive laser nephelometric assay for plasma pregnancy specific β1-glycoprotein levels

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Clinica Chimica Acta, 90 (1978) 87-91 @ Elsevier/North-Holland Biomedical Press

CCA 9854

A RAPID AND INEXPENSIVE LASER NEPHELOMETRIC ASSAY FOR PLASMA PREGNANCY SPECIFIC PI-GLYCOPROTEIN LEVELS

P.J. WOOD a**, DEBORAH COCKETT b and P. MASON b Departments of a Chemical Pathology Hospitals, Southampton (U.K.)

and b Human Reproduction,

Southampton

University

(Received June 3rd, 1978)

Summary A laser nephelometric method for the measurement of maternal plasma pregnancy specific /3,-glycoprotein levels is described. Fifty plasma specimens can be analysed within four hours at a low reagent cost. The assay has been used to determine the range of plasma pregnancy specific p,-glycoprotein concentrations in 212 normal pregnancies of from 31 to 41 weeks gestation. Introduction Pregnancy specific P1-glycoprotein (PS&G) is a P1-glycoprotein with a molecular weight of 90 000, which is synthesised by the placental syncytiotrophoblast cells. PS&G is secreted into the maternal circulation, where it has a half life of approximately 30 to 40 h [l]. The alternative names schwangerschaftsspezifisches protein 1 (SP,) and pregnancy associated plasma protein-C (PAPP-C) have been proposed by investigators in Germany [2] and America [ 31 who have isolated this glycoprotein. Studies of maternal plasma PSfllG levels may prove to be of value in the assessment of risk to the foetus, particularly in cases of growth retardation [4-61. Of the methods which are available for PS&G measurement, radial immunodiffusion [ 1,4] takes one to three days, while ‘rocket’ electroimmunodiffusion [ 1,7] requires a run of at least 14 h. Both these techniques have limited sample capacity. Recently, radioimmunoassays for PS&G have been developed [6,8]. These have advantages of increased sensitivity over existing methods, but require a total incubation time of 24 h or more. PSPIG concentrations in the maternal circulation may be as high as 30 mg/ * Correspondence

should be addressed to: Dr. P.J. Wood, Department of Chemical Pathology, Southampton General Hospital, Tremona Road, Southampton SO9 4XY. U.K.

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100 ml at term [4,5]. Circulating PSP,G levels are therefore well within the compass of the laser nephelometric methods which have recently been developed for the measurement of the immune globulins and other serum proteins [g-11]. It was therefore decided to develop an assay system for PSfl,G using the laser nephelometer. The method was based on the analysis of plasma rather than serum since it was intended to measure PSP,G levels in plasma specimens which had been collected for oestriol and human placental lactogen estimations. Problems with very high ‘blank’ nephelometry readings for some plasma specimens were overcome by the introduction of a protamine sulphate precipitation step into the assay protocol. Materials and methods Radial immunodiffusion

Radial immunodiffusion analyses for comparison metric method were performed with ‘M-Partigen’ Behringwerke AG) using a three day assay period.

with the laser nepheloplates (Code OTCE 03;

Laser nephelometer

PSPIG concentrations were determined with the Hyland ‘PDQ’ laser nephelometer. This instrument utilises a helium gas laser tube to produce a 0.9-mm diameter beam of radiation with wavelength 632.8 nm. Light, scattered by the immune complexes in the test specimen, is measured at a forward scattering angle of 31”. The use of this scattering angle reduces ‘background’ scatter readings to a minimum. The instrument was operated according to the manufacturer’s instructions, using sensitivity range 3 and a computation period of 15 sec. Nephelometer cuvettes, supplied by Hyland Ltd., were re-used after a rigorous washing in Decon 90 detergent (BDH Ltd.) tap water (3 rinses) and distilled water (3 rinses). Reagents

PS&G standard (Code OTFL 03) and rabbit anti-PSPIG antiserum (Code OTPI 05) were purchased from Behringwerke AG. The assay buffer consisted of phosphate buffer (0.05 mol/l, pH 7.4) containing sodium chloride (0.1 mol/l) and polyethylene glycol 6000 (40 g/l; BDH Ltd.). Protamine sulphate reagent for the treatment of plasma samples was prepared by the dilution of 1 volume of 1% protamine sulphate solution for injection (Boots Ltd.) with four volumes of assay buffer. Working antiserum reagent was prepared by making a 1 in 200 dilution of neat antiserum in assay buffer at least 30 min before use. Both the working antiserum reagent and the assay buffer, added to ‘tests’ and ‘blanks’ respectively, were filtered through a 0.22 (urn Millipore membrane filter before addition to assay cuvettes. Plasma specimens

Blood samples (10 ml) were obtained by venepuncture and dispensed into polystyrene lithium heparin tubes (10 I.U. heparin/ml plasma). After centrifugation, plasma was separated and stored at -20°C.

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Assay protocol Plasma aliquots (50 ~1) of specimens to be analysed were diluted with protamine sulphate reagent (500 ,ul) into 1.5 ml capacity polypropylene microcentrifuge tubes (Sarstedt Ltd.) with the aid of an autodiluter (LKB Instruments Ltd.; Type 2075 autodiluter). A 50-~1 aliquot of PSP,G standard (21 mg/ 100 ml) was diluted in the same way. Three doubling dilutions of this top standard were then made in the protamine sulphate reagent to give standards equivalent to 10.5, 5.25 and 2.63 mg/lOO ml plasma PSPIG. Test plasma and standard dilutions were vortex-mixed and centrifuged for 1 min on a ‘microfuge’ (Gelman Hawkesley Ltd.; microhaematocrit centrifuge). Aliquots (50 ~1) of the clear supernatant solutions were then dispensed into nephelometer cuvettes with 1.0 ml of working ~tiserum reagent (for ‘tests’) and 1.0 ml of assay buffer (for ‘blanks’), again using the LKB autodiluter. Cuvette contents were mixed thoroughly and incubated for 1 h at 37°C. After cooling, the cuvettes were vortex-mixed and the relative light scatter (RLS) reading for each specimen was obtained. Each ‘test’ result was corrected for the background scatter determined in the corresponding ‘blank’ cuvette. The working antiserum reagent was used to zero the instrument. Plasma samples with PSp,G results in excess of 21 mg/lOO ml were reanalysed using a 25 ~1 plasma aliquot. Validation of the laser ~ep~elo~e~ric method A study of incubation conditions has shown that the formation of the W&G antibody complex was almost complete after an incubation of 1 h at 37”C, with further small increases in complex formation for incubations of 2 and 3 h. A polyethylene glycol concentration of 40 g/l in the assay buffer gave an optimum ratio of test to blank RLS readings. The capacity of excess antigen to reduce the RLS level was investigated. A plasma PS&G concentration in excess of 45 mg/lOO ml would be required to reduce the RLS reading to that given by the 21 mg/lOO ml PSplG standard. The underestimation of test results due to excess antigen effects should not therefore be a problem with this assay system. Plasma PSPIG concentrations, measured in 6 normal men and 6 non-pregnant women were below the assay detection limit, which was approximately 1 mg/ 100 ml for the routine assay. Analysis of a test specimen, undiluted, and at dilutions of 1 in 2, 1 in 4 and 1 in 8 in male plasma gave results of 14.5, 15.0, 14.4 and 14.4 mg/lOO ml PS&G respectively. Results were therefore independent of the dilution of the test specimen. A comparison of PS&G levels determined in 27 plasma specimens by radial immunodiffusion and laser nephelometric methods demonstrated a close correlation between the two techniques (Fig. 1). The nephelometry calibration curve showed little variation from batch to batch. Fig. 2 illustrates the composite data for calibration curves from 12 consecutive batches of tests. Between-batch coefficients of variation for the analysis of two plasma samples in 14 assays were 5.1% (mean PSfl,G level 11.3 mgj 100 ml) and 5.7% (mean PS&G level 20.4 mg/lOO ml).

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Fig. 1. Correlation between plasma PSplG levels measured by radial immunodiffusion (X) and laser nephefometry (Y) in 27 subjects. Regression equation: Y = 0.978X + 1.446: Correlation coefficient (r) = 0.977.

Data for normal pregnancies were obtained from the analysis of blood samples taken at routine antenatal examination. Patients were followed-up and were excluded from the study if they developed any pregnancy complications, or were delivered of babies with a birth weight less than the 10th or greater than the 90th percentiles. The mean and 95th percentile range for plasma PS&G concentrations in 212 normal pregnancies of from 31 to 41 weeks gesta-

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Fig. 2, Reproduc~b6ity of the calibration curve. Means (t S.E.i’vf.) for the standard readings from 12 batches of tests, expressed as a percentage of the 21 m&l00 ml standard RLS reading. Fig, 3. Third trimester plasma PSPIG levels in 212 normal pregnancies expressed as the mean and 96% confidence range.

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tion are shown in Fig. 3. Plasma PS&G levels rose from a mean of 11.6 mgi 100 ml at 31 weeks gestation to a mean of 18.2 mg/lOO ml at 41 weeks. Discussion The heparin antagonist protamine sulphate has been successful in overcoming difficulties of occasional very high blank RLS readings when plasma specimens were analysed. The cause of the interference seen in some plasma specimens has not been identified, although the effectiveness of protamine sulphate treatment may be related to its ability to precipitate fibrinogen. The laser nephelomet~~ assay for plasma PSPIG levels has been shown to be precise and to give results in close agreement with those obtained by radial immunodiffusion, The adoption of a l-h incubation period at 37”C, and the use of autodilution and rapid centxifugation steps enable a batch of 50 plasma specimens to be analysed within 3-4 h. The cost of reagents for the assay is low, especially when batches of 20-50 tests can be performed. The method is thus well suited to the monitoring of plasma PS&G levels in the last trimester of pregnancy. Preliminary studies with the Beckman rate nephelometer indicate that the assay can be adapted to a rate nephelometric system which will provide a PSfllG result within l-2 min. The PS&G levels found in 212 normal pregnancies in the last trimester show a similar trend and range to those reported for radial immunodiffusion and radioimmuno~say methods 14-61. Research into the value of PS&G analyses in the assessment of foeto-placental function in high risk pregnancies is continuing. Acknowledgements We thank Dr. J. Smith and Mr. S. Redding of the Department of Immunology for their kind co-operation in the use of the Hyland laser nephelometer. References 1 2 3 4 5 6 7 8 9 10 11

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