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Determination of lysozyme in serum, urine, cerebrospinal fluid and feces by enzyme immunoassay
CIinica Chimica Acta, 142 (1984) 21-30 Elsevier
21
CCA 02934
Determination of lysozyme in seruni, urine, cerebrospinal fluid and feces by enzyme immunoassay Johan Department
Brouwer
*, Trudi van Leeuwen-Herberts Ruit
and Marjo
Otting-van
de
of Clinical Chemistry, Stichting Samenwerking Delftse Ziekenhuiren, Delft (The Netherlands) (Received April Znd, 1984) Key words: Lysozyme; Enzyme immunoassay; Reference intervals
Conjugates of human lysozyme and horseradish peroxidase (HRP) were prepared by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate. A conjugate containing 2 mol HRP/mol lysozyme was isolated by gel filtration and used as a labeled antigen in competitive enzyme immunoassays, in which anti-lysozyme rabbit IgG had been bound to wells of microtiter plates. The assay can detect as little as 1 pg lysozyme/l. The following reference intervals have been established: 950-2450 pg/l for serum, 1.7-123 pg/l for urine, 17.6-118 pg/l for cerebrospinal fluid and 0.04-1.5 pg/g for feces.
Introduction
Lysozyme or muramidase (EC 3.2.1.17) is an enzyme that catalyzes the hydrolysis of the peptidoglycan layer of bacterial cell walls. It is present in secretions and extracts of various organs. It originates from phagocytic cells and is actively secreted by monocytes and macrophages. The significance of lysozyme determinations in human serum, urine, cerebrospinal fluid (CSP) and feces has been reviewed recently by Dick [1,2]. Elevated levels of lysozyme have been found in serum during monocytic and monomyelocytic leukemia, sarcoidosis and chronic bacterial infections [3]; in urine during urinary tract infec* Address for correspondence: Dr. J. Brouwer, Dept. of Clinical Chemistry, S.S.D.Z., P.O. Box 5010, 2600 GA Delft, The Netherlands. 0009-8981/84/$03.00
0 1984 Elsevier Science Publishers B.V.
22
tions, proximal renal tubular damage, renal allograft rejection and excessive endogenous lysozyme synthesis whereby the reabsorption capacity of the proximal tubulus is exceeded [3]; in CSF during bacterial meningitis and tumors of the CNS [1,2] and in feces during inflammatory diseases of the gastrointestinal tract, e.g. M. Crohn, colorectal tumors and gastroenteritis due to bacterial or rotavirus infection [1,2]. Determination of the lysozyme concentration can be helpful in these cases in assessing progression of disease, monitoring efficacy of therapy and detection of relapse. Various methods for estimating lysozyme concentrations have been described. The enzyme activity can be measured turbidimetrically due to the lytic action of lysozyme on Micrococcus Iysodeikticus [4,5]. Immunochemically, lysozyme can be determined by rocket electrophoresis [6], laser nephelometry [7] and radioimmunoassay [8,9]. Radioimmunoassay is the only method so far that is sensitive enough for the determination of lysozyme in normal urine, CSF and feces. In this paper we describe a very sensitive competitive enzyme immunoassay (EIA) for lysozyme and its use in establishing reference intervals for normal serum, urine, CSF and feces. Human lysozyme has been coupled to HRP by means of Carlsson’s reagent [lo] or N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Use of this reagent results in the formation of discrete populations of conjugated molecules without intramolecular cross-linking. In order to construct an EIA as sensitive as possible, we isolated those lysozyme molecules which were labeled with HRP to the highest degree. Except for human lysozyme, which can be purified easily, all other reagents are commercially available. Materials and methods Materials CM-Sephadex C-50, DEAE-Sephadex A-50, Sephacryl S-200 and SPDP were obtained from Pharmacia (Uppsala, Sweden). Horseradish peroxidase (HRP, type VI) was obtained from Sigma (St. Louis, MO, USA). Enzyme immunoassays were carried out on flexible PVC microtiter plates (Flow Labs, Irvine, Scotland). Coating buffer contained 0.1 mol/l sodium carbonate, pH 9.6. As a wash buffer we used PBS, Tween: 0.05% Tween 20 in PBS (50 mmol/l sodium phosphate, pH 7.4, 0.15 mol/l sodium chloride). Dilutions of samples and standards were made in PBS, containing 1% bovine serum albumin (BSA), using a programmable diluter (Dilutrend, Clinicon, Mannheim, FRG). The chromogen solution consisted of 40 mg 0-phenylenediamine in 100 ml 24 mmol citric acid, 51 mmol/l disodium hydrogen phosphate. Before use 40 ~1 30% hydrogen peroxide was added. Absorbances were measured on an 8-channel photometer (Titertrek Multiskan, Flow Labs). The data were computed in a Hewlett Packard programmable calculator (HP 9815 A). Purification of human lysozyme Lysozyme was purified from urine of a patient with monocytic leukemia by the method of Johansson and Malmquist [6]. The procedure included adsorption to
23
CM-Sephadex C-50, elution with a linear gradient of 0.1-1.0 mol/l NaCl in 50 mmol/l Tris-HCl, pH 7.6 (lysozyme eluted at about 0.6 mmol/l NaCl), dialysis against distilled water, lyophihzation and crystallization in 1 mol/l NaCl at pH 4.5 and 4’C. The final preparation was pure as judged by SDS-polyacrylamide gel electrophoresis [ll] and amino acid analyses [12]. Pure crystallized lysozyme was dialyzed against distilled water and lyophilized. A 1% solution of the protein in PBS had an absorbance of 25.60 at 280 nm, in agreement with previously published values [13,14]. Preparation of lysozyme-HRP conjugates Lysozyme and HRP were treated with SPDP according to the instructions supplied by the manufacturer (Pharmacia). The initial ratios were 153 pg SPDP/mg lysozyme and 78 pg SPDP/mg HRP. The modified proteins contained 2.4 mol 2-pyridyl disulfide/mol lysozyme and 2.1 mol 2-pyridyl disulfide/mol HRP. Substituted HRP was reduced by dithiothreitol and a three-fold molar excess of modified HRP (8.4 mg) was mixed with 1.0 mg substituted lysozyme. The mixture of conjugated proteins was subjected to gel filtration on a 1.5 x 93 cm column of Sephacryl S-200 in PBS at a flow rate of 3.0 ml/h. Fractions of 1.0 ml were collected and monitored by measuring the absorbance at 230 nm. Portions of the eluted peaks were analyzed by electrophoresis [ll] on SDS slab gels containing a 6-20% gradient of polyacrylamide. Transferrin (80 000), catalase (60 000), aldolase (40 000) carbonic anhydrase (29 000), apoferritin (18 500) and lysozyme (14 300) were used as molecular mass marker proteins. The concentrations of HRP and lysozyme in the eluted fractions were calculated from absorbance measurements at 280 and 403 nm. A 1% solution of HRP had an absorbance of 25.0 at 403 nm and of 7.9 at 280 nm. Preparation of antibody Antiserum was raised in New Zealand white rabbits. For every injection 1 ml PBS, containing 0.5 mg lysozyme, was mixed with 1 ml Freund’s complete adjuvant. Animals received primary injections in popliteal lymph nodes. Booster injections were given intramuscularly and subcutaneously every month. Test bleeds were taken 10 days after each injection and the presence of antibody was detected by immunodiffusion against lysozyme. IgG was isolated by ammonium sulfate precipitation (2 X ) at 40% saturation, dialysis against 10 mmol/l Tris-HCl, pH 8.0 and chromatography on DEAE-Sephadex A-50. The anti-lysozyme rabbit IgG was dialyzed against PBS and stored at -7OOC. A solution containing 10 mg IgG/rnl had a titer of 0.172 mg/ml as determined by the single radial immunodiffusion method [15]. A solution of 10 pg IgG/ml in coating buffer was used for binding of IgG to microtiter plates. Preparation of samples for EIA Samples were stored at - 20 OC until use. Portions of urine and CSF were centrifuged for 10 min at 1500 X g to remove cells before they were frozen. Portions (0.5-4 g, in triplicate) of feces were homogenized in PBS so as to give a suspension
24
of 0.1-0.5 g feces/ml PBS. After centrifugation for 10 min at 1500 X g the supernatant was stored at - 20 ‘C. After thawing it was centrifuged for 5 min at 10000 X g and the final supernatant was used as a sample for EIA. Three dilutions in PBS, 1% BSA of each sample were assayed, each in duplicate. Serum was diluted 100, 200 and 400 times. Urine, CSF and feces-extract usually 2, 5 and 10 times, but had to be diluted further occasionally. Portions of a serum pool were stored at - 70 ‘C and used for the construction of standard curves by diluting 20, 50, 100, 200, 500, 1000 and 2000 times in PBS, 1% BSA. The standard serum pool was calibrated against human lysozyme by repeatedly assaying solutions containing known amounts of purified human lysozyme in PBS, 1% BSA. Concentrations of stock solutions of lysozyme in PBS were determined by absorbance measurements at 280 nm and dilutions were made in PBS, 1% BSA to give samples containing 20.0, 10.0 and 5.0 ng lysozyme/ml. Enzyme immunoassay Anti-lysozyme rabbit IgG was diluted to 10 p&/ml in coating buffer. Wells of PVC microtiter plates were filled with 150 ~1 of this solution, incubated for 16-20 h at room temperature and washed three times. 500 ~1 of diluted samples or standards were mixed with 60 ~1 diluted lysozyme-HRP conjugate. 150 ~1 of the sample solutions were added to the wells in duplicate. The standard serum solutions were assayed in triplicate. Wells of the first row of the plate contained only PBS, 1% BSA. Plates were incubated for 2 h at 37 OC and washed three times. Then 160 ~1 of chromogen solution was added and the reaction was allowed to proceed for 15 min in the dark at room temperature. The reaction was stopped by adding 40 ~1 of 2.5 mol/l sulfuric acid and the absorbance was measured at 492 nm against the wells of the first row of the plate, which served as blanks. Rt?!SUltS
Isolation of lysozyme-HRP conjugates The mixture of molecules obtained after conjugation of lysozyme and HRP was fractionated by gel filtration on Sephacryl S-200 for two reasons. Firstly, to remove excess modified HRP that had been used during coupling of the proteins and, secondly, to isolate the complex with the highest HRP: lysozyme ratio, because the use of these complexes will produce the most sensitive assay. The elution pattern is shown in Fig. 1. The mixture contained no uncoupled lysozyme. The molar ratio HRP: lysozyme was 1.1 in fraction I, 1.9 in fraction II and > 10 in fraction III. Portions of the eluted fractions were analyzed on a SDS polyacrylamide slab gel (Fig. 2). Fraction III contained uncoupled HRP (M, 40 000). Fraction II contained a 95 000 complex as the major component. As the ratio HRP: lysozyme is almost 2 in this peak, it can be inferred that the composition of this conjugate is (HRP)2-lysozyme. Fraction I contained a 165000 complex ((HRP)3-(lysozyme)3) as the major component. Other conjugates in this fraction were complexes with M,‘s values of
25
Fraction number
Fig. 1. Fractionation the eluate is shown.
of lysozyme-HRP
conjugates
on Sephacryl
S-200. Only the UV-absorbing
mol. massw10-3
165
i
Fig. 2. SDS polyacrylamide
40
gel electrophoresis
of peaks eluted from Sephacryl
S-200 (Fig. 1).
part of
26
95 000, 180 000, 220 000 and 275 000. The conjugate present in fraction II was used as a labeled antigen in competitive enzyme immunoassays for lysozyme. Enzyme immunoassay For competition in an immunoassay 500 ~1 of diluted serum, urine, CSF or feces-extract was mixed with 60 ~1 of diluted conjugate. The final content of conjugate in 1 ml of this mixture was 3 ng lysozyme coupled to 16 ng HRP. As a standard we used a serum pool that had been calibrated against solutions with known concentrations of purified human lysozyme in PBS, 1% BSA. This standard serum contained 1.60 pg lysozyme/ml (n = 30, coefficient of variation (CV) = 5.3%). A competition curve obtained with this serum is shown in Fig. 3. The detection limit is about 1 pg/l and the analytical range lies between 1 and 50 pg/l.
1
lldllutlon
of
standard
serum
pool
Fig. 3. Standard curve obtained by using dilutions Vertical bars represent f 2 SD of each value.
TABLE
of a serum pool containing
1.60 gg Iysozyme/mI.
I
Between-run Sample
Serum pool Urine pool CSF pool
variance No. of assays
54 20 41
Lysozyme concentration
(pg/I)
Mean
SD
cv
1620 17.1 56.2
103 1.2 4.6
6.4 7.0 8.2
(W)
27
Validity of the assay The within-run variance on 25 different microtiter plates was calculated from duplicate determinations of 12 to 16 samples which were run as non-adjacent pairs. The CV varied from 2.8 to 6.7% (mean 4.5, SD 1.13). The between-run variance was estimated by repeated analyses of pools of serum, urine and CSF (Table I). Portions of these pools were stored at - 20 o C. Dilutions were made for each assay as were the dilutions of the standard serum. The analytical recovery was measured in the serum pool (Table II) to which purified lysozyme had been added to a final concentration of 4.10 pg/ml. We found a recovery of 101.5% (n = 35, CV = 8, 4%). Part of this pool was used for investigating the effect of repeated freezing and thawing. The sample was stored at - 20 o C, thawed for every assay and frozen again. This procedure was repeated 30 times. The lysozyme concentration remained the same, within experimental error. Lysozyme concentrations were determined in 22 sera by both the described EIA and by rocket electrophoresis [6]. These sera covered the range 1120-4800 pg/l (mean 2438 pg/l). The correlation coefficient was 0.995 with a regression equation y = 0.77x + 412. Lysozyme in serum, urine, CSF and feces We have determined lysozyme in 123 sera and 155 urines from healthy blood donors (aged 18-60 yr) and in 48 portions of feces obtained from laboratory workers, aged 20-40 yr. CSF samples were from 106 patients without any detectable deviation in their CSF or serum [16]. The distributions are shown in Figs. 4, 5 and 6 for serum, urine and CSF. The mean values and reference limits are given in Table II. For serum the non-parametric 2.5 and 97.5 percentile limits are given [17]. The limits given for urine, CSF and feces represent the 0.95 fractions of log-Gaussian distributions. For urine and CSF these limits were nearly the same as the non-parametric 2.5 and 97.5 percentile limits. The number of feces samples analyzed was too low to permit non-parametric estimate of the reference interval. Urinary lysozyme was determined in 12 samples containing between 20 and 1000 leukocytes/pi. Portions of the urines were processed as described, that is cells were removed by centrifugation before freezing the supernatant, whereas other portions were frozen directly and centrifuged only before assay. In the latter cases lysozyme
TABLE II Lysozyme concentrations in serum, urine, CSF and feces Material
Mean
(Median)
Reference limits
Serum Urine CSF
1520 14.5 45.8
(1470) (15.4) (42.9)
950 -2450 1.7 - 123 17.6 - 118
Feces
0.25
(0.27)
0.04-
pg/l pg/l pg/l
1.5 P&Q3
Lysozyme , mg/l
Fig. 4. Distribution
1.6
of lysozyme
in serum.
16
160 Lysozyme
Fig. 5. Distribution of lysozyme detection limit (dotted line).
fig/l
,
in urine.
The concentration
t
Lysozyme
Fig. 6. Distribution
of lysozyme
in CSF.
,
pug/l
in eight
samples
(5.2%) was below
the
29
concentrations were raised by a factor 1.4-3.1 (mean 2.1). The effect of urinary pH on lysozyme was studied using a pool of 30 samples with above-normal concentrations of lysozyme. This pool contained 400 pg/l. Portions of 10 ml were adjusted to pH 3, 4, 5, 6, 7 and 8, and incubated at 37 ‘C. Fractions of 0.5 ml were withdrawn after 0.25, 0.5, 1, 2, 4, 8 and 24 h, diluted with 5 ml PBS/l% BSA and stored at - 20’ C until assay. At every pH the lysozyme concentration remained the same within experimental error.
Discussion The enzyme immunoassay described in this paper has been found to be very reliable for more than a year now. The sensitivity of the assay compares favorably with that of radioimmunoassays for lysozyme [8,9]. Once the microtiter plates have been coated, tests can be completed within 4 h. Although heterogeneity of human lysozyme has been described [18], the dilution curves obtained so far were all parallel to the standard curve, as was found also by Thomas et al [9] using radioimmunoassay. We have not compared the described EIA with the turbidimetric assay. Other authors, however, have described good correlations between immunochemical methods and the enzymatic assay [6,9]. Reference ranges for lysozyme in CSF and feces have not been reported before. The reference range found for normal serum agrees closely with those established by others [9,19] using radioimmunoassays. The concentrations of urinary lysozyme that we determined were considerably lower than those reported by Thomas et al [9]. These authors found a mean value of 450 pg/l in 18 subjects, which is 30 times higher than the mean concentration that we measured (Table II). This discrepancy is unexpected, the more so as their results for serum lysozyme agree closely with our data. Johansson and Malmquist [6], however, found that the level in normal urine is -Z 10% of the mean serum concentration, which was the detection limit in their assay. Increased levels of lysozyme in urine were found if cells had not been removed by centrifugation before freezing. This is, due to lysis of lysozyme-containing neutrophilic granulocytes upon freezing and thawing. Apart from that, we found no correlation between lysozyme values in urine and the number of leukocytes. This lack of correlation has been observed before [20,21]. Determination of urinary excretion of low molecular mass proteins is a well-known method for assessment of renal tubular damage. The most widely used test is that of Qnicroglobulin, because it is commercially available. However, the rapid degradation of this protein at 37OC, especially at pH below 6, is well-documented [22-241. Therefore the use of more stable low M, proteins, e.g. retinol-binding protein, has been advocated [22,24]. From our results it appears that lysozyme might also be a suitable marker, because it is very stable in urine at 37 o C and at pH between 3 and 8 for at least 24 h.
30
References 1 Dick W. Lysozym. Gnmdlagen und diagnostische bedeutung. Fortschr Med 1982; 100: 1230-1234. 2 Dick W. Lysozym-bestimmung. Klinische Signifikanz. Laboratoriumsblatt 1983; 33: 48-56. 3 Osserman EF. Lysozymuria in renal and non-renal diseases. In: Manuel Y, Revillard JP, Bethuel H, eds. Proteins in normal and pathological urine. Basel: S. Karger AG, 1970. 4 Litwack G. Photometric determination of lysozyme activity. Proc Sot Exp Biol Med 1955; 89: 401-403. 5 Osserman EF, Lawlor DP. Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med 1966; 124: 921-951. 6 Johansson BG, Malmquist J. Quantitative immunochemical determination of lysozyme (muramidase) in serum and urine. Stand J Lab Med 1971; 27: 255-261. 7 Goudswaard J, Virella G. Immunochemical determination of human lysozyme by laser nephelometry. Clin Chem 1977; 23: 967-970. 8 Peeters TL, Depraetere YR, Vantrappen GR. Radioimmunoassay for urinary lysozyme in human serum from leukemic patients. Clin Chem 1978; 24: 2155-2157. 9 Thomas MJ, Russo A, Craswell P, Ward M, Steinhardt I. Radioimmunoassay for serum and urinary lysozyme. Clin Chem 1981; 27: 1223-1226. 10 Carlsson J, Drevin H, Ax&r R. Protein thiolation and reversible protein-protein conjugation, N-succinimidyl 3-(2-pyridyldithio) propionate, a new heterobifunctional reagent. Biochem J 1978; 173: 723-737. 11 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London ) 1970; 227: 680-685. 12 Brouwer J, Pluyms WJM, Wamaar SO. Chemical characterization of Rauscher leukemia virus proteins. J Gen Virol 1979; 42: 415-421. 13 Parry RM, Chandan RC, Shahani KM. Isolation and characterization of human milk lysozyme. Arch Biochem Biophys 1969; 130: 59-65. 14 Canfield RE, Collins JC, Sobel JH. Human leukemia lysozyme. In: Osserman EF, Canfield RE, Beychock S, eds. Lysozyme. New York: Academic Press, 1974. 15 Becker W. Determination of antisera titres using the single radial immunodiffusion method. Immunochemistry 1969; 6: 539-546. 16 Brouwer J, Van Leeuwen-Herberts GHHM. Estimation of IgM in cerebrospinal fluid by enzyme immunoassay. Clin Chim Acta 1983; 131: 337-342. 17 Reed AH, Henry RJ, Mason WB. Influence of statistical method used on the resulting estimate of normal range. Clin Chem 1971; 17: 275-284. 18 Virella G. Electrophoresis of lysozyme into Micrococcus-containing agarose gel: quantitative and analytical applications. Clin Chim Acta 1977; 75: 107-115. 19 Venge P, Foucard T, Henriksen J, Hakansson L, Kreuger A. Serum-levels of lactoferrin, lysozyme and myeloperoxidase in normal, infection-prone and leukemic children. Clin Chim Acta 1984; 136: 121-130. 20 Wilkinson SP, Hirst D, Day DW, Williams R. Spectrum of renal tubular damage in renal failure secondary to cirrhosis and fulminant hepatic failure. J Clin Path01 1978; 31: 101-107. 21 Dick W, Theilmann L. Lysozymspiegel in urin bei Kindem mit akuten und chronisch rezidivierenden Harnweg infectionen. Paediatr Paedol 1980; 15: 345-350. 22 Bernard AM, Moreau D, Lauwerys R. Comparison of retinolbinding protein and µglobulin determination in urine for the early detection of tubular proteinuria Clin Chim Acta 1982; 126: l-7. 23 Davey PG, Gosling P. &Microglobulin instability in pathological urine. Clin Chem 1982; 28: 1330-1333. 24 Bastable MD. /3z-Microglobulin in urine: not suitable for assessing renal tubular function. Clin Chem 1983; 29: 996-997.
21
CCA 02934
Determination of lysozyme in seruni, urine, cerebrospinal fluid and feces by enzyme immunoassay Johan Department
Brouwer
*, Trudi van Leeuwen-Herberts Ruit
and Marjo
Otting-van
de
of Clinical Chemistry, Stichting Samenwerking Delftse Ziekenhuiren, Delft (The Netherlands) (Received April Znd, 1984) Key words: Lysozyme; Enzyme immunoassay; Reference intervals
Conjugates of human lysozyme and horseradish peroxidase (HRP) were prepared by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate. A conjugate containing 2 mol HRP/mol lysozyme was isolated by gel filtration and used as a labeled antigen in competitive enzyme immunoassays, in which anti-lysozyme rabbit IgG had been bound to wells of microtiter plates. The assay can detect as little as 1 pg lysozyme/l. The following reference intervals have been established: 950-2450 pg/l for serum, 1.7-123 pg/l for urine, 17.6-118 pg/l for cerebrospinal fluid and 0.04-1.5 pg/g for feces.
Introduction
Lysozyme or muramidase (EC 3.2.1.17) is an enzyme that catalyzes the hydrolysis of the peptidoglycan layer of bacterial cell walls. It is present in secretions and extracts of various organs. It originates from phagocytic cells and is actively secreted by monocytes and macrophages. The significance of lysozyme determinations in human serum, urine, cerebrospinal fluid (CSP) and feces has been reviewed recently by Dick [1,2]. Elevated levels of lysozyme have been found in serum during monocytic and monomyelocytic leukemia, sarcoidosis and chronic bacterial infections [3]; in urine during urinary tract infec* Address for correspondence: Dr. J. Brouwer, Dept. of Clinical Chemistry, S.S.D.Z., P.O. Box 5010, 2600 GA Delft, The Netherlands. 0009-8981/84/$03.00
0 1984 Elsevier Science Publishers B.V.
22
tions, proximal renal tubular damage, renal allograft rejection and excessive endogenous lysozyme synthesis whereby the reabsorption capacity of the proximal tubulus is exceeded [3]; in CSF during bacterial meningitis and tumors of the CNS [1,2] and in feces during inflammatory diseases of the gastrointestinal tract, e.g. M. Crohn, colorectal tumors and gastroenteritis due to bacterial or rotavirus infection [1,2]. Determination of the lysozyme concentration can be helpful in these cases in assessing progression of disease, monitoring efficacy of therapy and detection of relapse. Various methods for estimating lysozyme concentrations have been described. The enzyme activity can be measured turbidimetrically due to the lytic action of lysozyme on Micrococcus Iysodeikticus [4,5]. Immunochemically, lysozyme can be determined by rocket electrophoresis [6], laser nephelometry [7] and radioimmunoassay [8,9]. Radioimmunoassay is the only method so far that is sensitive enough for the determination of lysozyme in normal urine, CSF and feces. In this paper we describe a very sensitive competitive enzyme immunoassay (EIA) for lysozyme and its use in establishing reference intervals for normal serum, urine, CSF and feces. Human lysozyme has been coupled to HRP by means of Carlsson’s reagent [lo] or N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Use of this reagent results in the formation of discrete populations of conjugated molecules without intramolecular cross-linking. In order to construct an EIA as sensitive as possible, we isolated those lysozyme molecules which were labeled with HRP to the highest degree. Except for human lysozyme, which can be purified easily, all other reagents are commercially available. Materials and methods Materials CM-Sephadex C-50, DEAE-Sephadex A-50, Sephacryl S-200 and SPDP were obtained from Pharmacia (Uppsala, Sweden). Horseradish peroxidase (HRP, type VI) was obtained from Sigma (St. Louis, MO, USA). Enzyme immunoassays were carried out on flexible PVC microtiter plates (Flow Labs, Irvine, Scotland). Coating buffer contained 0.1 mol/l sodium carbonate, pH 9.6. As a wash buffer we used PBS, Tween: 0.05% Tween 20 in PBS (50 mmol/l sodium phosphate, pH 7.4, 0.15 mol/l sodium chloride). Dilutions of samples and standards were made in PBS, containing 1% bovine serum albumin (BSA), using a programmable diluter (Dilutrend, Clinicon, Mannheim, FRG). The chromogen solution consisted of 40 mg 0-phenylenediamine in 100 ml 24 mmol citric acid, 51 mmol/l disodium hydrogen phosphate. Before use 40 ~1 30% hydrogen peroxide was added. Absorbances were measured on an 8-channel photometer (Titertrek Multiskan, Flow Labs). The data were computed in a Hewlett Packard programmable calculator (HP 9815 A). Purification of human lysozyme Lysozyme was purified from urine of a patient with monocytic leukemia by the method of Johansson and Malmquist [6]. The procedure included adsorption to
23
CM-Sephadex C-50, elution with a linear gradient of 0.1-1.0 mol/l NaCl in 50 mmol/l Tris-HCl, pH 7.6 (lysozyme eluted at about 0.6 mmol/l NaCl), dialysis against distilled water, lyophihzation and crystallization in 1 mol/l NaCl at pH 4.5 and 4’C. The final preparation was pure as judged by SDS-polyacrylamide gel electrophoresis [ll] and amino acid analyses [12]. Pure crystallized lysozyme was dialyzed against distilled water and lyophilized. A 1% solution of the protein in PBS had an absorbance of 25.60 at 280 nm, in agreement with previously published values [13,14]. Preparation of lysozyme-HRP conjugates Lysozyme and HRP were treated with SPDP according to the instructions supplied by the manufacturer (Pharmacia). The initial ratios were 153 pg SPDP/mg lysozyme and 78 pg SPDP/mg HRP. The modified proteins contained 2.4 mol 2-pyridyl disulfide/mol lysozyme and 2.1 mol 2-pyridyl disulfide/mol HRP. Substituted HRP was reduced by dithiothreitol and a three-fold molar excess of modified HRP (8.4 mg) was mixed with 1.0 mg substituted lysozyme. The mixture of conjugated proteins was subjected to gel filtration on a 1.5 x 93 cm column of Sephacryl S-200 in PBS at a flow rate of 3.0 ml/h. Fractions of 1.0 ml were collected and monitored by measuring the absorbance at 230 nm. Portions of the eluted peaks were analyzed by electrophoresis [ll] on SDS slab gels containing a 6-20% gradient of polyacrylamide. Transferrin (80 000), catalase (60 000), aldolase (40 000) carbonic anhydrase (29 000), apoferritin (18 500) and lysozyme (14 300) were used as molecular mass marker proteins. The concentrations of HRP and lysozyme in the eluted fractions were calculated from absorbance measurements at 280 and 403 nm. A 1% solution of HRP had an absorbance of 25.0 at 403 nm and of 7.9 at 280 nm. Preparation of antibody Antiserum was raised in New Zealand white rabbits. For every injection 1 ml PBS, containing 0.5 mg lysozyme, was mixed with 1 ml Freund’s complete adjuvant. Animals received primary injections in popliteal lymph nodes. Booster injections were given intramuscularly and subcutaneously every month. Test bleeds were taken 10 days after each injection and the presence of antibody was detected by immunodiffusion against lysozyme. IgG was isolated by ammonium sulfate precipitation (2 X ) at 40% saturation, dialysis against 10 mmol/l Tris-HCl, pH 8.0 and chromatography on DEAE-Sephadex A-50. The anti-lysozyme rabbit IgG was dialyzed against PBS and stored at -7OOC. A solution containing 10 mg IgG/rnl had a titer of 0.172 mg/ml as determined by the single radial immunodiffusion method [15]. A solution of 10 pg IgG/ml in coating buffer was used for binding of IgG to microtiter plates. Preparation of samples for EIA Samples were stored at - 20 OC until use. Portions of urine and CSF were centrifuged for 10 min at 1500 X g to remove cells before they were frozen. Portions (0.5-4 g, in triplicate) of feces were homogenized in PBS so as to give a suspension
24
of 0.1-0.5 g feces/ml PBS. After centrifugation for 10 min at 1500 X g the supernatant was stored at - 20 ‘C. After thawing it was centrifuged for 5 min at 10000 X g and the final supernatant was used as a sample for EIA. Three dilutions in PBS, 1% BSA of each sample were assayed, each in duplicate. Serum was diluted 100, 200 and 400 times. Urine, CSF and feces-extract usually 2, 5 and 10 times, but had to be diluted further occasionally. Portions of a serum pool were stored at - 70 ‘C and used for the construction of standard curves by diluting 20, 50, 100, 200, 500, 1000 and 2000 times in PBS, 1% BSA. The standard serum pool was calibrated against human lysozyme by repeatedly assaying solutions containing known amounts of purified human lysozyme in PBS, 1% BSA. Concentrations of stock solutions of lysozyme in PBS were determined by absorbance measurements at 280 nm and dilutions were made in PBS, 1% BSA to give samples containing 20.0, 10.0 and 5.0 ng lysozyme/ml. Enzyme immunoassay Anti-lysozyme rabbit IgG was diluted to 10 p&/ml in coating buffer. Wells of PVC microtiter plates were filled with 150 ~1 of this solution, incubated for 16-20 h at room temperature and washed three times. 500 ~1 of diluted samples or standards were mixed with 60 ~1 diluted lysozyme-HRP conjugate. 150 ~1 of the sample solutions were added to the wells in duplicate. The standard serum solutions were assayed in triplicate. Wells of the first row of the plate contained only PBS, 1% BSA. Plates were incubated for 2 h at 37 OC and washed three times. Then 160 ~1 of chromogen solution was added and the reaction was allowed to proceed for 15 min in the dark at room temperature. The reaction was stopped by adding 40 ~1 of 2.5 mol/l sulfuric acid and the absorbance was measured at 492 nm against the wells of the first row of the plate, which served as blanks. Rt?!SUltS
Isolation of lysozyme-HRP conjugates The mixture of molecules obtained after conjugation of lysozyme and HRP was fractionated by gel filtration on Sephacryl S-200 for two reasons. Firstly, to remove excess modified HRP that had been used during coupling of the proteins and, secondly, to isolate the complex with the highest HRP: lysozyme ratio, because the use of these complexes will produce the most sensitive assay. The elution pattern is shown in Fig. 1. The mixture contained no uncoupled lysozyme. The molar ratio HRP: lysozyme was 1.1 in fraction I, 1.9 in fraction II and > 10 in fraction III. Portions of the eluted fractions were analyzed on a SDS polyacrylamide slab gel (Fig. 2). Fraction III contained uncoupled HRP (M, 40 000). Fraction II contained a 95 000 complex as the major component. As the ratio HRP: lysozyme is almost 2 in this peak, it can be inferred that the composition of this conjugate is (HRP)2-lysozyme. Fraction I contained a 165000 complex ((HRP)3-(lysozyme)3) as the major component. Other conjugates in this fraction were complexes with M,‘s values of
25
Fraction number
Fig. 1. Fractionation the eluate is shown.
of lysozyme-HRP
conjugates
on Sephacryl
S-200. Only the UV-absorbing
mol. massw10-3
165
i
Fig. 2. SDS polyacrylamide
40
gel electrophoresis
of peaks eluted from Sephacryl
S-200 (Fig. 1).
part of
26
95 000, 180 000, 220 000 and 275 000. The conjugate present in fraction II was used as a labeled antigen in competitive enzyme immunoassays for lysozyme. Enzyme immunoassay For competition in an immunoassay 500 ~1 of diluted serum, urine, CSF or feces-extract was mixed with 60 ~1 of diluted conjugate. The final content of conjugate in 1 ml of this mixture was 3 ng lysozyme coupled to 16 ng HRP. As a standard we used a serum pool that had been calibrated against solutions with known concentrations of purified human lysozyme in PBS, 1% BSA. This standard serum contained 1.60 pg lysozyme/ml (n = 30, coefficient of variation (CV) = 5.3%). A competition curve obtained with this serum is shown in Fig. 3. The detection limit is about 1 pg/l and the analytical range lies between 1 and 50 pg/l.
1
lldllutlon
of
standard
serum
pool
Fig. 3. Standard curve obtained by using dilutions Vertical bars represent f 2 SD of each value.
TABLE
of a serum pool containing
1.60 gg Iysozyme/mI.
I
Between-run Sample
Serum pool Urine pool CSF pool
variance No. of assays
54 20 41
Lysozyme concentration
(pg/I)
Mean
SD
cv
1620 17.1 56.2
103 1.2 4.6
6.4 7.0 8.2
(W)
27
Validity of the assay The within-run variance on 25 different microtiter plates was calculated from duplicate determinations of 12 to 16 samples which were run as non-adjacent pairs. The CV varied from 2.8 to 6.7% (mean 4.5, SD 1.13). The between-run variance was estimated by repeated analyses of pools of serum, urine and CSF (Table I). Portions of these pools were stored at - 20 o C. Dilutions were made for each assay as were the dilutions of the standard serum. The analytical recovery was measured in the serum pool (Table II) to which purified lysozyme had been added to a final concentration of 4.10 pg/ml. We found a recovery of 101.5% (n = 35, CV = 8, 4%). Part of this pool was used for investigating the effect of repeated freezing and thawing. The sample was stored at - 20 o C, thawed for every assay and frozen again. This procedure was repeated 30 times. The lysozyme concentration remained the same, within experimental error. Lysozyme concentrations were determined in 22 sera by both the described EIA and by rocket electrophoresis [6]. These sera covered the range 1120-4800 pg/l (mean 2438 pg/l). The correlation coefficient was 0.995 with a regression equation y = 0.77x + 412. Lysozyme in serum, urine, CSF and feces We have determined lysozyme in 123 sera and 155 urines from healthy blood donors (aged 18-60 yr) and in 48 portions of feces obtained from laboratory workers, aged 20-40 yr. CSF samples were from 106 patients without any detectable deviation in their CSF or serum [16]. The distributions are shown in Figs. 4, 5 and 6 for serum, urine and CSF. The mean values and reference limits are given in Table II. For serum the non-parametric 2.5 and 97.5 percentile limits are given [17]. The limits given for urine, CSF and feces represent the 0.95 fractions of log-Gaussian distributions. For urine and CSF these limits were nearly the same as the non-parametric 2.5 and 97.5 percentile limits. The number of feces samples analyzed was too low to permit non-parametric estimate of the reference interval. Urinary lysozyme was determined in 12 samples containing between 20 and 1000 leukocytes/pi. Portions of the urines were processed as described, that is cells were removed by centrifugation before freezing the supernatant, whereas other portions were frozen directly and centrifuged only before assay. In the latter cases lysozyme
TABLE II Lysozyme concentrations in serum, urine, CSF and feces Material
Mean
(Median)
Reference limits
Serum Urine CSF
1520 14.5 45.8
(1470) (15.4) (42.9)
950 -2450 1.7 - 123 17.6 - 118
Feces
0.25
(0.27)
0.04-
pg/l pg/l pg/l
1.5 P&Q3
Lysozyme , mg/l
Fig. 4. Distribution
1.6
of lysozyme
in serum.
16
160 Lysozyme
Fig. 5. Distribution of lysozyme detection limit (dotted line).
fig/l
,
in urine.
The concentration
t
Lysozyme
Fig. 6. Distribution
of lysozyme
in CSF.
,
pug/l
in eight
samples
(5.2%) was below
the
29
concentrations were raised by a factor 1.4-3.1 (mean 2.1). The effect of urinary pH on lysozyme was studied using a pool of 30 samples with above-normal concentrations of lysozyme. This pool contained 400 pg/l. Portions of 10 ml were adjusted to pH 3, 4, 5, 6, 7 and 8, and incubated at 37 ‘C. Fractions of 0.5 ml were withdrawn after 0.25, 0.5, 1, 2, 4, 8 and 24 h, diluted with 5 ml PBS/l% BSA and stored at - 20’ C until assay. At every pH the lysozyme concentration remained the same within experimental error.
Discussion The enzyme immunoassay described in this paper has been found to be very reliable for more than a year now. The sensitivity of the assay compares favorably with that of radioimmunoassays for lysozyme [8,9]. Once the microtiter plates have been coated, tests can be completed within 4 h. Although heterogeneity of human lysozyme has been described [18], the dilution curves obtained so far were all parallel to the standard curve, as was found also by Thomas et al [9] using radioimmunoassay. We have not compared the described EIA with the turbidimetric assay. Other authors, however, have described good correlations between immunochemical methods and the enzymatic assay [6,9]. Reference ranges for lysozyme in CSF and feces have not been reported before. The reference range found for normal serum agrees closely with those established by others [9,19] using radioimmunoassays. The concentrations of urinary lysozyme that we determined were considerably lower than those reported by Thomas et al [9]. These authors found a mean value of 450 pg/l in 18 subjects, which is 30 times higher than the mean concentration that we measured (Table II). This discrepancy is unexpected, the more so as their results for serum lysozyme agree closely with our data. Johansson and Malmquist [6], however, found that the level in normal urine is -Z 10% of the mean serum concentration, which was the detection limit in their assay. Increased levels of lysozyme in urine were found if cells had not been removed by centrifugation before freezing. This is, due to lysis of lysozyme-containing neutrophilic granulocytes upon freezing and thawing. Apart from that, we found no correlation between lysozyme values in urine and the number of leukocytes. This lack of correlation has been observed before [20,21]. Determination of urinary excretion of low molecular mass proteins is a well-known method for assessment of renal tubular damage. The most widely used test is that of Qnicroglobulin, because it is commercially available. However, the rapid degradation of this protein at 37OC, especially at pH below 6, is well-documented [22-241. Therefore the use of more stable low M, proteins, e.g. retinol-binding protein, has been advocated [22,24]. From our results it appears that lysozyme might also be a suitable marker, because it is very stable in urine at 37 o C and at pH between 3 and 8 for at least 24 h.
30
References 1 Dick W. Lysozym. Gnmdlagen und diagnostische bedeutung. Fortschr Med 1982; 100: 1230-1234. 2 Dick W. Lysozym-bestimmung. Klinische Signifikanz. Laboratoriumsblatt 1983; 33: 48-56. 3 Osserman EF. Lysozymuria in renal and non-renal diseases. In: Manuel Y, Revillard JP, Bethuel H, eds. Proteins in normal and pathological urine. Basel: S. Karger AG, 1970. 4 Litwack G. Photometric determination of lysozyme activity. Proc Sot Exp Biol Med 1955; 89: 401-403. 5 Osserman EF, Lawlor DP. Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med 1966; 124: 921-951. 6 Johansson BG, Malmquist J. Quantitative immunochemical determination of lysozyme (muramidase) in serum and urine. Stand J Lab Med 1971; 27: 255-261. 7 Goudswaard J, Virella G. Immunochemical determination of human lysozyme by laser nephelometry. Clin Chem 1977; 23: 967-970. 8 Peeters TL, Depraetere YR, Vantrappen GR. Radioimmunoassay for urinary lysozyme in human serum from leukemic patients. Clin Chem 1978; 24: 2155-2157. 9 Thomas MJ, Russo A, Craswell P, Ward M, Steinhardt I. Radioimmunoassay for serum and urinary lysozyme. Clin Chem 1981; 27: 1223-1226. 10 Carlsson J, Drevin H, Ax&r R. Protein thiolation and reversible protein-protein conjugation, N-succinimidyl 3-(2-pyridyldithio) propionate, a new heterobifunctional reagent. Biochem J 1978; 173: 723-737. 11 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London ) 1970; 227: 680-685. 12 Brouwer J, Pluyms WJM, Wamaar SO. Chemical characterization of Rauscher leukemia virus proteins. J Gen Virol 1979; 42: 415-421. 13 Parry RM, Chandan RC, Shahani KM. Isolation and characterization of human milk lysozyme. Arch Biochem Biophys 1969; 130: 59-65. 14 Canfield RE, Collins JC, Sobel JH. Human leukemia lysozyme. In: Osserman EF, Canfield RE, Beychock S, eds. Lysozyme. New York: Academic Press, 1974. 15 Becker W. Determination of antisera titres using the single radial immunodiffusion method. Immunochemistry 1969; 6: 539-546. 16 Brouwer J, Van Leeuwen-Herberts GHHM. Estimation of IgM in cerebrospinal fluid by enzyme immunoassay. Clin Chim Acta 1983; 131: 337-342. 17 Reed AH, Henry RJ, Mason WB. Influence of statistical method used on the resulting estimate of normal range. Clin Chem 1971; 17: 275-284. 18 Virella G. Electrophoresis of lysozyme into Micrococcus-containing agarose gel: quantitative and analytical applications. Clin Chim Acta 1977; 75: 107-115. 19 Venge P, Foucard T, Henriksen J, Hakansson L, Kreuger A. Serum-levels of lactoferrin, lysozyme and myeloperoxidase in normal, infection-prone and leukemic children. Clin Chim Acta 1984; 136: 121-130. 20 Wilkinson SP, Hirst D, Day DW, Williams R. Spectrum of renal tubular damage in renal failure secondary to cirrhosis and fulminant hepatic failure. J Clin Path01 1978; 31: 101-107. 21 Dick W, Theilmann L. Lysozymspiegel in urin bei Kindem mit akuten und chronisch rezidivierenden Harnweg infectionen. Paediatr Paedol 1980; 15: 345-350. 22 Bernard AM, Moreau D, Lauwerys R. Comparison of retinolbinding protein and µglobulin determination in urine for the early detection of tubular proteinuria Clin Chim Acta 1982; 126: l-7. 23 Davey PG, Gosling P. &Microglobulin instability in pathological urine. Clin Chem 1982; 28: 1330-1333. 24 Bastable MD. /3z-Microglobulin in urine: not suitable for assessing renal tubular function. Clin Chem 1983; 29: 996-997.