Automated Detection of Anti-Double-Stranded DNA Antibody in

Anal. Chem. 1999, 71, 1298-1302

Automated Detection of Anti-Double-Stranded DNA Antibody in Systemic Lupus Erythematosus Serum by Flow Immunoassay Tae-kyu Lim, Yumiko Komoda, Noriyuki Nakamura, and Tadashi Matsunaga*

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

We describe a novel automated flow immunoassay system for quantification of anti-double-stranded (ds) DNA autoimmune antibodies in the serum of patients suffering from systemic lupus erythematosus. dsDNA (360 bp) was covalently coupled with alkaline phosphatase (ALP) to form a novel analytical reagent (ALP-DNA). After immunoreaction, antibody-antigen complexes between ALPDNA and anti-dsDNA monoclonal antibody were separated from unreacted ALP-DNA by an ion-exchange column on the basis of the difference in isoelectric point. Antibody-antigen complexes were subsequently quantified by luminescence following addition of 3-(2′-spiroadamantane)-4-methoxy-4-(3′′-phosphoryloxy)phenyl-1,2-dioxetane. The assay yielded a linear relationship between signal and concentration of anti-dsDNA monoclonal antibody in the range of 0-300 µg/mL. This simple technique permits the assay of anti-dsDNA autoimmune antibodies within 25 min. The ion-exchange column was simply regenerated by occasional elution with eluent (20 mM N-methylpiperazine, pH 5.5) supplemented with 0.5 M NaCl, to remove unreacted ALP-DNA. Autoimmune diseases are characterized by biosynthesis of antibodies against one or more components of the patient. Systemic lupus erythematosus (SLE) is widely considered to be an autoimmune disease affecting several organs.1-3 It is typified by the biosynthesis of antibodies directed against self-DNA and other nuclear antigens4,5 such as histones6-8 and soluble nuclear proteins, e.g., Smith antigen and ribonucleoproteins.9,10 SLE is * Corresponding author: (fax) +81-42-385-7713; (e-mail) [email protected]. (1) Tan, E. M.; Cohen, A. S.; Fries, J. S.; Masi, A. T.; McShane, D. J.; Rothfield, N. F.; Schaller, J. G.; Talal, N.; Winchester, R. J. Arthritis Rheum. 1982, 24, 1271-1277. (2) Ravirajan, C. T.; Sarraf, C. E.; Anikumar, T. V.; Golding, M. C.; Alison, M. R.; Isenberg, D. A. Clin. Exp. Immunol. 1996, 105, 306-312. (3) Koutouzov, S.; Cabrespines, A.; Amoura, Z.; Chabre, H.; Lotton, C.; Bach, J. F. Eur. J. Immunol. 1996, 26, 472-486. (4) Termaat, R. M.; Assemann, K. J. M.; van Son, J. P. H. F.; Dijkman, H. B. P. M.; Koene, R. A. P.; Berden, J. H. M. Lab. Invest. 1993, 68, 164-173. (5) Sanchez-Guerrero, J.; Lew, R. A.; Fossel, A. H.; Schur, P. H. Arthritis Rheum. 1996, 39, 1055-1061. (6) Chabre, H.; Amoura, Z.; Pitte, J. C.; Godeau, P.; Bach, J. F.; Koutouzov, S. Arthritis Rheum. 1995, 38, 1485-1491. (7) Mizushima, N.; Kubota, T.; Nanki, T.; Koike, R.; Kohsaka, H.; Miyasaka, N. Rinsho Byori 1996, 44, 585-589. (8) Spronk, P. E.; Limburg, P. C.; Kallenberg, C. G. Lupus 1995, 4, 86-94. (9) Guialis, A.; Patrinou-Georgoula, M.; Tsifetaki, N.; Aidinis, V.; Sekeris, C. E.; Moutsopoulos, H. M. Clin. Exp. Immunol. 1994, 95, 385-389.

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further characterized by an increase in the level of natural polyreactive antibodies in serum, kidney eluates,11 and circulating immune complexes.3,12 The presence of anti-DNA antibodies in serum is strongly associated with SLE. Therefore, the detection of anti-DNA antibodies in serum is expected to be a main approach in SLE diagnosis. Numerous techniques have been developed for detection, characterization, and quantification of antibodies against doublestranded (ds) DNA (i.e., anti-dsDNA antibodies).12-15 These techniques may therefore be useful in clinical diagnosis of SLE. However, recent studies have revealed that antibodies produced against DNA are associated not only with SLE but with several other diseases.16 Detection of anti-DNA antibodies in serum is therefore not a specific diagnosis for SLE. The specificity for SLE may be regained by using pure dsDNA as the assay substrate. The Farr assay,17 which uses dsDNA as the assay substrate, and the indirect immunofluorescence technique,18,19 using Crithidia luciliae, currently offer the greatest diagnostic specificity for SLE. However, a comparison of these various techniques reveals discrepancies in their abilities to detect certain subpopulations of antibodies to DNA.20 Several approaches have been used for detection of anti-dsDNA antibodies by liquid chromatography.21-23 These techniques are time-consuming and demanding complicated (10) Williams, R. C. Jr.; Malone, C. C.; Cimbalnik, K.; Presley, M. A.; Roux, K. H.; Strelets, L.; Silvestris, F. Arthritis Rheum. 1997, 40, 109-123. (11) Amoura, Z.; Chabre, H.; Koutouzov, S.; Lotton, C.; Cabrespines, A.; Bach, J. F.; Jacob, L. Arthritis Rheum. 1994, 37, 1684-1688. (12) Adyel, F. Z.; Hentati, B.; Boulila, A.; Hachicha, J.; Ternynck, T.; Avrameas, S.; Ayadi, H. J. Clin. Lab. Anal. 1996, 10, 451-457. (13) Ter Borg, E. J.; Horst, G.; Hummel, E. J.; Limburg, P. C.; Kallenberg, C. G. M. Arthritis Rheum. 1990, 33, 634-643. (14) Budhai, L.; Oh, K.; Davidson, A. J. Clin. Invest. 1996, 98, 1585-1593. (15) Froelich, C. J.; Wallman, J.; Skosey, J. L.; Terodorescu, M. J. Rheumatol. 1990, 17, 192-200. (16) Tomer, Y.; Gilburd, B.; Sack, J.; Davies, T. F.; Meshorer, A.; Burek, C. L.; Rose, N. R.; Shoenfeld, Y. Clin. Immunol. Immunopathol. 1996, 78, 180187. (17) Wold, R. T.; Young, F. E.; Tan, E. M.; Farr, R. S. Science 1968, 161, 806809. (18) Aarden, L. A.; de Groot, E. R.; Feltkamp, T. E. W. N. Y. Acad. Sci. 1975, 254, 505-510. (19) Wigand, R.; Gottschalk, R.; Falkenbach, A.; Matthias, T.; Kaltwasser, J. P.; Hoelzer, D. Z. Rheumatol. 1997, 56, 53-62. (20) Smeenk, R.; Hylkema, M. Mol. Biol. Rep. 1992. 17, 71-79. (21) Kubota, T.; Akatsuka, T.; Kanai, Y. Clin. Exp. Immunol. 1985, 62, 321328. (22) Lafer, E. M.; Valle, R. P. C.; Msller, A.; Nordheim, A.; Schur, P. H.; Rich, A.; Stollar, B. D. J. Clin. Invest. 1983, 71, 314-319. (23) Miller, K. J.; Herman, A. C. Anal. Chem. 1996, 68, 3077-3082. 10.1021/ac980899r CCC: $18.00

© 1999 American Chemical Society Published on Web 02/20/1999

procedures that are difficult to automate. Therefore, a simple and safe method is required for the specific assay of anti-dsDNA antibodies. Recently, we developed an automated flow immunoassay system using an ion-exchange column for the detection of food allergens.24 Antibody-antigen complexes were subsequently separated from unreacted analytical substrate by ion-exchange column, on the basis of the difference in isoelectric point (pI). This approach can accommodate various immunoassay formats, offers precise control over assay conditions, and is readily automatable. This automated flow immunoassay system is equipped with an immunoreaction, anion exchange, reaction columns, and photodetector. In this paper, we have prepared alkaline phosphatase-conjugated dsDNA using a commercially available PCR digoxigenin labeling mix to form a novel analytical substrate. We report preliminary estimation of the diagnostic performance of our immunoassay using serum from patients suffering with SLE. Our novel approach does not require prior immobilization of antigen (or antibody) to a solid phase. Compared to solid-phase immunoassays, this approach offers a much shorter assay time. EXPERIMENTAL SECTION Reagents. Anti-digoxigenin (DIG) alkaline phosphatase (ALP) conjugated F(ab)2 fragment (IgG), and PCR DIG labeling mix were purchased from Boehringer Mannheim (Mannheim, Germany). CSPD (disodium 3-[4-methoxyspiro[1,2-dioxetane-3,2′-(5′chloro)tricyclo[3.3.1.1.3,7]decan]-4-yl]phenyl phosphate) was obtained from Tropix (Bedford, MA). Anti-dsDNA monoclonal antibody was purchased from Chemicon (Temecula, CA). Standard serum containing anti-dsDNA antibodies25 (World Health Organization, first international standard, WO/80, 200 IU/mL) was obtained from Medical and Biological Laboratories Co., Ltd. (MBL, Nagoya, Japan). Sephacryl S-300 was purchased from Pharmacia (Uppsala, Sweden). 3-(2′-spiroadamantane)-4-methoxy-4-(3′′-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). All other chemicals used were analytical-reagent or laboratory grade. Deionized-distilled water was used in all procedures. Samples. Standard serum containing anti-dsDNA antibodies (WO/80, 200 IU/mL) was diluted 2-fold with the sample running buffer (20 mM N-methylpiperazine, pH 5.5) for use in this study. Serum was drawn from five patients suffering with SLE. Control serum was drawn from healthy donors. All samples were purified by filtration through a 0.45-µm cellulose nitrate filter and then stored at -20 °C until required. The serum used was safe against hepatitis and AIDS in clinical test. Preparation of Alkaline Phosphatase-Conjugated DNA. This methodology is depicted in Figure 1. A DNA fragment (360 bp from Rhodobacter sphaeroides) was amplified by PCR (100-µL reaction volume containing 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 200 nM dNTP, 2.5 units of Taq DNA polymerase, and 0.2 µM of each oligonucleotide primer) using a Perkin-Elmer GeneAmp PCR system 9600 (25 cycles of denaturation at 95 °C for 0.5 min, annealing at 48 °C for 2 min, extension at 72 °C for 2 min, (24) Lim, T.-K.; Nakamura, N.; Matsunaga, T. Anal. Chim. Acta 1998, 370, 207214. (25) Feltkamp, T. E. W.; Kirkwood, T. B. L.; Maini, R. N.; Aarden, L. A. Ann. Rheum. Dis. 1988, 47, 740-746.

Figure 1. Schematic diagram showing immobilization of ALP on DIG-labeled DNA.

and final extension for a further 90 min). The sequence of the primers is presented as follows.26,27

forward primer: 5′-TGYGAYCCNAARGCNGA-3′ reverse primer: 5′-ADNGCCATCATYTCNCC-3′ Amplified DNA was coupled to DIG using a commercially available PCR DIG kit (250-µL reaction volume containing dATP, dCTP, and dGTP at 2 mM, 1.9 mM dTTP, and 0.1 mM DIG-11-dUTP, pH 7.0). DIG-labeled amplified DNA was electrophoresed on 1% agarose gel and stained with ethidium bromide. Fluorescent bands were clearly observed under UV light at a point on the gel corresponding to 360 bp. These bands were excised, transferred to 1.5-mL microtubes, and purified using the Prep-A-Gene DNA purification systems (Bio-Rad, Hercules, CA). DIG-labeled DNA (10 µL, 5 µg/mL) was spotted onto a piece of nylon membrane (Hybond-N, Amersham, Buckinghamshire, UK) and subsequently cross-linked by UV irradiation (3 min at 254 nm). The membrane was briefly rinsed in double-strength SSC (2×SSC: 0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) and shaken for 2-3 h at 42 °C in a solution containing 50% (v/v) formamide, 5×SSC, 0.02% (w/v) SDS, 0.1% (w/v) N-lauroylsarcosine, and 2% (w/v) blocking reagent (Boehriger Mannheim). The membrane was rinsed by agitation for 5 min at 24 °C in buffer 1 (0.1 M maleic acid, 0.15 M NaCl, 0.3% (w/v) Tween 20, pH 7.5) and blocked by incubation for 30 min at 24 °C in buffer 2 (buffer 1 supplemented with 1% (w/v) blocking reagent). The sheet was subsequently incubated in 10 mL of solution containing anti-DIG F(ab)2 fragment (10 000(26) Mevarech, M.; Rice, D.; Haselkorn, R. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6476-6480. (27) Zehr, J. P.; McReynolds, L. A. Appl. Environ. Microbiol. 1989, 55, 25222526.

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Figure 2. (A) Schematic diagram of automated flow immunoassay system and (B) principle of flow immunoassay. ALP-DNA and antibody are applied to immunoreaction column individually. Sample is incubated in a stream of buffer. The reaction mixture is passed through the anion-exchange column that selectively traps free ALP-DNA. The eluted species pass through the luminescent reaction column and luminescent detector, and the luminescence peak associated with the ALP-DNA antibody complex is monitored. Carrier pump (model 011-01, FLOM, Tokyo, Japan); microtube pump (MP-3, EYELA, Tokyo, Japan); peristaltic pump (U4-XV, Alitea, Stockholm, Sweden).

fold dilution in buffer 2 of an original stock solution at 150 units). For removal of unbound F(ab)2, the membrane was twice rinsed in buffer 1 for 15 min. The membrane was equilibrated for 5 min in buffer 3 (0.1 M Tris-HCl, 0.1 M NaCl, 50 mM MgCl2, pH 9.5) and incubated for 5 min under darkness in freshly prepared luminescence reagent (10 mL of buffer 3 containing 100 µL CSPD). The membrane was sealed in a polyester/polyethylene hybridization bag (Gibco BRL, Paisley, UK) and exposed to Kodak X-OMAT AR for 1 h at ambient temperature under darkness (unless otherwise stated). ALP-conjugated DNA was judged to be homogeneous by gel filtration using a column packed with Sephacryl S-300. Protein concentrations were determined by the Bradford method (Bio-Rad).28 Standard Equipment and Method for Flow Immunoassay. The flow immunoassay system (shown in Figure 2) consisted of the immunoreaction, anion exchange, luminescent reaction columns, and luminescent detector, and four pumps. The immunoreaction column (0.5-mm i.d., 5-m length) was used to incubate antidsDNA antibody and ALP-DNA. The luminescent reaction column (stainless steel coil; 1-mm i.d., 16-m length) was used to incubate luminescence reagent and ALP-DNA-anti-dsDNA antibody. These columns were maintained at 37 °C in a thermostatic incubator. The luminescence detector was interfaced to a photosensor amplifier (C2719, Hamamatsu-Photonics, Hamamatsu, Shizuoka, Japan). All tubing except the stainless steel coil was Tefzel tube (0.5-mm i.d.) from Pharmacia. Luminescence intensities were calculated by an IBM computer running the MPCA-30s software package from DKK Corp. (Tokyo, Japan). The anion-exchange column was equilibrated with 20 mM N-methylpiperazine (pH 5.5) prior to each run of flow immunoas-

says. ALP-DNA (100 µL, 50 µg/mL) and 100 µL of sample (antidsDNA monoclonal antibody, standard serum, or patient serum, respectively) were applied to the immunoreaction column in a stream of N-methylpiperazine buffer (flow rate 50 µL/min) for immunoreaction. The effluent from the immunoreaction column was fed onto an anion-exchange column (16-mm i.d., 25-mm length) packed with Sepharose Fast Flow (quaternary ammonium type, Pharmacia) in a stream of 20 mM N-methylpiperazine buffer (pH 5.5, flow rate 1.0 mL/min). The effluent from the anionexchange column was mixed with a luminescent substrate (AMPPD) and passed through the luminescent reaction column for incubation at a flow rate of 1 mL/min. After incubation, the combined effluent was passed through the luminescent detector for quantification of ALP-labeled antibody-antigen complexes. The anion-exchange column was regenerated by occasional elution with 20 mM N-methylpiperazine supplemented with 0.5 M NaCl, to remove accumulated unreacted ALP-DNA. Furthermore, serum from a woman (47 years old) suffering from SLE was routinely assayed for anti-dsDNA antibodies by both flow immunoassay system and ELISA at a Japanese hospital and monitoring exacerbation of the disease condition was assessed.

(28) Bradford, M. Anal. Biochem. 1976, 72, 248-254.

(29) Lanzillo, J. J. Anal. Biochem. 1991, 194, 45-53.

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RESULTS AND DISCUSSION Preparation of ALP-Conjugated DNA. A 360-bp fragment of the gene from R. sphaeroides was amplified by PCR and labeled with DIG. The time course of immunoreaction between the DIGcoupled dsDNA fragment (360 bp) and commercially available ALP-conjugated F(ab)2 fragment was investigated by chemiluminescence.29 Our data indicated that equilibrium was reached within 30 min. The number of ALP molecules incorporated per 360-bp

dsDNA fragment was estimated from the sequence of the gene (180 thymine residues per double-stranded molecule of the 360bp DNA fragment) and the ratios of DIG-11-dUTP to dTTP (1: 20) in the PCR DIG labeling. We estimated that 9 molecules of ALP were incorporated per 360-bp dsDNA fragment. Optimization of the Separation Conditions for the Flow Immunoassay. We previously showed that antigen-antibody complex may be successfully separated from unreacted antibody and antigen by the ion-exchange column, on the basis of a difference in pI.24,30,31 We analyzed the isoelectric points of antidsDNA monoclonal antibody, ALP-DNA and ALP-DNA-antidsDNA monoclonal antibody complex by isoelectric focusing on a PhastGel IEF 3-9. Electrophoresis and silver staining were carried out using the Pharmacia Phastsystem. Three species were found to have isoelectric points of 6.8 (anti-dsDNA monoclonal antibody), 4.5 (ALP-DNA), and 6.0 (ALP-DNA-anti-dsDNA monoclonal antibody complex). The antibody-antigen complex arising from immunoreaction between ALP-DNA and anti-dsDNA monoclonal antibody was therefore successfully separated from unreacted ALP-DNA under the desired conditions by anion exchange on Sepharose Fast Flow (quaternary ammonium type) buffered at pH 5.5 with 20 mM N-methylpiperazine. When only free ALP-DNA was applied to the anion-exchange column in N-methylpiperazine buffer (20 mM, pH 5.5), no peaks were observed. The effect of applying ALP-DNA and anti-dsDNA monoclonal antibody to the flow immunoassay system, on signal from the luminescent detector, was subsequently investigated. Commercially available anti-dsDNA monoclonal antibody was made into a solution (200 µg/mL) in running buffer (20 mM N-methylpiperazine, pH 5.5). ALP-DNA (100 µL, 50 µg/mL) and anti-dsDNA monoclonal antibody (100 µL, 200 µg/mL) were simultaneously applied at 25-min intervals (three injections) to the immunoreaction column, and the column was eluted with running buffer (20 mM N-methylpiperazine, pH 5.5). This yielded a series of three equivalent-sized peaks from the luminescent detector, spaced ∼25 min apart, after an initial lag of 25 min (Figure 3). To optimize the signal output, the effect of varying the duration of the immunoreaction between ALP-DNA and anti-dsDNA monoclonal antibody was investigated. The two parameters affecting the duration of the immunoreaction were the flow rate of the running buffer and the length of the immunoreaction column. A combination of the longest immunoreaction column (0.5-mm i.d., 5-m length) and slowest flow rate (50 µL/min) yielded the largest peak height from the luminescent detector (data not shown). The duration of the immunoreaction between ALP-DNA and anti-dsDNA monoclonal antibody was ∼10 min given these optimized parameters of length and flow rate. Automated Flow Immunoassay. In a subsequent experiment, the response of the immunoassay was investigated when the concentration of anti-dsDNA monoclonal antibody was varied. Varying amounts of anti-dsDNA monoclonal antibody (100 µL, 0-300 µg/mL) were applied alongside a constant amount of ALPDNA (100 µL, 50 µg/mL). The signal from the luminescent detector increased with increasing concentration of anti-dsDNA monoclonal antibody. AMPPD did not yield a significant background signal. This suggested that unreacted ALP-DNA was (30) Lim, T.-K.; Nakamura, N.; Matsunaga, T. Anal. Chim. Acta 1997, 354, 2934. (31) Lim, T.-K.; Nakamura, N.; Matsunaga, T. Biotechnol. Tech., in press.

Figure 3. Separation of ALP-DNA-anti-dsDNA-mAb complex using the anion-exchange column. ALP-DNA (100 µL, 50 µg/mL) and anti-dsDNA-mAb (100 µL, 200 µg/mL) were incubated in the immunoreaction column at a flow rate of 0.1 mL/min. Arrows indicate the sample application.

retained by the anion-exchange resin. A linear relationship (R ) 0.995) was obtained between the signal from the luminescent detector (mV) and concentration of anti-dsDNA monoclonal antibody in the range of 0-300 µg/mL (n )10 replicate samples per point). This method was faster (25 min) and simpler to use than conventional ELISAs involving microtiter plates. The reproducibility of this flow immunoassay was examined using five different concentrations of anti-dsDNA monoclonal antibody (5, 10, 50, 100, and 200 µg/mL). The coefficients of variation for each of these sets were all within the range 1.5-3%. The data obtained using this system showed good correlation (R ) 0.993, n ) 15). The anion-exchange column was successfully regenerated by occasional elution with running buffer (20 mM N-methylpiperazine, pH 5.5) containing 0.5 M NaCl, to remove accumulated unreacted ALP-DNA. ALP-DNA recovered in this manner was treated on a HiTrap desalting column. This is referred to as recycled ALP-DNA. This ability to use recycled ALP-DNA for immunoassay of anti-dsDNA monoclonal antibody was investigated. A fixed amount of anti-dsDNA monoclonal antibody (100 µL, 200 µg/mL) and recycled ALP-DNA (100 µL, 50 µg/mL) was submitted for flow immunoassay and the response of the immunoassay monitored. Our data suggest that results indicated that signal was obtained from the ALP-DNA-dsDNA antibody complex. The anion-exchange column was rapidly regenerated with N-methylpiperazine buffer containing 0.5 M NaCl and efficient reuse of ALP-DNA. These results indicated that this flow immunoassay system is suitable for diagnosis of SLE in clinical practice. Therefore, this flow immunoassay was applied using serum from five patients having anti-dsDNA antibodies. Detection of Anti-dsDNA Antibody in the Serum of SLE Patients. Standard serum containing anti-dsDNA antibodies (WO/80) was used to make a standard calibration curve (Figure 4). The immunoassay yielded a linear relationship between signal from the luminescent detector (mV) and concentration of the standard anti-dsDNA antibody, in the range of 0-400 IU/mL (n ) 20 replicates per point). The signal was reproducible with a correlation coefficient of 0.999. Serum was drawn from each patient Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

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Figure 4. Relationship between output and dsDNA antibody concentration of standard serum. ALP-DNA, 50 µg/mL.

Figure 6. Anti-dsDNA antibody levels determined by the flow immunoassay, in relation to disease activity in a patient with SLE.

Figure 5. Comparison of anti-dsDNA antibody concentration obtained from flow immunoassay and ELISA.

suffering from SLE over a period of one year and having antidsDNA antibodies (45, 62, 70, 110, and 210 IU/mL) as a result of diagnosis with a commercially available kit, MESACUP DNA-II test [ds] ELISA (MBL). The values of dsDNA antibody concentration were estimated from signal output using a calibration curve. A good correlation was found between the response of the flow immunoassay described in this paper and the conventional ELISA (Figure 5). Detection of anti-dsDNA antibodies by ELISA requires that dsDNA be coupled to the surface of wells in a plastic ELISA plate. This is difficult due to electrostatic repulsion between negatively charged dsDNA and the negatively charged surface of the plastic well. This can be overcome by precoating the wells with poly(L-lysine) or protamine sulfate. However, this causes IgM antibodies to bind to protamine and poly(L-lysine), leading to unacceptable background noise when assaying the patient samples.20,32 By contrast, the novel flow immunoassay the described in this paper does not require prior immobilization of dsDNA to a solid phase. Hence, problems associated with adhesion of in vivo antibody-antigen complexes to a precoat are avoided. (32) Brinkman, K.; Termaat, R.-M.; Brink, H. G.; van den Berden, J. H. M.; Smeenk, R. J. T. J. Immunol. Methods 1991, 139, 91-100.

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Measurement of Anti-dsDNA Antibody Levels as a Predictor of Disease Exacerbation in a SLE Patient Using an Automated Flow Immunoassay System. A long-term analysis of serum anti-dsDNA antibody levels in a patient suffering from SLE and undergoing treatment with prednisolone revealed a biphasic pattern (Figure 6). The first phase consisted of a rapid decrease in serum antibody levels upon increase in the dosage of prednisolone from 7 to 27.5 mg/day. The patient progressively recovered and did not exhibit any signs of disease by September 1996. The second phase consisted of a gradual increase in serum anti-dsDNA antibody levels. By April 1997 (6 months later), the patient was exhibiting arthralgia, a typical symptom of SLE. CONCLUSIONS For diagnosis of SLE, we have demonstrated a novel flow immunoassay which does not require immobilization of antibody (or antigen) to a solid phase. It revolves around the separation of antibody-antigen complexes from unreacted antibody and antigen by ion exchange column, on the basis of a difference in isoelectric point. The flow immunoassay approach offers greater control over immunoreaction conditions compared with solid-phase immunoassay formats. In studies conducted with serum from human donors, we determined that adsorption of undefined serum components by the anion-exchange column did not adversely effect the assay. This was probably because of the very high capacity of resin. This immunoassay exhibited a throughput of 2 samples/ h, excellent reproducibility, and a reliable detection limit of 3 µg/ mL using the anti-dsDNA monoclonal antibody. Received for review August 12, 1998. Accepted December 22, 1998. AC980899R