Journal of Clinical LaboratoryAnalysis 6:24-29 (1992)
Microparticle-Enhanced Nephelometric lmmunoassay for Human C-Reactive Protein R Montagne,' I? Laroche,' M.L. Cuilliere,' R Varcin,2 B. P ~ uand , ~ J. Duheille' 'Immunology Laborafory, Faculty of Medicine, Nancy, France; 'Sanofi Research, Monfpellier, France; 3/mmunologyLaboratory, Faculty of Pharmacy, Monfpellier, France. Polyfunctional hydrophilic microspheresof 125-nm diameter can be produced by copolymerization of acrylic monomers. Purified c-reactive protein (CRP) was covalently bound to these new micropheres,and the conjugate obtained was used as reagent in a microparticle-enhanced nephelometric immunoassay for human CR!? This assay was based on the measure, with a specially designed nephelometer, of the light scattered by aggregates formed during the immunoagglutinationof the conjugate with anti-CRP antiserum. Sensitive inhibition of this agglutination by free CRP (6 ngiml) allowed CRP quantitation in highly
Key words:
microparticleagglutination, immunoassay, immunonephelometry,c-reactive protein
INTRODUCTION C-reactive protein (CRP) is constituted by five identical subunits (molecular mass: 23,000), is noncovalently bound (1-3), and reacts with polysaccharides of some bacteria, allowing their phagocytosis (4) with activation of the classical pathway of complement (5,6). Its normal seric concentration is lower than 0.008 g/L (7) but is strongly increased during the acute-phase response of an inflammatory reaction (8). CRP is considered an essential marker protein in inflammation (9, lo); numerous methods of CRP quantitation during inflammation have been described (11). Some of these methods use latex agglutination: slide or tube test (1 2), quantified with a cell counter (13), turbidimeter, or nephelometer (14). Radioimmunological (15,16) and immunoenzymological (17,18) methods have also been proposed. Although they are highly sensitive, such immunoassays are often cumbersome and time-consuming (numerous washings, repeated incubations). Previous reports (19,20) have shown the great interest of microparticle-enhanced nephelometric immunoassay using new hydrophilic and polyfunctional microspheres (MS) specially synthesized and covalently coated with proteins (MSprotein conjugate). These MS-protein conjugates are agglutinated by specific antibodies, and their agglutination can be inhibited by free protein, permitting quantitation by measurement with a nephelometer of the light scattered by MS-protein conjugate agglutinates. 0 1992 Wiley-Liss, Inc.
diluted serum samples (1/500-1/2,000), excluding any interference or sample pretreatment. The CRP assay was easy to perform (no washing or phase separation), reliable (coefficients of variation ranged from 1.3% to 9.3% for within-run and between-rundeterminations), and accurate (mean percentage of recovery: 104%; correlation coefficients with accepted analytical methods 20.97) over a large range of concentrations. The inhibition mode excluded errors in the antigen excess zone and provided total security at high concentrations.
We describe here the obtention and nephelometric study of MS-CRP conjugate and its use to devise a microparticleenhanced nephelometric immunoassay for CRP. The validity of this new assay was assessed and its results compared with accepted methods for CRP estimation.
MATERIALS AND METHODS
Chemical and Biochemical Reagents 2-Hydroxyethyl methacrylate, acrolein, and methacrylic acid were purchased from Merck (Darmstad, Germany), and N,N'methylene-bis-acrylamide from Eastman Kodak (Rochester, NY). Reagent-grade sodium dodecyl sulfate (SDS), hydroquinone, sodium dihydrogenophosphate, disodium hydrogenophosphate, sodium chloride, 2-aminoethanol, sucrose, and sodium azide were obtained from Prolabo (Paris, France). The buffer for nephelometry from Diagnostics Pasteur (Marnes, France) was used for the immunonephelometric assays. Purified human CRP (5 g/L; immunoelectrophoretically pure), anti-CRP goat antiserum, CRP standard serum (0.1500 g/L), and CRP control sera (0.013 & 0.002 g/L; 0.0525 -+ 0.0079 g/L; 0.1050 0.0158 g/L) were Diagnostics Pasteur products. LN standard CRP (0.075 g/L) from Behring
*
Received July 16, 1991; accepted September 6, 1991 Address reprint requests to Dr. P. Montagne, Immunology Laboratory, Faculty of Medicine, BP 184, F-54505Vandoeuvre les Nancy CEDEX, France.
Microparticle NephelometricCRP Assay
(Marburg, Germany) was also used. Human serum albumin (HSA) was obtained from Sigma (St. Louis, MO). Human sera used for precision, accuracy, and correlation studies, chosen as far as possible from patients with documented infectious or rheumatic diseases, had been frozen at - 20°C or stored at 4°C for a short period of time.
+
Preparation of Microspheres Commercial monomers, stabilized by 50-200 ppm hydroquinone monomethyl ether, were distilled prior to use: methacrylic acid (60"C, 0.133 kPa), 2-hydroxyethyl methacrylate (95"C, 1.33 kPa), acrolein (53"C, atmospheric pressure). Distilled monomers (total concentration: 80 giL) and SDS (0.8 g/L) were mixed in deionized and deaerated water, saturated with argon, in the following conditions: 2-hydroxyethylmethacrylate 29.85 g, acrolein 28.20 g, methacrylic acid 1.20 g, and N,N'-methylene-bis-acrylamide0.75 g, for a total volume of 0.75 L. The mixture, conditioned under vacuum in a 1.5-L borosilicated glass vessel, was y-irradiated oC@ '( source, CENG, France) for 3 h at a flux of 75 h a d cmP2,under rotating agitation. The polymerized MS suspension was quickly transferred into 0.75 L of an aqueous solution of hydroquinone (1 g/L), saturated with argon to prevent postpolymerization, and stored at 4°C. MS characterization (shape, dispersion, size, concentration)was performed as previously reported (21).
Preparation of MS-CRP Conjugate CRP (3 mg) was mixed with 10 mg of MS in 1 ml of 0.1 M phosphate, 0.2 M NaC1, pH 7.2 buffer. After stirring for 2 h at room temperature and 18 h at 4"C, 50 p.1 of 2-aminoethanol (2.4 M) or human serum albumin (20 g/L) pH 8 buffered solution was added, and the mixture was incubated for a further 4 h at room temperature. MS-CRP conjugate was then separated from uncoupled reagents by centrifugation (1 h, 12,OOOg, Beckman SW50 rotor, Spinco L ultracentrifuge) on a discontinuous sucrose gradient (200/800 g/L in 0.1 M phosphate buffer pH 7.2). The MS-CRP conjugate, recovered at the interface of sucrose solutions, was stored at 4"C, at 2 g/L, in 0.1 M phosphate buffer pH 7.2 containing 2 g/L of sodium azide. CRP and HSA were measured by conventional immunonephelometry in the binding supernatant, thereby permitting estimation of the binding yields.
lmmunonephelometric Study of MS-CRP Conjugate Immunonephelometric study of the MS-CRP conjugate was performed by nephelometric measurement of light scattering during their reaction with serial dilutions (1/1,600 to 1/204,800)of the anti-CRP antiserum (agglutination reaction) and with previously chosen dilutions (1/12,800 and 1/14,400) of this antiserum in the presence of known concentrations (0
25
-
to 100 ng/ml) of purified CRP or CRP in standard sera (inhibition reaction). Measurement of light-scattering intensity was performed with the Diagnostics Pasteur nephelometer (Nephelia N 600) specially conceived for that use. The incident light, coherent and collimated, of an He-Ne Laser (power = 2 mW, wavelength = 632.8 nm) was passed across a disposable microcuvette (minimal volume of measurement: 250 ~ 1 light ; path 1 cm; Nephelia cuvettes, Diagnostics Pasteur), which contained the reaction mixture. The scattered light was collimated on a light-sensitive silicium diode at a fixed angle of 10" with regard to the incident beam. The nephelometer was fitted out with an automatic microcuvette changing and a reading device permitting positive sample and assay identification.
Determinationof CRP in Human Serum Microparticle-enhanced nephelometric immunoassay was performed as a one-step immunoassay: unknown or control sera, MS-CRP conjugate, anti-CRP antiserum, and buffer for nephelometry were mixed together in the reaction microcuvette according to the procedure described in Table 1. All predilutions were performed in the buffer for nephelometry, with a Hamilton (Bonaduz, Switzerland) dilutor. Two reaction protocols were used, differing from one another by the dilution of the serum sample in the reaction cuvette: 1/2,000 for the "high values protocol" and M O O for the "low values protocol.'' The calibration curve was plotted by using data from six serial dilutions of CRP standard serum (0.1500 g/L). After 1 h at room temperature, light scattering was measured by the nephelometer at a rate of 10 s between two successive measures. Reproducibility of the calibration curves was estimated by five assays of each dilution of the CRP standard serum repeated on each of 5 days. Precision of the assay was assessed by measuring CRP concentration (low, middle, and high) in human sera, 10-30 times with one calibration curve on the same day (within-runprecision) and on 10-30 different assays (between-run precision). Recovery study was performed by adding a known amount (0.100 g/L) of purified CRP to six different human sera and five or six known increasing amounts (0.015-0.125 g/L) to TABLE 1. Procedure for Serum CRP Determination Reagent Prediluted CRP standard serum (0.15 g/L) 1/150- 1/24OO
Reaction vol (ul)
Reagent concm in reaction cuvette
50
100-6.25 ng/ml
OR
Prediluted sample or control 11200 (high values protocol) 1/50(low values protocol) MS-CRP conjugate (2 g/L) Buffer for nephelometry Prediluted anti-CRP antiserum (1/720)
50
50 375 25
112,000 1/500 200 kg/ml 1114,400
Montagneet al.
26
three different sera. The initial CRP concentration in all overloaded sera was
Microparticle-Enhanced Nephelometric lmmunoassay for Human C-Reactive Protein R Montagne,' I? Laroche,' M.L. Cuilliere,' R Varcin,2 B. P ~ uand , ~ J. Duheille' 'Immunology Laborafory, Faculty of Medicine, Nancy, France; 'Sanofi Research, Monfpellier, France; 3/mmunologyLaboratory, Faculty of Pharmacy, Monfpellier, France. Polyfunctional hydrophilic microspheresof 125-nm diameter can be produced by copolymerization of acrylic monomers. Purified c-reactive protein (CRP) was covalently bound to these new micropheres,and the conjugate obtained was used as reagent in a microparticle-enhanced nephelometric immunoassay for human CR!? This assay was based on the measure, with a specially designed nephelometer, of the light scattered by aggregates formed during the immunoagglutinationof the conjugate with anti-CRP antiserum. Sensitive inhibition of this agglutination by free CRP (6 ngiml) allowed CRP quantitation in highly
Key words:
microparticleagglutination, immunoassay, immunonephelometry,c-reactive protein
INTRODUCTION C-reactive protein (CRP) is constituted by five identical subunits (molecular mass: 23,000), is noncovalently bound (1-3), and reacts with polysaccharides of some bacteria, allowing their phagocytosis (4) with activation of the classical pathway of complement (5,6). Its normal seric concentration is lower than 0.008 g/L (7) but is strongly increased during the acute-phase response of an inflammatory reaction (8). CRP is considered an essential marker protein in inflammation (9, lo); numerous methods of CRP quantitation during inflammation have been described (11). Some of these methods use latex agglutination: slide or tube test (1 2), quantified with a cell counter (13), turbidimeter, or nephelometer (14). Radioimmunological (15,16) and immunoenzymological (17,18) methods have also been proposed. Although they are highly sensitive, such immunoassays are often cumbersome and time-consuming (numerous washings, repeated incubations). Previous reports (19,20) have shown the great interest of microparticle-enhanced nephelometric immunoassay using new hydrophilic and polyfunctional microspheres (MS) specially synthesized and covalently coated with proteins (MSprotein conjugate). These MS-protein conjugates are agglutinated by specific antibodies, and their agglutination can be inhibited by free protein, permitting quantitation by measurement with a nephelometer of the light scattered by MS-protein conjugate agglutinates. 0 1992 Wiley-Liss, Inc.
diluted serum samples (1/500-1/2,000), excluding any interference or sample pretreatment. The CRP assay was easy to perform (no washing or phase separation), reliable (coefficients of variation ranged from 1.3% to 9.3% for within-run and between-rundeterminations), and accurate (mean percentage of recovery: 104%; correlation coefficients with accepted analytical methods 20.97) over a large range of concentrations. The inhibition mode excluded errors in the antigen excess zone and provided total security at high concentrations.
We describe here the obtention and nephelometric study of MS-CRP conjugate and its use to devise a microparticleenhanced nephelometric immunoassay for CRP. The validity of this new assay was assessed and its results compared with accepted methods for CRP estimation.
MATERIALS AND METHODS
Chemical and Biochemical Reagents 2-Hydroxyethyl methacrylate, acrolein, and methacrylic acid were purchased from Merck (Darmstad, Germany), and N,N'methylene-bis-acrylamide from Eastman Kodak (Rochester, NY). Reagent-grade sodium dodecyl sulfate (SDS), hydroquinone, sodium dihydrogenophosphate, disodium hydrogenophosphate, sodium chloride, 2-aminoethanol, sucrose, and sodium azide were obtained from Prolabo (Paris, France). The buffer for nephelometry from Diagnostics Pasteur (Marnes, France) was used for the immunonephelometric assays. Purified human CRP (5 g/L; immunoelectrophoretically pure), anti-CRP goat antiserum, CRP standard serum (0.1500 g/L), and CRP control sera (0.013 & 0.002 g/L; 0.0525 -+ 0.0079 g/L; 0.1050 0.0158 g/L) were Diagnostics Pasteur products. LN standard CRP (0.075 g/L) from Behring
*
Received July 16, 1991; accepted September 6, 1991 Address reprint requests to Dr. P. Montagne, Immunology Laboratory, Faculty of Medicine, BP 184, F-54505Vandoeuvre les Nancy CEDEX, France.
Microparticle NephelometricCRP Assay
(Marburg, Germany) was also used. Human serum albumin (HSA) was obtained from Sigma (St. Louis, MO). Human sera used for precision, accuracy, and correlation studies, chosen as far as possible from patients with documented infectious or rheumatic diseases, had been frozen at - 20°C or stored at 4°C for a short period of time.
+
Preparation of Microspheres Commercial monomers, stabilized by 50-200 ppm hydroquinone monomethyl ether, were distilled prior to use: methacrylic acid (60"C, 0.133 kPa), 2-hydroxyethyl methacrylate (95"C, 1.33 kPa), acrolein (53"C, atmospheric pressure). Distilled monomers (total concentration: 80 giL) and SDS (0.8 g/L) were mixed in deionized and deaerated water, saturated with argon, in the following conditions: 2-hydroxyethylmethacrylate 29.85 g, acrolein 28.20 g, methacrylic acid 1.20 g, and N,N'-methylene-bis-acrylamide0.75 g, for a total volume of 0.75 L. The mixture, conditioned under vacuum in a 1.5-L borosilicated glass vessel, was y-irradiated oC@ '( source, CENG, France) for 3 h at a flux of 75 h a d cmP2,under rotating agitation. The polymerized MS suspension was quickly transferred into 0.75 L of an aqueous solution of hydroquinone (1 g/L), saturated with argon to prevent postpolymerization, and stored at 4°C. MS characterization (shape, dispersion, size, concentration)was performed as previously reported (21).
Preparation of MS-CRP Conjugate CRP (3 mg) was mixed with 10 mg of MS in 1 ml of 0.1 M phosphate, 0.2 M NaC1, pH 7.2 buffer. After stirring for 2 h at room temperature and 18 h at 4"C, 50 p.1 of 2-aminoethanol (2.4 M) or human serum albumin (20 g/L) pH 8 buffered solution was added, and the mixture was incubated for a further 4 h at room temperature. MS-CRP conjugate was then separated from uncoupled reagents by centrifugation (1 h, 12,OOOg, Beckman SW50 rotor, Spinco L ultracentrifuge) on a discontinuous sucrose gradient (200/800 g/L in 0.1 M phosphate buffer pH 7.2). The MS-CRP conjugate, recovered at the interface of sucrose solutions, was stored at 4"C, at 2 g/L, in 0.1 M phosphate buffer pH 7.2 containing 2 g/L of sodium azide. CRP and HSA were measured by conventional immunonephelometry in the binding supernatant, thereby permitting estimation of the binding yields.
lmmunonephelometric Study of MS-CRP Conjugate Immunonephelometric study of the MS-CRP conjugate was performed by nephelometric measurement of light scattering during their reaction with serial dilutions (1/1,600 to 1/204,800)of the anti-CRP antiserum (agglutination reaction) and with previously chosen dilutions (1/12,800 and 1/14,400) of this antiserum in the presence of known concentrations (0
25
-
to 100 ng/ml) of purified CRP or CRP in standard sera (inhibition reaction). Measurement of light-scattering intensity was performed with the Diagnostics Pasteur nephelometer (Nephelia N 600) specially conceived for that use. The incident light, coherent and collimated, of an He-Ne Laser (power = 2 mW, wavelength = 632.8 nm) was passed across a disposable microcuvette (minimal volume of measurement: 250 ~ 1 light ; path 1 cm; Nephelia cuvettes, Diagnostics Pasteur), which contained the reaction mixture. The scattered light was collimated on a light-sensitive silicium diode at a fixed angle of 10" with regard to the incident beam. The nephelometer was fitted out with an automatic microcuvette changing and a reading device permitting positive sample and assay identification.
Determinationof CRP in Human Serum Microparticle-enhanced nephelometric immunoassay was performed as a one-step immunoassay: unknown or control sera, MS-CRP conjugate, anti-CRP antiserum, and buffer for nephelometry were mixed together in the reaction microcuvette according to the procedure described in Table 1. All predilutions were performed in the buffer for nephelometry, with a Hamilton (Bonaduz, Switzerland) dilutor. Two reaction protocols were used, differing from one another by the dilution of the serum sample in the reaction cuvette: 1/2,000 for the "high values protocol" and M O O for the "low values protocol.'' The calibration curve was plotted by using data from six serial dilutions of CRP standard serum (0.1500 g/L). After 1 h at room temperature, light scattering was measured by the nephelometer at a rate of 10 s between two successive measures. Reproducibility of the calibration curves was estimated by five assays of each dilution of the CRP standard serum repeated on each of 5 days. Precision of the assay was assessed by measuring CRP concentration (low, middle, and high) in human sera, 10-30 times with one calibration curve on the same day (within-runprecision) and on 10-30 different assays (between-run precision). Recovery study was performed by adding a known amount (0.100 g/L) of purified CRP to six different human sera and five or six known increasing amounts (0.015-0.125 g/L) to TABLE 1. Procedure for Serum CRP Determination Reagent Prediluted CRP standard serum (0.15 g/L) 1/150- 1/24OO
Reaction vol (ul)
Reagent concm in reaction cuvette
50
100-6.25 ng/ml
OR
Prediluted sample or control 11200 (high values protocol) 1/50(low values protocol) MS-CRP conjugate (2 g/L) Buffer for nephelometry Prediluted anti-CRP antiserum (1/720)
50
50 375 25
112,000 1/500 200 kg/ml 1114,400
Montagneet al.
26
three different sera. The initial CRP concentration in all overloaded sera was
