Journal oflmmunological Methods, 144 (1991) 49-55 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50

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JIM 07000

Latex immunoassay of transferrin in urine A. Bernard, K.S. Chia a n d R. L a u w e r y s Unit~ de Toxicologie Industrielle et de M~decine du Travail, Universit~ Catholique de Louvain, Brussels, Belgium (Received 28 February 1991, revised received 20 May 1991, accepted 28 June 1991)

A fully automated assay based on latex particle agglutination has been developed for the determination of transferrin in urine. The assay consists of incubating urine samples for 30 min at 50 ° C with latex particles on which an anti-transferrin antibody has been adsorbed and then quantifying the residual unagglutinated particles with an optical particle counter. The measurable concentration range is 0.5-10 /~g/1. Intra- and interassay coefficients of variation varied from 2.1 to 11.8% and the recovery averaged 102.5% (SD 13.8). Correlation coefficients between transferrinuria and albuminuria were 0.79 in 100 healthy subjects and 0.97 in 90 subjects with established or suspected glomerular involvement. Transferrin and albumin were stable in urine over the pH range 5 - 9 for 24 h at 37 o C, or for 2 weeks at 4 o C or room temperature. Transferrin was, however, less stable than albumin during prolonged storage of urine at 4 ° C. The present latex immunoassay of transferrin may also be adapted to give a turbidimetric reading in which agglutination is quantified by measuring the decrease of absorbance at 360 nm. Key words: Transferrinuria; Albuminuria; Latex agglutination test; Particle counting assay; Turbidimetry

Introduction An abnormally increased urinary excretion of albumin in the absence of elevated proteinuria (micro- or paucialbuminuria) is now widely used for the early detection of glomerular involvement in diseases such as diabetes, systemic lupus or hypertension (Viberti et al., 1982; Mogensen, 1987; Terai et al., 1987). Evidence has been presented recently that transferrinuria is a more sensitive index than albuminuria of the early renal alterations associated with diabetes mellitus in humans (Bernard

Correspondence to: A. Bernard, Unit~ de Toxicologie Industrielle et de M6decine du Travail, Universit6 Catholique de Louvain, Clos Chapelle-aux-Champs, 30.54., 1200 Brussels, Belgium.

et al., 1988, 1990a; Cheung et al., 1989; McCormick et al., 1990a) and also in experimental animals (Bernard et al., 1988; McCormick et al., 1990b; Cardenas et al., 1991). This higher sensitivity of urinary transferrin to increased glomerular leakiness is probably also valid for other types of nephropathies such as that caused by chronic cadmium poisoning (Bernard et al., 1990b). Transferrin occurs in urine at concentrations which are about ten times lower than that of albumin. Transferrin is, therefore, much less easily quantifiable in urine than albumin. The immunoturbidimetric methods described thus far (Rifai et al., 1988; McCormick et al., 1989) lack sensitivity and do not permit the accurate determination of transferrin in urine without preliminary concentration. In this paper, we describe a sensitive and fully automated immunoassay for urinary transferrin and we report data on the

50

distribution, stability and correlations of transferrin and albumin in normal and pathological urines.

Materials and methods

Urine specimens Spot urine samples were collected from the following groups of subjects (all males, ages 20-60 years): 100 subjects in good health, 50 diabetics, ten workers exposed to cadmium and 17 workers exposed to solvents. These samples were stored at 4 °C until analysis.

Reagents Latex.

Polystyrene latex particles (0.845 + 0.014 (SD) /~m in diameter, E S T A P O R K080) were kindly donated by Dr. J.C. Daniel (Rh6nePoulenc Industrie, Aubervilliers, France). These particles were supplied as an aqueous suspension at a concentration of 100 g/1. They were used directly without washing. Antibodies. The immunoglobulin fractions of rabbit antibodies against human transferrin (A061) and human albumin (A001) were purchased from Dako, Glostrup, Denmark. Standards. The assays for transferrin and albumin were calibrated with purified proteins from Sigma Chemical Co., St. Louis, MO 69178. Buffers. The buffer used for diluting the standards and urines consisted of a glycine-buffered saline comprising (per liter) 0.1 mol of glycine, 0.17 mol of NaC1, 7.6 mmol of NaN 3 and 1 g of bovine serum albumin (Sigma) and adjusted to pH 9 with NaOH.

Preparation of latex reagent The antibody coating of latex particles and their stabilization with bovine serum albumin was performed as follows: 10 lzl of Dako antibody were dispersed in 1 ml of a glycine-buffered saline containing per liter 10 mmol of glycine and 17 mmol of NaC1 per litre. After adjusting the pH to 9, 50 Ixl of the latex suspension were added. After 7 min of incubation, the latex suspension was dispersed in 10 ml of distilled water containing 50 mg of bovine serum albumin (BSA, Calbiochem, San Diego, CA 92112, no. 126609). This

preparation was then sonicated for 20 s with a Virsonic 300 Ultrasonifier (power 3) (Virtis Company, NY, 15525) before adding 1 ml of a glycine-NaOH buffer, 1 mol/1, pH 10.1. This preparation which was sufficient for 100 assays was stable for 1 day at room temperature. It was also possible to store it for a few days at 4 ° C but in that case, it was resonicated as above before use.

Assay The assay was carried out automatically in a continuous flow system which has been described in detail previously (Bernard and Lauwerys, 1983). This system was modified to include a Tecan 505 manipulator (Tecan US, Chapell Hill, NC 27514) which automatically diluted the urine samples and the standards. Urine samples were routinely analyzed at two different dilutions (20 and 40fold) to detect a possible postzone effect due to an excess of antigen. Aliquots of the latex reagent were incubated with the protein standard or the diluted urine for 25 min in a mixing coil heated at 50 ° C. After dilution of the reagent stream, residual unagglutinated latex particles were counted with a Technicon Autocounter. Turbidimetry was also investigated as an alternative to particle counting. For this mode of reading, the incubation was carried out as follows. 50 Izl of standard or diluted urine were pipetted in duplicate into 5 ml glass test tubes. Tubes containing 5 0 / z l of GBSBSA were regularly spaced in each series of determinations (zero standards). Then, 50 /zl of latex reagent were added to each tube, rapidly mixed and the mixture incubated at 45 ° C in a shaking water bath (60 cycles/min, 5.5 cm amplitude). After 60 min incubation, the tubes were removed from the water bath and the agglutination was stopped by gently dispersing the latex particles in 1 ml of a solution containing 0.27 mol of NaCI and 1 ml of Tween 20 per litre. The latex aggregates formed during incubation were sufficiently stable to permit the storage of the tubes for several hours at room temperature or for 24 h at 4 °C without significant change in the agglutination pattern. The decrease in the absorbance at 360 nm of the latex suspension was measured with a Beckman speetrophotometer. Each tube

51 TABLE I

individually was inverted 2 - 3 times before transferring its contents to a cuvette (5 mm light path). The agglutination was expressed as the difference between the absorbance of the 'zero standard' and that of the latex incubated with transferrin.

B E T W E E N R U N P R E C I S I O N OF L A T E X I M M U N O A S SAY F O R T H E D E T E R M I N A T I O N O F M I C R O T R A N S FERRINURIA Urines were diluted 40 times, n = 7 determinations over 2 weeks

Other methods Albumin was also determined by latex immunoassay as reported previously (Bernard and Lauwerys, 1983a,b). Creatinine was measured in urine by Jaffe's method (Henry, 1965).

Mean (/2g/1)

SD

CV(%)

155 384 612 876 1,450

12.2 45.4 32.8 84.4 64.9

7.9 11.8 5.4 9.6 4.5

Results

Assay performance Fig. 1 shows composites of seven standard curves run successively over 2 h or of 20 curves obtained over a period of 2 months with different batches of antibody-coated latex particles. The working range extended from about 0.5 to 50 /xg/1. As urine samples must be diluted at least five times for an accurate determination, the limit of detection was in practice around 2.5/zg/1. The higher between-run variability of the agglutination curves was due mainly to variations in the agglutinability of the latex reagent (prepared

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freshly) and to the changes which unavoidably occurred with time in the flow rates of the manifold. The within-run precision was tested by assaying five replicates of four urines containing 34, 53, 250 and 440 /~g of transferrin per liter. The CVs were 10.5, 2.1, 6 and 4.8%, respectively. The between run precision was assessed on five urines analyzed seven times over 2 weeks using on each occasion a freshly prepared latex reagent. The CVs ranged from 4.5 to 11.8% (Table I).

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Concentretlon of trensferrln (pgll) Fig. 1. Standard curve of transferrin measured by latex immunoassay (particle counting reading). The curve is a composite of seven curves obtained sequentially during the same run (within-run) or of 20 curves obtained over 2 m o n t h s with different batches of antibody-coated latex particles (between-run). Vertical bars show _+ 1 SD.

52 0.35

To test the accuracy of the assay, purified transferrin was added to eight different urines in order to increase the transferrin concentration by 1 mg/1. The recovery averaged 102.5% (SD, 13.8). The turbidimetric reading produced standard curves which were less sensitive than those obtained by particle counting (Fig. 2). However, the limit of detection (25 /zg/1 on five-fold-diluted urines) remained sufficiently low to quantify transferrin in all normal urine samples. The between assay CVs calculated on four urines analyzed five times over 2 weeks ranged from 6.1 to 9.5%.

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Latex immunoassay of transferrin in urine.

A fully automated assay based on latex particle agglutination has been developed for the determination of transferrin in urine. The assay consists of ...
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