Development of a rapid and sensitive immunofluorometric assay for glutathione S-transferase A.
The limitations associated with using aminotransferases as serum markers of liver damage may be ameliorated by the inclusion of an additional complementary marker with a short plasma half-life and an even distribution throughout the liver lobule. The glutathione Stransferase (GST) gene superfamily encodes a plethora of enzymes involved primarily in the conjugation of reduced glutathione to active electrophilic species. Cytosolic GSTs occur as dimeric globular proteins composed of subunits with relative molecular masses of 23-29 kDa. In vertebrates, seven classes of soluble GSTs can be distinguished based on genetic and protein structure: [alpha], [kappa], [mu], [pi], [sigma], [theta], and [zeta] (2, 3). Although all human tissues contain GST enzymatic activity, each tissue displays a unique pattern of GST isoenzymes. In humans, the [alpha]-class GSTs (GSTAs) are composed of at least four distinct subunits (A1, A2, A3, and A4) (4, 5). However, in hepatic cytosol, they occur principally as GSTA1-1 and GSTA2-2 homodimers or as GSTA1-2 heterodimers. These isoenzymes are uniformly distributed throughout the liver and are released into the circulation during impairment of hepatocellular integrity. The short plasma half-life of GSTA (~1 h) combined with its even distribution throughout the liver lobule (6) suggests that it may be a useful serum liver marker complementary to the aminotransferases.
The usefulness of GSTA as an indicator of acute and chronic liver damage in patients has been investigated in several clinical studies involving pregnant women with the hemolysis, increased liver enzymes, and low platelet counts syndrome (7,8); neonates of women with hemolysis, increased liver enzymes, and low platelet counts syndrome (9); birth asphyxia (10,11); liver transplant rejection (12, 13); acute alcohol intoxication (14); alcoholic cirrhosis (15); acetaminophen (paracetamol)-induced liver damage (16-19); and acute (20,21) and chronic hepatitis (22-24).
Several groups have described radioimmunoassays for the measurement of GSTA concentrations in biological fluids (10, 21, 25, 26). More recently, several two-site enzyme immunoassays (EIAs) have been developed based on polyclonal anti-GSTA antibodies alone (27-29) or in combination with a monoclonal capture antibody (30). In general, these assays display a short dynamic range and require a prolonged incubation period (usually overnight). A commercially available EIA method (31) is available, and although it can be performed within 4 h, this assay has a short dynamic range. A time-resolved immunofluorometric assay (TR-IFMA) based on polyclonal anti-GSTA antibodies has been described (32).
We recently developed a panel of monoclonal antibodies with high specificity for human GSTAs. In this report, we describe their use in the development of a sensitive and rapid one-step TR-IFMA for measuring total GSTA concentrations in human serum.
Materials and Methods
All general laboratory reagents were purchased from Sigma or Merck unless stated otherwise. Aqueous reagents were prepared in water purified by reversed osmosis followed by filtration with a MilliQ OF-PLUS system (Millipore Corp.). All pH measurements were undertaken at room temperature. Protein concentrations were determined by the Bradford method (33) using a bovine serum albumin (BSA) calibrator (Bio-Rad protein assay; Bio-Rad Laboratories). Hybridomas were maintained in RPMI 1640 (Gibco, Life Technologies), supplemented with 150 mL/L fetal bovine serum (Biological Industries).
PRODUCTION AND PURIFICATION OF GSTs
Recombinant human GSTA1-1 was expressed in Escherichia coli using the plasmid construct pTacGST2 (34). The plasmid was a gift from Dr. P.G. Board (Australian National University, Canberra, Australia). Shake cultures were grown at 37 [degrees]C in Luria-Bertani broth to an absorbance of 0.2 at 600 nm. Protein expression was induced by the addition of 1 mmol/L isopropyl-D-thiogalactoside, and cells were then harvested after 3 h by centrifugation. GSTA1-1 was purified from the bacterial sonicate by gel filtration on Sephadex G50 (Pharmacia) followed by affinity chromatography on S-hexylglutathione (35) coupled to epoxy-activated Sepharose 6B (Pharmacia). Purified recombinant human GSTA1-1 showed GST catalytic enzyme activity, displaying a specific activity of 70 U/mg of protein (1-chloro-2,4-dinitrobenzene as substrate). Recombinant [pi]-class GST (GSTP) was produced using a cDNA clone encoding GSTP1-1 (ATCC). The cDNA clone was expressed in Sf9-insect cells using a baculovirus approach (Invitrogen) and purified by affinity chromatography to a specific activity of 35 U/mg (1-chloro-2,4dinitrobenzene as substrate).
Human liver GSTA and [alpha]-class GST (GSTM) were purified from resection specimens essentially as described by Meyer and Ketterer (36). In brief, total GST was isolated from the 105 000g cytosolic fraction by chromatography on glutathione-Sepharose 4B (Pharmacia), and the [alpha]- and [mu]-class isoenzymes were resolved by hydroxyapatite chromatography. The [alpha]-class isoenzymes GSTA1-1 and GSTA1-2 were isolated by an additional ion-exchange fast protein liquid chromatography step using a mono-Q column (Pharmacia). The acquisition of all human materials used in this study adhered strictly to approved institutional guidelines. Recombinant GSTA1-1 and liver-derived GSTA were iodinated using the indirect Iodogen method.
Female BALB/c mice (6-8 weeks of age; Harlan Olac Ltd.) were primed by subcutaneous injection of 25 [micro]g of GSTA emulsified in Freund's complete adjuvant. Booster immunizations of 50 [micro]g of GSTA (subcutaneous) in Freund's incomplete adjuvant were given at 1 and 3 months after the initial priming dose. Five months after the original priming dose, four daily intraperitoneal boosts of 100 [micro]g of GSTAs in saline were given immediately before fusion (37). Hybridomas were produced by the polyethylene glycol-facilitated fusion of splenocytes to the nonsecreting NSO myeloma cell line (Medical Research Council of Molecular Biology, University Postgraduate Medical School, Cambridge, England) (38).
Cell culture supernatants were screened for the anti-GSTA monoclonal antibody using an antigen-capture assay in 96-well microtiter wells with [sup.125.I]-radiolabeled GSTA as the tracer. Selected parental hybridomas were subcloned twice by limiting dilution and injected into mice for ascites production. Monoclonal antibodies were purified from ascites fluid by protein A-Sepharose 4B (Pharmacia) chromatography. The purified antibodies were sterile filtered and stored in phosphate-buffered saline at 4 [degrees]C. Monoclonal antibodies were isotyped using the Isostrip method (Roche Diagnostics GmbH).
PREPARATION OF ASSAY SOLID PHASE
The solid-phase monoclonal antibody L1 was incubated for 10 min at room temperature in 0.1 mol/L glycine (pH 2.5) at a concentration of 150 mg/L. The acid-treated antibody was then diluted to 5 mg/L in 0.2 mol/L Na[H.sup.2]P[O.sub.4] (pH 4.3) and stirred at room temperature for 10 min. Microtiter plates (96-well; Nunc-Immuno Maxisorp C12 microtitration strips; Nunc) containing 1 [micro]g (0.2 mL) of acid-treated L1 per well were incubated at 37 [degrees]C for 24 h and then rinsed twice with wash buffer (0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, 1 g/L Germall, 0.5 mL/L Tween 20, pH 7.3). The plate surface was blocked at room temperature for 24 h with 300 [micro]L/well of a buffer containing 1 g/L BSA, 0.05 mol/L Tris-HCl, 60 g/L sorbitol, 0.2 mmol/L diethylenetriamine pentacetic acid, and 0.5 g/L Na[N.sub.3] (pH 7.3). After aspiration of the blocking solution, plates were dried at room temperature and stored over desiccant at 4 [degrees]C.
CONJUGATION OF ANTIBODIES WITH [Eu.sup.3+] CHELATES
The monoclonal antibody LD45 was conjugated to a europium chelate using the Delfia Eu-Labeling reagent set (Wallac). The antibody was incubated with a 12.5 molar excess of [Eu.sup.3+]-labeling reagent in 0.1 mol/L sodium borate buffer (pH 8.6) at room temperature for 48 h. Free label was separated from the conjugate by gel filtration using an elution buffer containing 0.5 mol/L NaCl, 0.05 mol/L Tris-HCl, 0.5 g/L Na[N.sub.3] (pH 7.8). The stock conjugate was stored at 4 [degrees]C.
CALIBRATORS AND CONTROLS
Calibrators were prepared by dilution of purified recombinant GSTA1-1 in matrix buffer (0.05 mol/L Tris-HCl, 0.1 mol/L NaCl, 1 g/L Germall containing 60 g/L BSA) to 0, 1, 5, 25, 125, and 625 [micro]g/L; the dilutions were then calibrated against the purified liver GSTA calibrators contained in the Hepkit-Hm EIA (Biotrin). Two control samples containing heat-inactivated normal human serum (60 [degrees]C for 30 min) supplemented with liver GSTA (2 and 50 [micro]g/L) were included in every assay. Calibrators and controls were stable at 4 [degrees]C for 2 weeks and at -70 [degrees]C for at least 12 months.
For assaying samples, each well received 200 ng of [Eu.sup.3+]-LD45 antibody in 150 [micro]L of assay buffer [0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, 0.02 mol/L diethylenetriamine pentacetic acid, 0.5 g/L Na[N.sub.3], 0.1 mL/L Tween 20, 20 mg/L Amaranth, 0.5 g/L BSA, 0.5 g/L Bovine IgG, and 15 mg/L MAK33-IgG (Roche Molecular Biochemicals), pH 7.8]. Twenty-five microliters of each calibrator or sample was then added to duplicate wells, followed by continuous shaking at room temperature for 30 min. The plates were washed six times with wash buffer before 200 [micro]L of Delfia-enhancement solution (Wallac) was added. After incubation with shaking at room temperature for 5 min, fluorescence was measured in a time-resolved fluorometer (VICTOR 1220 multilabel counter; Wallac), and GSTA concentrations were calculated by the Wallac Multicalc program.
Reference values for GSTA in serum were determined in a group of blood donors, of whom 104 were female (age range, 21-66 years; median, 41 years) and 104 were male (age range, 21-69 years; median, 41.5 years). Serum samples from a group of colon and rectum cancer (n = 20) and osteosarcoma (n = 76) patients were used in studying the correlation between GSTA measurements by our assay and the Hepkit-Hm EIA (human GSTA EIA). Serum samples (n = 88) from osteosarcoma patients were used to study the stability of GSTA in serum stored at -20 [degrees]C. Statistical calculations were performed using Microsoft Excel 97 software.
The analytical detection limit (i.e., the lowest detectable GSTA concentration that could be distinguished from zero using statistical criteria) was calculated by the Wallac Multicalc program (Wallac). Between-assay imprecision (mean CV) was determined from analysis of 585 calibrators and serum samples in duplicate in 15 subsequent assays. Within-assay imprecision was determined from 24 replicates of three different sera in one assay and 4 replicates of two control samples in 15 subsequent assays.
Spleen cells from a mouse immunized with recombinant human GSTA were fused with the NSO myeloma cells and gave 19 positive clones when screened in an antigen capture assay using 125I-radiolabeled recombinant human GSTA. Two of the antibodies (L1 and L3) performed well in a two-site immunoassay for recombinant human GSTA1-1, but they performed poorly in assays of patient sera or human liver-derived GSTA. A second fusion was undertaken using human liver GSTA as the immunogen. Hybridomas from this fusion were tested in a sandwich assay using human liver GSTA with 1251-radiolabeled monoclonal antibody L1 as tracer. This second fusion gave 62 positive clones, 5 of which were subcloned twice, expanded, and then injected into mice for ascites production.
Pair combinations of the six selected monoclonal antibodies were evaluated in two-site immunoradiometric assays. The monoclonal antibody L1 performed particularly well as the solid-phase antibody together with LD45 as the tracer antibody. This antibody combination did not detect GSTP1-1 or GSTM concentrations as high as 350 [micro]g/L, but it gave identical titration curves for GSTA from human liver, recombinant GSTA1-1, and purified human liver GSTA1-1 and GSTA1-2 (data not shown). Isotyping demonstrated that both antibodies were of the IgG1 ([kappa]) class.
PREPARATION OF [Eu.sup.3+]-LABELED ANTIBODY
Europium labeling of the tracer antibody LD45 led to a calculated incorporation of four to five europium chelate molecules per antibody molecule. In the TR-IFMA, this degree of incorporation gave a signal of 5 000 000 cps with the highest GSTA calibrator (625 [micro]g/L) with a background of 400 cps. The tracer was stable at 4 [degrees]C for at least 12 months and consistently gave low nonspecific binding.
KINETICS AND ASSAY FORMAT
Calibrators containing 1-625 [micro]g/L GSTA and serum controls were used for comparing a two-step and a one-step assay. In the two-step assay, calibrators and controls were incubated with the solid-phase antibody for 30 min, which was then washed before incubation for 30 min with a [Eu.sup.3+]-labeled detector antibody. In the onestep assay, the sample and [Eu.sup.3+]-labeled antibody were incubated simultaneously with the solid phase for 30 min. The sensitivities and the slopes of the dose-response curves with GSTA calibrators were similar in both the one-step and two-step procedures (data not shown). However, in the one-step assay, nonspecific binding was lower and the calibrators and serum samples gave higher counts than in the two-step assay. Consequently, only the one-step assay was evaluated further. Calibrators and serum controls were used to determine assay kinetics. Both the calibrators and serum samples reached plateau values within 30 min. A dose-response curve using recombinant GSTA1-1 of 20-20 000 [micro]g/L in a series of twofold dilutions revealed a response maximum at 2500 [micro]g/L GSTA, with only a slight high-dose "hook effect". Assay linearity was assessed using three different serum samples. These were diluted 2- to 32-fold in matrix buffer and produced linear slopes parallel with the calibration curve.
DETECTION LIMITS AND PRECISION
The between-assay imprecision (mean CV) was <5% in a series of 585 calibrators and patient samples analyzed in 15 independent assays. The dose-response of the TRIFMA was nearly linear for calibrators containing 1-625[micro]g/L GSTA (Fig. 1) with an analytical detection limit of 0.07 [micro]g/L. Within-run imprecision was studied by measuring three different sera (311, 111, and 25 [micro]g/L GSTA) and was 1.8-2.6% (mean, 2.1%). Additionally, between-and within-run CVs were 2.2% and 1.4%, respectively, for two 50 [micro]g/L GSTA control samples and 2.9% and 1.8%, respectively, for 2.5 [micro]g/L GSTA control samples. GSTA recovery was studied using serum specimens containing a known amount of GSTA to which 2 or 60[micro]g/L human liver GSTA was added. Apparent mean recoveries for GSTA were 97% [+ or -] 3% and 93% [+ or -] 2%, respectively.
Assay interference by hemolysis or increased bilirubin concentrations was investigated using normal serum supplemented with purified human hepatic GSTA to a concentration of 150 [micro]g/L. Whole blood was lysed by repeated freezing and thawing, giving a homogeneous lysate with a hemoglobin concentration of 133 g/L. One sample of the GSTA-supplemented serum received whole-blood lysate; bilirubin was added to the second and third serum samples, giving total bilirubin concentrations of 136 and 14 mg/L, respectively. The recoveries of supplemented GSTA in these serum samples were then measured. There was no interference by hemolysis (mean recovery, 101%) or bilirubin (mean recoveries, 105% and 103% for 136 and 14 mg/L bilirubin, respectively).
Interference of lipemia was investigated using serum samples from three patients with high triglyceride concentrations (4.09-5.40 mmol/L). Normal human serum and the three patient sera were supplemented with purified human GSTA to a target concentration of 100 [micro]g/L. GSTA concentrations were determined in both the supplemented sera and the patient sera without added GSTA. The mean recovery of the added GSTA in these serum samples was 96%.
SAMPLE STORAGE AND STABILITY
Repeated freezing and thawing (four times) of 11 serum samples did not cause any significant change in the measured GSTA concentration. There were no significant changes in GSTA concentrations (range, 1-3330 F[micro]g/L) in 88 serum samples from osteosarcoma patients determined before and after 12 months of storage at -20 [degrees]C.
[FIGURE 1 OMITTED]
The concentration of GSTA in the sera of 104 healthy females was 0.6-11.7 [micro]g/L, with a median of 2.0 [micro]g/L. The concentration of GSTA in sera of 104 males was 0.7-12.4 [micro]g/L, with a median of 2.6 [micro]g/L. The GSTA concentrations followed a gaussian distribution on a logarithmic scale in both sexes. On the logarithmic scale, the reference intervals (mean [+ or -] 1.96 SD) were 0.6-7.2 [micro]g/L for women (mean, 2.0 [micro]g/L) and 0.7-9.8 [micro]g/L for men (mean, 2.6 [micro]g/L). The differences between sexes were statistically significant (P = 0.01) by a two-tailed Mest. We assayed 76 serum samples from osteosarcoma patients shortly after high-dose chemotherapy and 20 serum samples from patients with colon and rectum cancer, using both the TR-IFMA and Hepkit-Hm EIA. GSTA values measured in the two assays correlated well ([r.sup.2] = 0.98; y = 0.82x - 0.38), as shown in Fig. 2. High serum concentrations of GSTA were also noticed 2 h after the start of high-dose intravenous chemotherapy in osteosarcoma patients. In one of these patients, serum GSTA concentrations >3000 [micro]g/L were seen (Fig. 3).
Increased serum GSTA is a sensitive indicator of acute liver damage in humans (1). To date, no assay for GSTA has been described that possesses the speed and simplicity required for use in the routine clinical laboratory setting. Immunoassays have been produced that use polyclonal antibodies alone or in combination with monoclonal antibody-based solid phases (10,21,25,29-32). However, these methods are characterized by either a requirement for prolonged incubation times (often overnight) or cumbersome methodologies. A single commercially available polyclonal antibody-based EIA method for GSTA is on the market, but it requires multiple incubation steps and has a poor dynamic range. A common problem with using polyclonal antibodies as reagents in immunoassays is that different batches of antibodies may vary markedly in their specificity and affinity for antigen and hence require extensive pretesting. This is of particular concern when the antigen is a member of a closely related gene family. In this regard, it is surprising that no immunoassay for GSTA that is based entirely on monoclonal antibodies has been described to date. This may be attributable to difficulties in raising monoclonals to GSTA that perform well as antibody pairs. In the present study, >6000 hybridoma clones were screened before we obtained antibodies suitable for use in a sandwich-type immunoassay for GSTA.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Initially, we constructed panels of hybridomas using splenocytes from mice immunized with recombinant GSTA1-1. Monoclonal antibodies were selected on their ability to bind recombinant GSTA1-1 in an antigen capture assay. Several antibody pairs were subsequently chosen for the construction of an immunoradiometric assay. During the development of the assay, it became apparent that all of the antibody pairs selected reacted preferentially with the recombinant antigen. This observation suggested that, despite similar specific enzymatic activities, the recombinant GSTA1-1 is structurally dissimilar from the liver-derived enzyme. A second series of fusions were undertaken using human liver GSTA as immunogen. These fusions were screened in an immunoradiometric assay using human liver-derived GSTA and one of our antirecombinant GSTA1-1 antibodies (L1) as the tracer. In this way, only those antibodies with good reactivity to natural GSTA and the ability to form a pair with monoclonal L1 were detected. Positive clones from this initial screen were further selected based on their specificity (no cross-reactivity with GSTP and GSTM) and equal reactivity with human liver-derived GSTA1-1 and GSTA1-2.
The L1 and LD45 monoclonal antibodies were finally selected and used to develop a one-step TR-IFMA for measurement of GSTA in serum. The L1 antibody was chosen as the solid-phase antibody. The L1 antibody is of the IgG class and hence avoids problems of loss of solid-phase capacity through activation of the complement system (39). Before plate coating, L1 was acid-treated to increase adsorption efficiency. This is a routine procedure in our laboratory and presumably acts by exposing hydrophobic protein domains by partial denaturation. After blocking and drying, the resulting solid phase was stable for at least 12 months when stored desiccated at 4 [degrees]C. Europium labeling of the tracer antibody LD45 was straightforward with the use of a commercially available europium chelate reagent. The tracer was stable at 4 [degrees]C for at least 15 months and did not suffer from aggregation problems, as revealed by consistently low nonspecific binding.
All of the GSTA isoenzymes appear to be expressed in human liver, but the proportions are variable (40, 41). Because of this interindividual variation, we chose to develop an assay using a pair of monoclonal antibodies selected originally for their ability to bind to both GSTA subunits. The commercially available EIA for measuring GSTA in biological fluids (Hepkit) is reported by the manufacturers to detect total GSTA. A comparison of our TR-IFMA and the Hepkit showed good correlation ([r.sup.2] = 0.98), further indicating that our method detects total GSTA.
The TR-IFMA has a wide measuring range, 0.07-625 [micro]g/L, and a 10-fold sample predilution would be expected to cover most GSTA concentrations in patient samples. Furthermore, a dose-response curve revealed only a minimal high-dose hook effect at GSTA concentrations much higher than that of the highest TR-IFMA GSTA calibrator. Analytical recovery of GSTA was good, and the assay displayed good within- and between-run CVs.
Although we observed differences in immunoreactivity between recombinant and liver-derived GSTA, both antibodies used in our TR-IFMA bind to epitopes present on recombinant GSTA. This allowed the use of recombinant GSTA1-1 to prepare assay calibrators. The use of recombinant GSTA prevents the ethical and practical difficulties associated with the acquisition of human liver tissue.
Our reference intervals were calculated from measurements in serum samples from 208 blood donors. The upper reference limit of 7.2 [micro]g/L for females (n = 104) was in agreement with the 6 [micro]g/L for women (n = 48) reported by Tiainen and Karhi (32), whereas our corresponding value for males, 9.8 [micro]g/L (n = 104), was somewhat different from theirs (14 [micro]g/L; n = 48). This could be attributable to differences between populations and factors such as the pattern of alcohol consumption. The overall upper reference value reported for the Hepkit (31) is given as 8.0 [micro]g/L (n = 219), but no information on the differences in GSTA concentrations between men and women is provided. Measurements of GSTA in serum samples from osteosarcoma patients taken at multiple time points during the first 3 days after high-dose intravenous methotrexate chemotherapy showed markedly increased values a few hours after the infusion was started. GSTA measurements may provide new information of possible hepatotoxic effects of high-dose chemotherapy.
In conclusion, we describe the development of a new TR-IFMA for GSTA in human serum. The method is novel because it is monoclonal antibody-based, requires 35 min of incubation, and is standardized using a recombinant antigen expressed in E. coli. This assay may be of use in evaluating the potential of GSTA monitoring in the routine clinical setting.
This work was supported by the Norwegian Cancer Society. The skilled technical assistance of Anders Andersen, Tone Varaas, and Kari Hauge Olsen is greatly appreciated. We also thank Dr. Kirsten Sundby Hall (Department of Oncology, The Norwegian Radium Hospital) for providing the patient serum samples. The NSO cell line was obtained from the Medical Research Council of Molecular Biology, University Postgraduate Medical School, Hills Road, Cambridge, CB2 2QH, England [Clark MR, Wright BW, Milstein C (developers); described by Galfre and Milstein (38)].
Received November 28, 2000; accepted February 8, 2001.
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(41.) Hayes JD, Kerr LA, Cronshaw AD. Evidence that glutathione Stransferases 131131 and 132132 are the products of separate genes and that their expression in human liver is subject to inter-individual variation. Molecular relationships between the 131 and B2 subunits and other a class glutathione Stransferases. Biochem J 1989;264:437-45.
 Nonstandard abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GSTA, -M, and -P, [alpha]-, [mu]-, and [pi]-class glutathione S-transferase; ElA, enzyme immunoassay; TR-IFMA, time-resolved immunofluorometric assay; and BSA, bovine serum albumin.
LAILA K. DAJANI,* ELISABETH PAUS, and DAVID J. WARREN
Section for Clinical Pharmacology, Central Laboratory, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
*Author for correspondence. Fax 47-22-93-46-86; e-mail firstname.lastname@example.org.
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|Title Annotation:||Enzymes and Protein Markers|
|Author:||Dajani, Laila K.; Paus, Elisabeth; Warren, David J.|
|Date:||May 1, 2001|
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