Clin Biochem, Vol. 25, pp. 245-249, 1992 Printed in the USA. All rights reserved.
0009-9120/92 $5.00 + .O0 Copyright © 1992 The Canadian Society of Clinical Chemists.
Measurement of Serum 132-Microglobulin by a Latex Nephelometric Immunoassay F. IGUAZ, 1 J. NAVAL, 2 and L. BORQUE 1 1Laboratorio Central, Hospital "San Millan," Logrofio, Spain and 2Departamento de Bioqufmica, Facultad de Ciencias, Zaragoza, Spain A rapid and simple technique for the measurement of beta2microglobulin on the Behring nephelometer was developed using a commercially available latex-anti [32-microglobulin. This is a fully automated assay and no pretreatment of sample is necessary. An excellent correlation with radioimmunoassay (r = 0.972) and time-resolved fluoroimmunoassay (r = 0.914) was obtained. Haemolysis, lipemia, bilirubin, and rheumatoid factor do not cause interference because of the high dilution of samples and the use of Fab fragments of the antibody. The analytical range extends from 0.6 to 10.7 mg/L. Between-run imprecision (CV) ranged from 6 to 8%.
KEY WORDS: B2-microglobulin; nephelometric assay; latex; reference values; rheumatoid factor. Introduction
eta2-microglobulin ([32-M) is a single polypep-
tide chain of 100 aminoacids, M r 11,815, first B isolated by Bergg&rd and Bearn (1). It comprises the
light chain of histocompatibility antigens class I (2). The protein is released to the extracellular compartment and is found in low concentrations in serum, urine, and other body fluids (3,4). [32-M is eliminated by glomerular filtration and almost totally reabsorbed and catabolized by the proximal tubule cells (3). Determination of B2-M is of clinical interest in patients with decreased glomerular filtration rate, hemodialysed subjects (5), renal transplantation (68), and in inflammatory (9-14), malignant (15-17), and immunological disorders (18-22). Increased concentrations of ~ 2 - M in urine may be due to increased filtered load or to tubular cell damage. A variety of techniques including radioimmunoassay (RIA) (23), ELISA (24), time-resolved fluoroimmunoassay (25), nephelometry (26), and turbidimetry (27-29) have been used. The method reported in this paper has been developed using a commercially available latex-anti[32-M manufac-
Correspondence: Fernando Iguaz, Hospital "San Millan" Laboratorio Central, c/Autonomia, 3, 26004 Logrofio, Spain. Manuscript received November 28, 1991; revised March 13, 1992; accepted March 18, 1992. CLINICAL BIOCHEMISTRY, VOLUME 25, A U G U S T 1992
tured for a turbidimetric system (LA-2000 Eiken Chemical Co Ltd Tokyo, Japan). We used an instrument that is often available in clinical laboratories. Only 40 ~L of reagent/test are required, at a cost of less than $1 per test. Materials and methods INSTRUMENTATION
We used a Behring Nephelometer Analyzer (BNA, B e h r i n g w e r k e AG, M a r b u r g , G e r m a n y ) for all nephelometric measurements. This is a fully automated system, equipped with a light diode t h a t emits at 840 nm. The scattered light is measured at a solid angle of 12-24 °. SAMPLES
Serum was separated within 3h of collection, at room temperature, after centrifugation at 1000 x g for 6 min and stored at - 2 0 °C. Samples from 53 male and 40 female blood donors, 18-64 years old, were used to develop a reference range. Sera from patients with different diseases (lymphomas, n = 24; chronic renal insufficiency, n = 25; seropositive HIV, n = 55) were also tested. REAGENTS
Polystyrene latex particles (0.1% w/v) of 0.126 ~m diameter coated with anti-[32-M (Fab fragments), and the lyophilized standard were obtained from Eiken Chemical Co Ltd, Tokyo, Japan. The latex particles were stored at 4 °C. We used a 0.15 mmol/L NaC1 solution for automatic dilutions of samples and standards. Haemoglobin was obtained from freshly lysed red blood cells. Bilirubin was purchased from Sigma (St Louis, MO, USA) and Intralipid from Kabivitrum, Stockholm, Sweden. Rheumatoid factor, IgM type, was isolated from a pool of patients with a high level of RF by a classical method involving precipitation, dialysis, gel filtration, and lyophilization techniques (29). TM
245
IGUAZ, NAVAL, AND BORQUE TABLE 2 Within-Run and Between-Run Reproducibility of the Immunonephelometric Method
TABLE 1 Settings of the Immunonephelometric Method for ~2-Microglobulin Sample vol, ~L Reagent 1 vol, IxL Reagent 2 vol, jxL Reaction buffer vol (1) Reaction buffer vol (2) Measuring time, min Standard No. of standard points First dilution Deviation allowed, % Validity, days Units Measuring range Lower level Upper level
n
Mean (mg/L)
CV (%)
Within-run
20 20 20
0.96 2.26 6.32
6.3 2.7 3.3
Between-run
10 10 10
0.98 2.15 5.86
7.7 6.1 5.8
Sample dilution 1:100 Minimal dilution 1:20 ~2M-Latex
40 40 0 60 60 6
NaC1, 0.15 mmol/L NaC1, 0.15 mmol/L Fixed time ~2-microglobulin
5 1:20 5 7 mg/L
within a single calibration run. Inter-assay imprecision was calculated from 10 different calibration runs (in 10 consecutive weeks).
0.6 10.7
LINEARITY ASSAY PROCEDURE
The latex suspension was warmed for 30 min at 37 °C before assay. The calibration curve was obtained by automatic two-fold dilutions (from 1/20 to 1/320) of the standard to produce a concentration range from 0.6 to 10.7 mg/L. The instrument program is shown in Table 1. The sample is automatically prediluted to 1:100. Diluted samples or standards (40 ~L) are added to 120 ~L of saline and 40 ~L of latex reagent and mixed. The scattered light is measured after 10 s and after 6 min. The instrument plots incremental signal of standards versus their concentrations, using a logit-log calibration curve. Incremental signals of the samples are converted into concentrations by interpolation. IMPRECISION
Three pools of sera at different levels of ~2-M were prepared for imprecision studies. Intra-assay imprecision was determined by replicate testing (n = 20)
Linearity was studied by serial two-fold dilution of a pool of sera with saline, over the range of 0.8 mg/L to 30 mg/L. INTERFERING SUBSTANCES
The interference study was developed according to Glick e t a/.(30). The maximum concentrations of interfering substances added to a pooled serum were: 12 g/L of haemoglobin, 1.71 mmol/L of bilirubin, Intralipid 2% (v/v), and 1100 IU/mL of rheumatoid factor. COMPARISON PROCEDURES
For comparative studies, we used an RIA method (Phadebas ~2-microglobulin, Pharmacia, Uppsala, Sweden) and a time-resolved fluoroimmunoassay procedure (DELFIA ~2-micro, Pharmacia, Uppsala, 35 --J
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0009-9120/92 $5.00 + .O0 Copyright © 1992 The Canadian Society of Clinical Chemists.
Measurement of Serum 132-Microglobulin by a Latex Nephelometric Immunoassay F. IGUAZ, 1 J. NAVAL, 2 and L. BORQUE 1 1Laboratorio Central, Hospital "San Millan," Logrofio, Spain and 2Departamento de Bioqufmica, Facultad de Ciencias, Zaragoza, Spain A rapid and simple technique for the measurement of beta2microglobulin on the Behring nephelometer was developed using a commercially available latex-anti [32-microglobulin. This is a fully automated assay and no pretreatment of sample is necessary. An excellent correlation with radioimmunoassay (r = 0.972) and time-resolved fluoroimmunoassay (r = 0.914) was obtained. Haemolysis, lipemia, bilirubin, and rheumatoid factor do not cause interference because of the high dilution of samples and the use of Fab fragments of the antibody. The analytical range extends from 0.6 to 10.7 mg/L. Between-run imprecision (CV) ranged from 6 to 8%.
KEY WORDS: B2-microglobulin; nephelometric assay; latex; reference values; rheumatoid factor. Introduction
eta2-microglobulin ([32-M) is a single polypep-
tide chain of 100 aminoacids, M r 11,815, first B isolated by Bergg&rd and Bearn (1). It comprises the
light chain of histocompatibility antigens class I (2). The protein is released to the extracellular compartment and is found in low concentrations in serum, urine, and other body fluids (3,4). [32-M is eliminated by glomerular filtration and almost totally reabsorbed and catabolized by the proximal tubule cells (3). Determination of B2-M is of clinical interest in patients with decreased glomerular filtration rate, hemodialysed subjects (5), renal transplantation (68), and in inflammatory (9-14), malignant (15-17), and immunological disorders (18-22). Increased concentrations of ~ 2 - M in urine may be due to increased filtered load or to tubular cell damage. A variety of techniques including radioimmunoassay (RIA) (23), ELISA (24), time-resolved fluoroimmunoassay (25), nephelometry (26), and turbidimetry (27-29) have been used. The method reported in this paper has been developed using a commercially available latex-anti[32-M manufac-
Correspondence: Fernando Iguaz, Hospital "San Millan" Laboratorio Central, c/Autonomia, 3, 26004 Logrofio, Spain. Manuscript received November 28, 1991; revised March 13, 1992; accepted March 18, 1992. CLINICAL BIOCHEMISTRY, VOLUME 25, A U G U S T 1992
tured for a turbidimetric system (LA-2000 Eiken Chemical Co Ltd Tokyo, Japan). We used an instrument that is often available in clinical laboratories. Only 40 ~L of reagent/test are required, at a cost of less than $1 per test. Materials and methods INSTRUMENTATION
We used a Behring Nephelometer Analyzer (BNA, B e h r i n g w e r k e AG, M a r b u r g , G e r m a n y ) for all nephelometric measurements. This is a fully automated system, equipped with a light diode t h a t emits at 840 nm. The scattered light is measured at a solid angle of 12-24 °. SAMPLES
Serum was separated within 3h of collection, at room temperature, after centrifugation at 1000 x g for 6 min and stored at - 2 0 °C. Samples from 53 male and 40 female blood donors, 18-64 years old, were used to develop a reference range. Sera from patients with different diseases (lymphomas, n = 24; chronic renal insufficiency, n = 25; seropositive HIV, n = 55) were also tested. REAGENTS
Polystyrene latex particles (0.1% w/v) of 0.126 ~m diameter coated with anti-[32-M (Fab fragments), and the lyophilized standard were obtained from Eiken Chemical Co Ltd, Tokyo, Japan. The latex particles were stored at 4 °C. We used a 0.15 mmol/L NaC1 solution for automatic dilutions of samples and standards. Haemoglobin was obtained from freshly lysed red blood cells. Bilirubin was purchased from Sigma (St Louis, MO, USA) and Intralipid from Kabivitrum, Stockholm, Sweden. Rheumatoid factor, IgM type, was isolated from a pool of patients with a high level of RF by a classical method involving precipitation, dialysis, gel filtration, and lyophilization techniques (29). TM
245
IGUAZ, NAVAL, AND BORQUE TABLE 2 Within-Run and Between-Run Reproducibility of the Immunonephelometric Method
TABLE 1 Settings of the Immunonephelometric Method for ~2-Microglobulin Sample vol, ~L Reagent 1 vol, IxL Reagent 2 vol, jxL Reaction buffer vol (1) Reaction buffer vol (2) Measuring time, min Standard No. of standard points First dilution Deviation allowed, % Validity, days Units Measuring range Lower level Upper level
n
Mean (mg/L)
CV (%)
Within-run
20 20 20
0.96 2.26 6.32
6.3 2.7 3.3
Between-run
10 10 10
0.98 2.15 5.86
7.7 6.1 5.8
Sample dilution 1:100 Minimal dilution 1:20 ~2M-Latex
40 40 0 60 60 6
NaC1, 0.15 mmol/L NaC1, 0.15 mmol/L Fixed time ~2-microglobulin
5 1:20 5 7 mg/L
within a single calibration run. Inter-assay imprecision was calculated from 10 different calibration runs (in 10 consecutive weeks).
0.6 10.7
LINEARITY ASSAY PROCEDURE
The latex suspension was warmed for 30 min at 37 °C before assay. The calibration curve was obtained by automatic two-fold dilutions (from 1/20 to 1/320) of the standard to produce a concentration range from 0.6 to 10.7 mg/L. The instrument program is shown in Table 1. The sample is automatically prediluted to 1:100. Diluted samples or standards (40 ~L) are added to 120 ~L of saline and 40 ~L of latex reagent and mixed. The scattered light is measured after 10 s and after 6 min. The instrument plots incremental signal of standards versus their concentrations, using a logit-log calibration curve. Incremental signals of the samples are converted into concentrations by interpolation. IMPRECISION
Three pools of sera at different levels of ~2-M were prepared for imprecision studies. Intra-assay imprecision was determined by replicate testing (n = 20)
Linearity was studied by serial two-fold dilution of a pool of sera with saline, over the range of 0.8 mg/L to 30 mg/L. INTERFERING SUBSTANCES
The interference study was developed according to Glick e t a/.(30). The maximum concentrations of interfering substances added to a pooled serum were: 12 g/L of haemoglobin, 1.71 mmol/L of bilirubin, Intralipid 2% (v/v), and 1100 IU/mL of rheumatoid factor. COMPARISON PROCEDURES
For comparative studies, we used an RIA method (Phadebas ~2-microglobulin, Pharmacia, Uppsala, Sweden) and a time-resolved fluoroimmunoassay procedure (DELFIA ~2-micro, Pharmacia, Uppsala, 35 --J
3000
28
E = .,a
2500
e-=!
2000 E o •
21
o oh .
1500
14
O O c
.~
1ooo 500
I O~ I ¢o.
-
7
