Journal of Immunological Methods, 129 (1990) 97-103 Elsevier

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JIM05548

A rapid and robust particle-enhanced turbidimetric immunoassay for s e r u m ~ 2 microglobulin E.A. Medcalf 1, D.J. N e w m a n 1, A. Gilboa 1, E.G. G o r m a n 2 and C.P. Price 1 t Department of Clinical Biochemistry, The London Hospital Medical College, Turner Street, Whitechapel, London El 2AD, U.K., and 2 E.L du Pont de Nemours& Co., Building 700, Glasgow Site, Wilmington, DE 19898, U.S.A. (Received 14 August 1989, revised received 30 November 1989, accepted 5 January 1990)

A rapid particle-enhanced turbidimetric immunoassay (PETIA), for the measurement of serum fl2-microglobulin is described. The method has a working range of 0.2-40 mg/l, with good precision and a correlation coefficient of 0.97 when compared with an established radioimmunoassay method. One of the major advantages of this assay is the stability of the calibration curve (up to at least 20 months). This, and the fact that no pretreatment of serum samples is necessary, makes the assay ideally suited for all types of routine determination. Key words: Particle-enhanced turbidimetric immunoassay; Serum f12 microglobulin; Calibration stability

Introduction The protein /32 microglobulin (fl2M), was first isolated from the urine of patients with Wilson's disease and chronic cadmium poisoning, by Bergg~d and Beam (1968). f12M is a low molecular weight protein (MW = 11,815), consisting of 100 amino acid residues. It is found on the surface of nearly all nucleated cells, non-covalently linked to the heavy chains of the class I histocompatability antigens. The protein on the plasma membrane surface is in equilibrium with the surrounding medium, and can be found at low concentrations in serum, urine and other body fluids (Cunningham et al., 1973; Plesner, 1980; Sadler et al., 1987). Free B2M is filtered at the glomerulus and subsequently almost totally reabsorbed and

Correspondence to: D.J. Newman, Department of Clinical Biochemistry, The London Hospital Medical College, Turner Street, Whitechapel, London E1 2AD, U.K.

catabolised by the proximal tubule cells. Normal levels of serum f12M are in the range 0.9-2.5 mg/1 (Bernard et al., 1981), elevations being found in patients with: (a) increased nucleated cell turnover, as in malignancy, e.g., multiple myelomatosis and in non-malignant disorders, e.g., acquired immune deficiency syndrome (AIDS), and (b) reduced glomerular filtration rate. Increases in urinary f12M may be due to increased load, thereby exceeding the renal tubule cell capacity to reabsorb the protein, or to tubular cell damage, whereby little can be reabsorbed. Urinary fl2M is rapidly degraded at acidic pHs, and so is now rarely measured due to the problems involved in ensuring that all urine collections are maintained at pH > 6.0 (Plesner, 1980). Assays already developed for the determination of serum fl2M, include a variety of techniques including radioimmunoassay (RIA), radial immunodiffusion (RID) and more recently nephel0metric, turbidimetric, fluorescent and enzyme immunoassay (Fujirebio Inc., 1984; Tachibana et al., 1984; Playford et al., 1985; Bjerrum and Birgens,

0022-1759/90/$03.50 © 1990 Elsevier Science ~ublishers B.V. (Biomedical Division)

98 1986; Tillyer and Rawal, 1988). The method described in this paper has the advantage of being non-isotopic, and previous experience with C-reactive protein (CRP) (Price et al., 1987) using the same particles resulted in a very robust assay with good reagent stability. A turbidimetric rather than nephelometric method was chosen in order to avoid the necessity to pretreat the samples and to take account of the availability of suitable instrumentation in a standard laboratory.

Materials and instrumentation

Latex particles The latex particles were based on a poly-vinyl naphthalene core, with a chemically reactive shell of chloro-methyl-styrene (Litchfield et al., 1984) and were kindly provided by E.I. du Pont de Nemours (Wilmington, DE, U.S.A.), as a 10% (w/v) suspension of 40 nm, 70 nm and 150 nm diameter particles. Throughout the development work a 0.5% concentration of these particles was used for the preparation of the antibody/particle reagent.

Antibody Affinity purified rabbit anti-human fl2M (code no: A072) was a gift from Dakopatts (Copenhagen, Denmark). Prior to use the antibody was dialysed in 15 mmol/l phosphate buffer pH 7.4, over 24 h.

Buffers All material used was AnalaR grade (BDH, Dagenham Essex, U.K.) unless otherwise specified. Coupling buffer. 15 mmol/l phosphate buffer pH 7.4 containing 0.05% (v/v) GAFAC RE610 (GAF, Wythenshaw, Manchester, U.K.) and 0.1% sodium azide.

Stock buffer for antibody/particle reagent. 200 retool/1 glycine pH 7.4 containing 0.1% sodium azide. Wash buffer for antibody/particle reagent. 50 mmol/l glycine pH 7.4.

Working antibody/particle reagent diluent. 5 mmol/l glycine pH 7.4 containing 0.1% sodium azide.

Final assay buffer.. 340 mmol/l borate/KCl pH 10.0 containing 3.38% PEG 8000 (Union Carbide, Danbury, CN), 1% bovine serum albumin (Sigma, Poole, U.K.), and 0.1% sodium azide. Standards Purified ]~2M was obtained from SCIPAC (Sittingbourne, Kent, U.K.), and diluted in either 0.9 % saline (containing 0.001% BRIJ 35 (BDH)), or horse serum (Sigma) to give concentrations of 20, 10, 5, 2.5, 1.25 and 0.625 mg/l.

Samples Serum samples were collected from healthy adults and from patients with either decreased glomerular function, increased immunoglobulin, or increased rheumatoid factor concentrations.

Instrumentation All the immunoassay reactions were monitored on the Multistat 111 microcentrifugal analyser (CFA), (Instrumentation Laboratory Warrington, U.K.), using a 340 nm filter at a temperature of 30 + I°C. A cuvette with a pathlength of 0.5 cm was used in the Multistat analyser rotors. Curve fitting was carried out manually.

Results

Synthesis and optimisatwn of antibody/particle reagent The synthesis of the antibody/particle reagent was optimised for maximum aggregation. This process involved (i) determining the optimum ratio of antibody to particle, to provide the most sensitivity within the required assay range, and (ii) determining the optimum size of particle (from those available), for use in the coupling and to provide absorbance changes over a short assay time, within the optical capability of the instrument (Figs. 1 and 2). It would be necessary to undertake the antibody : particle optirnisation procedure for each new batch of antisera used. In each case the antibody and particles were mixed at their respective concentrations (diluted in the coupling buffer), e.g., 1 mg antibody per mt of 0.5% particles, and coupled overnight at 37 °C

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b o r a t e / K C l buffer p H 10.0 at a final concentration of 100 m m o l / l in the reaction cuvette.

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A rapid and robust particle-enhanced turbidimetric immunoassay for serum beta 2 microglobulin.

A rapid particle-enhanced turbidimetric immunoassay (PETIA), for the measurement of serum beta 2-microglobulin is described. The method has a working ...
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