A rapid ELISA for measurement of antibodies to nucliec acid antigens using UV-treated polystyrene microplates

Journal oflmmunologicalMethods, 90 (1986) 105-110 Elsevier

105

JIM03936

A rapid ELISA for measurement of antibodies to nucleic acid antigens using UV-treated polystyrene microplates Moncef Zouali and B . David Stollar * Tufts University Health Science Campus, Department of Biochemistry and Pharmacology, Boston, MA 02111, U.S.A. (Received 20 December 1985, accepted 24 January 1986)

Pretreatment of polystyrene microplate wells with certain doses of UV light enhances their capacity for binding to single-stranded DNA, double stranded DNA and various synthetic polynucleotides. The use of UV-irradiated plates to immobilize nucleic acid antigens provides a simple, rapid, and specific ELISA for measuring anti-nucleic acid antibodies. The assay is at least as sensitive as the more complex method of precoating plates with poly(L-lysine). It is useful for detection of anti-DNA antibodies in sera of systemic lupus erythematosus patients, as well as in culture fluids of murine and human anti-DNA-secreting hybridomas. Key words: EL1SA; Anti-DNA antibodies," Systemic lupus erythematosus," UV irradiation

Introduction

One of the most important serological features of SLE is the presence of high levels of antibodies to the nucleic acid antigen DNA. While the origin of this hyperproduction remains unresolved, it is generally accepted that antibodies to DNA are involved in the pathogenesis of the glomerulonephritis associated with this autoimmune disease (Koffler, 1974; Ebling and Hahn, 1980). Furthermore, these serological markers are of diagnostic and prognostic value: a rise in antibody levels frequently precedes an exacerbation of the disease, and their decrease is associated with an improvement in the clinical condition (Swaak et al., 1979; Hochberg et al., 1985). A number of methods have been reported for the detection of anti-DNA antibodies, including * To whom correspondence should be addressed. Abbreoiations: BSA, bovine serum albumin; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; SLE, systemic lupus erythematosus.

counter-immunoelectrophoresis (Davis and Winfield, 1974), complement fixation (Stollar, 1977), indirect hemagglutination (Koffler et al., 1971), immunofluorescence using nuclei of Crithidia luciliae as the antigenic substrate (Aarden et al., 1975), radioimmunoassay (Wold et al., 1968; Aarden et al., 1976) and enzyme immunoassays (Aotsuka et al., 1979; Klotz et al., 1979). The technique using enzyme-labelled antibodies (ELISA) is a widely used method that permits detection of antibodies to single stranded (ssDNA), double-stranded DNA (dsDNA) and synthetic polynucleotides. A critical step for the ELISA is the immobilization of nucleic acid antigens on the solid phase. Efficient immobilization may require precoating with poly(L-lysine) (Aotsuka et al., 1979), or linking sonified DNA to methylated bovine serum albumin (BSA) carrier (Rubin et al., 1983). To circumvent the use of DNA-protein conjugates as a coating antigen, which might alter the conformation of DNA, and to avoid the use of poly(L-lysine) precoating which is a tedious step,

0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

106 we developed a simple method for efficient immobilization of nucleic acid antigens on the solid phase. This procedure involves UV pretreatment of polystyrene plates. We examined the sensitivity of this assay compared to a conventional ELISA using poly(L-lysine) pretreated plates, a method we have been using routinely in our laboratory during the last few years. We also evaluated its applicability to lupus sera and to murine and human monoclonal antibodies generated by hybridomas. Materials and methods

Sera A reference human serum monospecific for dsDNA was obtained from the AF-CDC ANA Reference Laboratory (Center for Disease Control, Atlanta, GA). SLE sera were from previously described panels, and had been shown to react with D N A in c o m p l e m e n t fixation, immunofluorescence, or radioimmunoassays (Miniter et al., 1979; Papalian et al., 1980; Lafer et al., 1983).

Murine and human monoclonal antibodies Generation and characterization of the monoclonal antibodies used in this study have been described in detail (Andrzejewski et al., 1981; Shoenfeld et al., 1983b). Human myeloma proteins were also used (Zouali et al., 1984a).

Nucleic acid antigens Calf thymus dsDNA was from PL-Biochemicals (now Pharmacia Molecular Biologicals, Piscataway, N J) and was purified further as described (Papalian et al., 1980). ssDNA was obtained by heating the native form at 100°C for 10 min and then quickly chilling it in an ice-water bath. Synthetic polynucleotides were also from PL Biochemicals.

Coating procedures Three coating procedures were compared for immobilization of nucleic acid antigens on polystyrene plates. (1) Poly(L-lysine) precoating procedure. Polystyrene plates (Immulon type 1, Dynatech Labs., Virginia) were incubated with a poly(L-lysine)

solution (50 /~g/ml in distilled water) for 30 min at RT. The plates were washed three times with Tris-buffered saline (10 mM Tris, 150 mM NaC1, pH 7.4; TBS) and then coated with 100 /~1 of nucleic acid antigen at a concentration of 5/~g/ml in TBS for 2 h at RT. They were washed with TBS, saturated by incubation with poly(L-glutamate) (50 /Lg/ml in TBS) for 2 h at RT, and finally washed with TBS. (2) Dry coating procedure. Plates were incubated with 100/~1 of the nucleic acid solution (5 /zg/ml in TBS) for 18 h at 37°C, and then washed with TBS to remove unbound antigen. (3) UV-pretreatment procedure. Polystyrene microtiter plates were irradiated with the UV germicidal lamp (Atlantic UV Corp., New York, G36T6L, 2537A, 120 /~W at 1 m) of a laminar flow biosafety tissue culture hood at a distance of 65 cm for periods varying from 15 min to 15 h. They were then incubated with 100 #l of the nucleic acid solution (5/~g/ml in TBS) for periods varying from 15 rain to 12 h at RT, after which they were washed with TBS.

Solid-phase enzyme immunoassay for nucleic acidbinding antibodies After they were coated with antigen and washed, the plates were saturated by incubation with phosphate-buffered saline (10 mM sodium phosphate, 140 mM NaCI, pH 7.4; PBS) containing 1% BSA (RIA grade, Sigma Chemical Corp., St. Louis, MO) (PBS-BSA) for 2 h at RT. A volume of the antibody or serum to be tested, diluted in PBSBSA, was added. After 1 h of incubation at RT, the plates were washed with PBS containing 0.1% Tween 20 (Fisher). Bound antibodies were revealed with an appropriate anti-immunoglobulinalkaline phosphatase conjugate, with the substrate p-nitrophenyl phosphate (Sigma) 0.1% diluted in a 0.05 M carbonate buffer, pH 9.5 containing 2 mM MgC12. The absorbance at 405 nm was read with a multiscan automatic reader (Dynatech, Alexandria, VA). To ensure the specificity of this assay, antibody samples were simultaneously assayed against plates treated in the same manner, but to which no nucleic acid antigen was added. All samples were tested in duplicate and arithmetic means of absorbance values were calculated.

107 Results

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Development of the coating procedure

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Considering the possibility that UV light irradiation may lead to formation of reactive groups in polystyrene, possibly by oxidative mechanisms (Rabek, 1985), we tested the ability of UV to increase the binding of ssDNA to polystyrene ELISA wells. As a first step, plates were irradiated with various doses of UV, to determine the optimal UV dose for nucleic acid binding to the solid phase that allows accurate measurements of autoantibodies. The second step was to determine the minimal period of incubation period with nucleic acid antigen required to give maximal sensitivity in the ELISA assay. In these experiments we used both murine and h u m a n monoclonal lupus antibodies. When the ELISA plates were irradiated for varying times before they were coated with a s s D N A solution for 2 h, optimal absorbance readings were reached after UV irradiation of the plates for 12 h (Fig. 1). It is also notable that untreated wells did not bind detectable amounts of D N A under these conditions. We next tested ELISA plates that were irradiated overnight and then incubated with a s s D N A solution (5 /~g m1-1 in TBS) for varying times at RT. We found that the optimal A405 n m was reached after a 2 h incubation period of the plates with s s D N A (Fig. 2). During the course of these experi-

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ments we also found that the background readings, i.e., the a b s o r b a n c e o b t a i n e d with sham-treated wells, were virtually negative. Based on these results we selected two parameters for coating: a minimum of 12 h as the period for UV treatment of the plate and a minimum of 2 h as the incubation period of the irradiated plate with the nucleic acid antigen. In addition we showed that after UV irradiation the polystyrene solid phase remains active for D N A binding for at least 10 days.

Comparison of UV irradiation to poly(L-lysine) pretreatment of the plates To assess the accuracy and validity of the values obtained in the ELISA using UV pretreatment, compared to that obtained using poly(Llysine) pretreatment of the plates, we tested a series of 11 human anti-DNA secreting hybridomas at various dilutions for binding to ssDNA immobilized by either of the two procedures. As shown in Fig. 3, most of the samples gave higher absorbance values at 405 nm when tested against ssDNA immobilized on UV-irradiated polystyrene plates. In other experiments, we found that a series of 15 human myeloma proteins of various isotypes exhibited no binding to UVtreated DNA-coated plates. Similarly, 20 monoclonal antibodies against 4(-hydroxy-3-nitrophenyl)acetyl generated from various mouse strains (from Dr. T. Imanishi-Kari) were negative on DNA-coated plates.

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Applicability of the UV-coating procedure to nucleic acid antigens other than ssDNA Because autoantibodies to D N A also react with polynucleotides (Andrzejewski et al., 1981; Shoenfeld et al., 1983b), we tested whether synthetic polynucleotides could be immobilized on UVtreated polystyrene plates. We found that the procedure described above for s s D N A is also applicable to poly(I), poly(dT) and poly(G) (not shown). We also were able to use the UV-treated, s s D N A coated plates for competition assays, where the binding of a monoclonal a n t i - D N A antibody is inhibited by preincubation in liquid phase with nucleic acid antigen competitors.

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Fig. 5. Binding of the international reference C D C anti-dsDNA human serum ( A ) and serum of SLE patient 'AND' ( B ) to dsDNA.

109

immobilized on polystyrene plates by dry coating, poly(L-lysine) pretreatment, or UV irradiation of the solid phase. As represented in Fig. 4, pretreatment of the plates with poly(L-lysine) gave similar results as pre-irradiation with UV. In contrast, immobilization of dsDNA by dry coating yielded lower absorbance readings at 405 nm. A critical concern was then to ensure that coating with dsDNA after pre-irradiation of the plate did not result in denaturation of the dsDNA. This was excluded by the finding that monoclonal antibodies to ssDNA did not interact with dsDNA immobilized on UV-treated polystyrene plates. To determine whether anti-dsDNA antibodies could be measured in SLE sera without interference by unrelated immunoglobulins, we tested a reference human anti-dsRNA serum and sera obtained from SLE patients during active periods of their disease. We found that the UV treatment and coating procedure was at least as sensitive as the procedure using poly(L-lysine) pretreatment of the plates. The dry-coating method was again much less sensitive (Fig. 5).

Discussion

In recent years, many studies have focused on generation and characterization of monoclonal anti-DNA antibodies in relation to SLE. The establishment of murine (Andrzejewski et al., 1981) as well as human (Shoenfeld et al., 1983b) secreting hybridomas represents a powerful approach to understanding the origins of autoimmunity (Schwartz and Stollar, 1985). The ELISA assay has been used for screening hybridomas for examining the binding specificities of monoclonal lupus antibodies toward nucleic acid antigens (Andrzejewski et al., 1981; Shoenfeld et al., 1983b), and for isotypic (Gavalchin et al., 1985) and idiotypic (Shoenfeld et al., 1983a; Zouali et al., 1984b),characterization of autoantibodies. It is also routinely used for clinical evaluation of anti-DNA antibody in autoimmune rheumatic diseases. Investigators have had variable results in immobilizing DNA on the solid phase for immunoassay purposes. Because binding of labelled DNA to polystyrene wells is generally low (Fish

and Ziff, 1981), pretreatment of polystyrene with poly(L-lysine) was introduced (Aotsuka et al., 1979). In the present paper we have outlined a simple, rapid and reproducible method for detection of anti-DNA antibodies using UV-pre-irradiated polystyrene wells. The ELISA methodology described herein produced measurements of antiDNA antibodies that were similar to those obtained by using poly(L-lysine) plates, a tedious technique that has been used in our laboratory for several years. The two methods were comparable in their selectivity, the UV-coating procedure being slightly more sensitive. This procedure requires an overnight irradiation of the plates, followed by a 2 h incubation with the nucleic acid antigen at RT. It gives virtually negative background readings at 405 nm in irradiated wells blocked with BSA and lacking nucleic acid antigen. It is applicable to purified monoclonal antibodies, culture fluid containing antibodies, and whole sera. It permits measurement of antibodies to ssDNA, dsDNA and various synthetic polynucleotide antigens. The mechanism whereby UV irradiation increases the binding capacity of polystyrene wells is unknown. Hydrocarbon polymers with saturated backbone chains are susceptible to oxidation involving singlet 02 (Rabek, 1985). UV treatment of polystyrene in the presence of air has been associated with ring opening oxidation and formation of chemically reactive (Rabek, 1985) muconic dialdehyde structures. This may occur through formation of a dioxetane intermediate. During photooxidation of polystyrene, polyene structures in the main chain can also be generated. In this case 02 oxidation may generate reactive peroxides on the polymer: The ELISA described in this paper utilizes a common germicidal lamp and as far as possible commercially available reagents. The use of alkaline phosphatase as the enzyme permits the presence of azide in the reagents and has produced very reproducible assays in several laboratories. Acknowledgements Dr. M. Zouali was supported by a National Institutes of Health John E. Fogarty International

110

Fellowship (1 F05 TW-1862). This research was supported in part by Grant AM31151 from the National Institutes of Health and by Seragen Inc.

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