Quantitation of C-reactive protein in cerebrospinal fluid and serum by zone immunoelectrophoresis assay (ZIA)

Journal of Immunological Methods, 100 (1987) 191-195

191

Elsevier JIM 04375

Quantitation of C-reactive protein in cerebrospinal fluid and serum by zone immunoelectrophoresis assay (ZIA) L.-O. Hansson 1, L. Lindquist 2, .T. Linn6 3 and E. Sego 1 t Department of Clinical Chemistry, Danderyd Hospita~ 2 lnslitutio n of Infectious Diseases, Raslagstull Hospita~ and ~ Institution of Pediatrics, St. G~ran Hospital,t Karolinska Institute, Stockolm, Sweden

(Roceived 15 January 1987, ac.~pted 16 February 1987)

The zone immunoelectrophoresis assay (ZIA) for C-reactive protein (CRP) determinations is easy to perform and requires only small amount of antiserum, e.g., 25-100 and 0.5-1.0 #1 anti-CRP antibody/20 serum and CSF samples, respectively. For quantitating CSF-CRP the immunoprecipitates formed were stained using alkaline phosphatase-conjugated secondary antibodies and the lowest standard concentration used was 30 #g/1. The immunoprecipitates formed when measuring CRP in serum were stained by Coomasie brilliant blue R250 with a detection limit of about 300 #g/l. CRP was determined in cerebrospinal fluid in 27 patients with bacterial meningitis (range < 0.03-23.0 rag/l) and in 25 patients with viral meningitis (range < 0.03-0.23 rag/l). CRP was quantitated in 52 sera by both the CRP ZIA method ( y ) and by electroimmunoassay (x). The correlation coefficient was r = 0.992 with the regression line y = 1.024 x + 0.855. Key words: C-reative protein; Cerebrospinal fluid; Meningitis

Introduction

C-reactive protein (CRP) is an acute-phase protein in man. Increased levels are seen in different types of disease such as infections, rheumatoid arthritis, neoplasia and in tissue necrosis (Morley and Kuslmer, 1982). CRP quantitation in cerebrospinal fluid (CSF), and serum has been used and also recommended for differentiating between viral and bacterial meningitis (Sindic et al., 1984; Abramson et al., 1985; Donald et al., 1985; Tanner et al., 1985; Virji et al., 1985). Unfortunately, however, the CSF concentration of CRP is often below the detection limits for single radial immunodiffusion,

Correspondence to: L.-O. Hans.son, Department of Clinical

Chemistry, Dand©ryd Hospital, S-182 88 Danderyd, Sweden.

electroimmunoassay, turbidometric or nephelometric assay (Gray et al., 1986). In the present study, we present a simple and inexpensive zone immunoelectrophoresis assay (ZIA) (Vesterberg, 1980) for CRP determinations. The usefulness of the method is exemplified by CSF-CRP determinations in 52 patients with different type of menigitis but it has not been our aim to evaluate the diagnostic value of CSF-CRP determinations in meningitis.

Materials and methods

Zone immunoelectrophoresis assay (ZIA ) ZIA was performed using Quantiphor equipment (De.saga, Heidelberg, F.R.G.) as described by Vesterberg (1980). The rabbit anti-human CRP

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192 (Code A 073) and the alkaline phosphatase-conjugated swine anti-rabbit IgO (code 306) antisera were purchased from Dakopatts (Copenhagen, Denmark). A CRP standard serum (code ORCE 02/03) was purchased from Behring (Marburg, F.R.G.). Agarose type HSA from Litex (Glostrup, Denmark) was used. Gel Bond foil was from Marine Colloids (Rockland, ME). All other chemicals were purchased from Sigma (St. Louis, MO). The Quantiphor tube set comprises 20 small glass tubes (OD 2 mm). It is essential that the tubes are cleaned (e.g., with 0.1% Tween 80) and rinsed carefully before each use. We have found that the assay system is more stable when the tubes are coated with 5% bovine albumin solution for 2 h.

Electrophoresis buffer Tris- Tricine p H 8. 6 with BaCi 2, 2 mmol/L The buffer solution (2 liters) was made by dissolving 13.6 g N-tris-(hydroxymethyl)methylglycine (Tricine) and 0.98 g bariumchloride in deionized water. About 140 ml 1 tool/1 tris(hydroxy-methyl)aminomethane (Tris) were added to obtain pH 8.6. The final volume was adjusted with deionized water to 2 liters. The final Tricine and BaCI 2 concentration was 40 and 2 mmol/l, respectively. Bovine serum albumin solution Bovine serum albumin (BSA), fraction V, 5 g was dissolved in 100 ml of the electrophoresis buffer. Serum-CRP ZIA A 1% (w/v) solution of agarose in electrophoresis buffer was prepared by boiling. 25 or 100 #1 of the CRP antisera and 50 #1 of the bovine albumin solution were mixed carefully with 10 ml of the agarose solution at 56 o C. The tube set was filled according to the Quantiphor instruction manual. 25 #1 CRP antibody and 50 #1 sample volume were used for CRP quantitation over the range 0.30-5.0 mg/l. 100 pl CRP antibody and 10 pl sample volume were used for CRP quantitation over the range 5-80 mg/l. CSF-CRP ZIA A 0.8% (w/v) solution of agarose in electro-

phoresis buffer, containing 5% (w/v) dextran T10 (Pharmacia Fine Chemical, Uppsala, Sweden) was prepared by boiling. The CRP antisera was diluted 1 : 10 in electrophoresis buffer. 10 #1 of the diluted CRP antisera and 50 #1 of the bovine albumin solution were mixed carefully with 10 ml of the agarose solution (56 o C). A sample volume of 50 #1 was used, and the CFS samples were diluted 1:2 to 1:8 with electrophoresis buffer containing BSA, 50 g/l and sucrose, 100 g/l. The CRP quantitation range was 30-500 pg/l.

Sample application and electrophoretic conditions The gel rods, in the tube set, were allowed to solidify for at least 30 min before use. The tube set was then placed in the Quantiphore electrophoresis cell, and the system filled with electrophoresis buffer. The samples were carefully layered on top of each gel rod. Both the CSF and the serum samples were run overnight (20 h) at room temperature at 25 V. The cathode was in the lower buffer chamber since CRP migrates towards the cathode in this buffer system. Staining of the immunoprecipitates Serum-CRP ZIA. When the electrophoresis was completed, the gel rods were pushed out of the glass tubes onto the hydrophilic side of a Gel Bond foil. Two filter papers, an absorbant pad, and a weight of 1 kg were placed on top of the gel rods for 15 min. The pressed gel rods were then washed twice for 30 min in phosphate-buffered saline (PBS) pH 7.4 with 0.5% Tween 20. After each wash the gels were pressed for 10 rain (see above). The immunoprecipitates were stained with Coomasie brilliant blue R 250 (Weeke, 1973). CSF-CRP ZIA. The gel rods were processed as for serum CRP-ZIA. After washing twice in PBS-Tween the gels were incubated for 1-2 h with alkaline phosphatase-conjugated swine anti-rabbit IgG antisera diluted 1:2000 in PBS-Tween. Thereafter the gels were pressed and washed twice for 10 rain in PBS, and once in veronal-acetate buffer, 0.15 mol/l, pH 9.6 for 10 min. The gels were then incubated for 45 rain at 37 °C in 18 ml veronal-acetate buffer (0.15 mol/l, pH 9.6) containing 40 pl MgCi 2 (2 mol/l), 2.5 ml nitro blue tetrazolium (NBT, 0.1% in veronal-acetate buffer),

193 and 0.2 ml fl-indoxylphosphate (5 mg dissolved in 1 ml dimethylformamide and 1-2 drops HCI, 1 m o l / l ) (Blake et al., 1984). Finally the gels were pressed and washed three times and allowed to dry. The lengths of the precipitates were measured and the concentrations of the samples were read from the standard curve.

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Electroimmunoassay Serum CRP was also measured by electroimmunoassay according to Laurell (1966) except that barium chloride (2 mmol/l) was used instead of calcium lactate in the veronal buffer (Kindmark and Thorell, 1972).

Patients CFS-CRP was analyzed with ZIA in 27 patients with culture proven bacterial meningitis (Haemophilus influenzae 13, Streptococcus pneumoniae 8, and Neisseria meningitidis 6) and in 25 patients with viral meningoencephalitis (enterovirus 11, mumps 8, tick born encephalitis 3, varicella zoster 2, Herpes simplex 1). The viral etiologies were established by either CSF virus isolation, serology, or a clinical picture of mumps. All patients with viral meningocephalitis had a CSF leukocyte count of 10 x 106/1 or more. Serum samples, analyzed for CRP both by ZIA and electroimmunoassay, were taken from 52 patients with various acute infections (upper respiratory tract infections 21, pneumonia 10, urinary tract infections 5, varicella zoster 3, septicemia 2, miscellaneous viral infections 8, and miscellaneous bacterial infections 3).

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Results

Fig. 1. Typical results of CRP quantitation by zone immunoeIcctrophoresis after staining the immunoprecipitates using Coomassie brilliant blue R250 (,4) and alkaline phosphataseconjugated secondary antibodies (B), respectively.

After .staining with either alkaline phosphataselabelled second antibody or Coomassie the immunoprecipitates formed by ZIA were distinct and easy to measure (Fig. 1A and B). Within and between series precision for CRP-ZIA at various concentrations of CRP are presented in Table I. The correlation between CRP-ZIA and electroimmunoassay (EIA) calculated from patient sera was r = 0.992, y = 1.024x + 0.855 (Fig. 2). The 95%

confidence interval for the mean difference (EIAZIA) was - 4 . 8 - 0 . 0 8 mg/l, and did not differ significantly from zero. In Fig. 3 the individual values are shown for CSF-CRP measured by ZIA in patients with bacterial and viral meningoencephalitis.

194 TABLE i

MENING

PRECISION OF ZONE IMMUNOELECTROPHORES1S ASSAY OF C-REACTIVE PROTEIN AT VARIOUS CONCENTRATIONS OF Nc PROTEIN C-reactive protein, mean concentration

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,10.5 32.5 13.7 3.9 0.10 0.07

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We have described a sensitive method for C-reative protein quantitation which can be used both for serum and C S F samples. The lower limit of detection for C R P quantitation by Z I A was 30 # g / l which is about 100 times more sensitive c o m p a r e d to ordinary immunochemical methods for C R P determination. A similar sensitivity has

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Fig. 2. Comparison between quantitation of C-reactive protein by electroimmunoassay (EIA) and zone immunoelectrophoresis (ZIA) in patients with acute infectious diseases (n = 52). The equation of the regression line was ZIA ~ 1.024X EIA+0.855 with a correlation coefficient of 0.992.

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Fig. 3. CSF quantitation of C-reactive protein by ZIA in patients with bacterial (n = 27) and viral (n = 25) meningitoencephalitis. The dotted line represents the lower detection limit (0.03 rag/l).

been found for the method in the quantitation of other proteins (Vesterberg, 1983). A n excellent agreement was found for C R P levels in serum samples analyzed by Z I A and E I A (Fig. 2). At the mean concentration of 3.9 m g / l a between-series coefficient of variation of 12.6% was found (Table I). Although this may seem high the variation of the individual mean values for each series (3.0, 4.0, 4.0, 4.0, 4.0, and 4.5 m g / l ) is acceptable for practical use. As an example of the application of C-reactive protein determination by Z I A we have analyzed C S F samples from patients with bacterial and viral meningoencephalitis. Our aim has not been to evaluate the diagnostic value of C R P determination in meningitis. However, a large range was found for C S F - C R P ( < 0.03-23.0 m g / l ) in bacterial meningitis. In viral meningoenccphalitis the range was < 0.03-0.23 mg/1, partially overlapping with values in the bacterial group. A b o u t 50% of the values for patients with bacterial meningitis were below 1.0 m g / l , and should not be measurable with ordinary routine C R P assays.

195

Our results are in close agreement with those obtained by Gray et al. (1986) and by Shaltout et al. (1986). Other suitable methods for the quantitation of low concentrations of CRP are radioimmunoassay, enzyme immunoassay, and particle-counting immunoassay, but unlike zone immunoelectrophoresis these assays require expensive laboratory equipment.

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Gray, B.M., Simmons, D.R., Mason, H., Barnum, S. and Volanaki~ J.E. (1986) J. Pediatr. 108, 665. Kindmark, C.-O. and ThoreU, J.l. (1972) Scand. J. Clin. Lab. Invest. 29 (suppl. 124), 49. Laurell, C.-B. (1966) Anal. Biochem. 15, 235. Morley, J.J. and Kushner, J. (1982) Ann. N.Y. Acad. Sci. 389, 406. Shahout, A., [] Shirbiny, A., KJHander, J., Ragheb, A. and [] Heir, S.A., 1986, Ann. Trop. Paediatr. 6, 31. Sindic, CJ.M., CoUet-Cassart, D., Laterre, E.C. and Masson" P.L. (1984) J. Neurol. Sci. 63, 339. Tanner, A.R., Collins, A.L. and Bull, F.G. (1985) Ciin. Chim. Acta 147, 267. Vesterberg, O. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 617. Vesterberg, O. (1983) Clin. Chim. Acta 132, 209. Virji, M.A., Diven" W.A. and Kelly, R.H. (1985) Clin. Chim. Acta 148, 31. Weeke, B. (1973) Scand. J. Immunol. Suppl. 2, 15.