ARTICLES
Flow Injection Solid-Phase Chemiluminescent Immunoassay Using a Membrane-Based Reactor Hanjiu Liu, Jim C. Yu,' Dilbir S. Bindra, Richard S. Givens, and George S. Wilson* Center for Bioanalytical Research, University of Kansas, Lawrence, Kansas 66045
A flow Injectlon analysls (FIA) sandwlch Immunoassay (ELISA) using a polyvlnylldene dlfluorlde polymer based membrane lmmunosorbent has been proposed. The membrane to whlch the antigen (bovine IgG) Is attached Is mounted In a flow cell (5-pL dead volume). Analyte In the form of mouse antlbovlne IgG Is Injected Into the flowing stream followed by a goat antl-mouse IgG horseradish peroxidase (HRP) conjugate. The HRP was used to catalyze the enhanced lumlnol reaction, resultlng In chemllumlnescence, whlch was detected directly wlthln the immunoreactor. A detectlon limit of 1 fmol was obtalned whlle spiked serum samples gave precision of 8.7 YO.
INTRODUCTION Flow injection analysis (FIA) is well-known for its rapidity, precision, and ability to handle small sample volumes. Its analytical parameters can be well controlled and reproducibly maintained. These characteristics make it especially suitable for enzyme immunoassays. The combination of the extraordinarily high selectivity of the immunoassay and the versatility of FIA is particularly useful in clinical and pharmaceutical applications. Miller and Lim (1)described an application of FIA utilizing a homogeneous immunoassay with energy-transfer fluorometry. Christian and Kelly (2) determined human IgG by means of a homogeneous FIA enzyme immunoassay. Recently, Meyerhoff and Lee (3)described an air-segmented continuous-flow homogeneous enzyme immunoassay. FIA can also be applied to solid-phase immunoassays. Generally, the solid-phase immunoassay has much better sensitivity when compared with homogeneous assays due to reduced interference from the sample matrix. Also, when combined with FIA, the procedure consumes less reagent because normally one of the analytical reagents (e.g. the antibody) is immobilized on a solid support and may be reusable. de Alwis and Wilson ( 4 )reported the first application of FIA in a solid-phase sandwich enzyme immunoassay. In their system, a reusable immunoreactor containing an immobilized
* To whom correspondence should be addressed. Present address: Department of Chemistry, Central Missouri State University, Warrensburg, MO 64093. 0003-2700/9 1/0383-0666$02.50/0
antibody was used on-line with an FIA system. The system was capable of continuously carrying out each of the steps involved in solid-phase sandwich immunoassays, including the immune reaction, the washing, the sandwich reaction, and the enzymatic reaction, in about 10 min. Later, de Alwis and Wilson (5) described an FIA-based solid-phase competitive immunoassay. More recently, Mattiasson and co-workers (6) and Lee and Meyerhoff (3)reported their work in the field of FIA solid-phase enzyme immunoassays. FIA-based solidphase immunoassays generally offer better speed (less than 10 min) and precision (better than 5%) than conventional assays such as the ELISA using microtiter plates or plastic tubes. This improvement is due to the much better reproducibility associated with reactions of solid-phase components and soluble species. Postcolumn detection has been employed almost exclusively for FIA solid-phase immunoassays resulting in smaller signals not only because of the dilution of the product before reaching the detector but also due to the high blank signal from the nonspecifically adsorbed enzyme-labeled antibodies on the injector, the connection tubing, and the packing material. The use of chemiluminescent labels in immunoassays (7-10) has become increasingly popular in recent years because of the high sensitivity and wide dynamic range afforded by this method. Three classes of successfully generated chemiluminescence compounds are acyl hydrazides (luminol), acridinium esters, and trichlorophenyl oxalate esters. The oxidation of luminol and its derivatives by hydrogen peroxide in the presence of catalyst (e.g., horseradish peroxidase) has received considerable attention. In aqueous solution at neutral pH in the presence of a catalyst such as horseradish peroxidase (HRP), typically the reaction proceeds with an emission efficiency of 1 %. Whitehead and co-workers (11)have reported that certain phenol derivatives (e.g., p-iodophenol) enhance the intensity and prolong the length of the chemiluminescent reaction. This phenomenon has been commonly referred to as enhanced chemiluminescence. Although the exact mechanism of the enhancement is not well understood, the reaction has been increasingly used in immunoassays (12-14). Both isoluminol and HRP have been widely used for labeling. The feasibility of using chemiluminescence to achieve oncolumn detection in FIA has been demonstrated by Hool and Nieman (15-17). In their system hydrogen peroxide is the analyte, and either luminol or HRP is immobilized on controlled-pore glass, which is then packed into a transparent flow 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
cell. The flow cell is placed directly in front of a photomultplier tube (PMT). Recently, Shellum and Gubitz (18) reported a similar FIA system for carrying out a sandwich immunoassay. In this latter system a Teflon tube, which contains antibodies covalently immobilized to a rigid beaded support, was used as an immunoreactor. Acridinium ester was used as the label for the second antibody. There are some major disadvantages associated with using packed tubing as an immunoreactor for the solid-phase FIA procedure. First, light-scattering by the packing material seriously compromises the detection of chemiluminescence (15, 16). Second, the small-diameter tubing usually desired for FIA (e.g. 1 mm) often cannot be packed uniformly. This usually causes channeling, which greatly reduces the performance of the reactor (5,15,16).Third, it is difficult to pack the tubing consistently every time, resulting in significant variation particularly of reactor sensitivity (19). In this paper we report our study of an FIA solid-phase chemiluminescent sandwich immunoassay system using a thin-layer flow cell as the immunoreactor with antigen immobilized on a membrane. The analytical performance of the membrane-based reactor was evaluated by using a model sandwich assay of bovine IgG, mouse monoclonal anti-bovine IgG, and HRP-labeled goat anti-mouse IgG. By using this type of reactor, the light-scattering problem associated with the tubing reactors was eliminated and a universal calibration curve was obtained for different pieces of membrane coupled to the protein under identical conditions. A minimum detectable quantity of l fmol of analyte was achieved with an assay time of about 10 min. EXPERIMENTAL SECTION Materials. Bovine IgG (bIgG),Cohn fraction V, was purchased from Fluka (Hauppauge, NY). Affinity-purified mouse monoclonal anti-bovine immunoglobin G (IgG) was a donation from Rohan Wimalasena. Goat anti-mouse IgG conjugated with HRP was purchased from Organon Teknika (Lot No. 3611-0081, West Chester, PA). Bovine serum albumin (BSA) was obtained from Sigma (St. Louis, MO). (3-Glycidoxypropyl)trimethoxysilane (GOPS) was purchased from Aldrich. Luminol and p-iodophenol were purchased from Sigma and were used without further purification. Horseradish peroxidase (HRP) 65-80% isoenzyme C (2355 IU/mg) was obtained from Biozyme (SanDiego, CA). The activated membrane was purchased from Millipore (Bedford, MA) under the trade name of Immobilon AV affinity membrane. All other materials were reagent grade chemicals unless specified. Distilled-deionized water was used in the preparation of buffers. Immobilization Procedures. A sheet (12 X 10 cm) of the activated membrane was soaked for 18 h at 4 "C in 10 mL of 0.1 M phosphate buffer (pH 7.4) containing 5 mg/mL of bovine IgG, as recommended by the manufacturer. It was then deactivated by immersing in 10% (v/v) ethanolamine in 1.0 M carbonate buffer (pH 9.5) for 1 h, followed by washing with water. The antibody-coupled membrane was allowed to dry at 4 "C and was then stored at 4 "C. Instrumentation. The apparatus for studying chemiluminescence in static solution has been described previously (20). The sample housing has a rubber septum so that solutions may be directly injected to the sample cell under lighbtight conditions. A 3-mL quartz cell with dimensions of 1.0 X 1.0 X 3.0 cm was used for the chemiluminescent kinetic studies. The FIA system used in this work is shown schematically in Figure 1. A Rainin peristaltic pump was used to deliver either of the two buffers, which were selected by using a three-way stream switching valve (Rainin, Woburn, MA). The assay buffer was 0.1 M phosphate buffer (PB, pH 7.4) containing 0.3% bovine serum albumin (BSA) and 5 X lod M p-iodophenol. The washing buffer was 0.1 M phosphate solution buffered at pH 2.2. The flow rate was 0.1 mL/min unless otherwise specified. A Pharmacia ACT100 automatic injector with a 25 pL sample loop was used. Teflon tubing of 0.8 mm i.d. was used between the injector and the immunoreactor. The immunoreactor was placed directly in front of a PMT (IP28A, RCA). The PMT anode current was converted
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to voltage and amplified by a standard circuit. The output signals were recorded by both a strip chart recorder and an integrator (Hewlett-Packard 3390A). RESULTS AND DISCUSSION A sensitive chemiluminescent label is highly desirable in order to gain maximum detection capability. This label must be the limiting reagent such that its concentration is directly proportional to the emission output. There are three possible chemiluminescent labels associated with the luminol reaction. Isoluminol may be used as a label, but it has a rather low emission efficiency (typically 1% ), especially when conjugated to a protein. A peroxide-generating enzyme such as glucose oxidase (GOx) may be used as a label. In that case hydrogen peroxide, one of the reagents necessary for the chemiluminescence reaction can then be generated by adding glucose and oxygen. Horseradish peroxidase (HRP), a catalyst for the luminol reaction may also be used as a label under such conditions. The dependence of the enhanced chemiluminescence on H R P and hydrogen peroxide was studied. Enhanced Chemiluminescence of Luminol. The chemiluminescent reaction of luminol was studied in static solutions of 0.1 M phosphate buffer (pH 7.4). The lifetime of the emission is significantly prolonged by addition of p-iodophenol (10). The emission intensity first increases with increasing concentration of p-iodophenol, reaching a maximum when the concentration is approximately 2.5 x M, and then decreases with further increase of p-iodophenol. The emission intensity is directly proportional to the concentration of H202down to approximately lo-' M under the following conditions: 3.3 X lo4 M luminol, 1.5 X M p-iodophenol, and 1.2 X M HRP. Significant light emission was observed when luminol, p-iodophenol, and HRP were mixed without any H202. In contrast, the chemiluminescent intensity is directly proportional to the amount of HRP down to about M under the following conditions: lo4 M luminol, 0.01 M HzOz, and 2.5 X M p-iodophenol. This high sensitivity made HRP the choice as label for the subsequent study. The rate constant for the decay of the chemilumenescence emission increases with increasing concentrations of HRP, as shown in Figure 2. These data show that the long-lived, p-iodophenol enhanced chemiluminescence decay rates are directly proportional to the H R P concentration, i.e., that H R P catalyzes the chemiluminescence decay with a rate constant of 2.47 x 105 M-' Thin-Layer Flow Cell Immunoreactor. The thin-layer flow cell immunoreactor is shown in Figure 3. It consists of a piece (0.5 cm x 3.0 cm) of bovine IgG-coupled membrane and a Teflon spacer, which are sandwiched between two transparent Plexiglas plates. The spacer creates a 0.15 cm wide, 0.01 cm thick, and 2.5 cm long flow channel with a volume of approximately 5 wL. The surface area of the membrane exposed to the flowing steam is approximately 0.4 cm2. The two Plexiglas plates are held together by four screws. The assembly is then directly mounted in front of the P M T window.
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antibody. larger than that of mouse anti-bovine injected in order to ensure that all the retained mouse anti-bovine antibody is “sandwiched”. The injection of buffer (pH 7.4) serves two purposes here: (1)to prevent possible carry-over of the enzyme conjugate by the injector sample loop and (2) to give sufficient washing time for the nonsepcifically adsorbed labeled antibody. The substrate solution contains 0.01 M H20z,5 X M p-iodophenol, and 2.5 X M luminol. After the calibration was completed, the reactor was regenerated by washing with pH 2.2 buffer for 10 min. The reactor was then ready for another calibration. Typical signals from the experiments are shown in Figure 4. The reproducibility of the CL detection and the stability of the sandwich complex were demonstrated by reinjections of the enzyme substrate luminol reagents, which correspond to “interrogating” the sandwich already formed. The signals indicated by an asterisk showed that the complex was stable for short periods of time (10 min) and that the CL signal intensity was reproduced by introducing the labeled antibody and the enzyme substrate but without injecting any additional antibodies or antigen. This demonstrates that the sandwich formed is stable. The numbers above the peaks indicate the accumulated mass of analyte. Triplicate injection cycles with the same analyte solution yield peak areas with a relative standard deviation of 5%. A calibration curve was constructed by plotting the increment of the peak areas vs femtomoles of mouse anti-bovine injected each time. The calibration curve is linear with a slope of 0.028, an intercept of 0.0, and a correlation coefficient of 0.99. It was observed that washing with pH 2.2 buffer to regenerate the immunosorbent resulted in a loss of membrane capacity. This could be due to denaturation of immobilized antibody or to the loss of antibody, which was initially adsorbed rather than covalently attached. After three regenerations, the output signal decreases about 20%. The membrane may then be replaced with a fresh one if desired. The replacement procedure is simple and rapid, usually taking only about 2 min. Different pieces of membrane from the same sheet coupled with bovine IgG gave a series of calibration curve slopes with standard deviations of about 10%. This deviation is believed to be caused by the inconsistent tightness of the two Plexiglas plates when the membrane is replaced, which may change the thickness of the flowing channel and consequently change the hydrodynamic properties of the flowing stream, and is not due to membrane itself. The detection limit of the system with a fresh membrane is approximately 1 fmol at a S I N ratio of 3. The saturation of the reactor sets the upper limit of the dynamic range of the calibration. With repeated mouse anti-bovine injections
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The membrane is made from polyvinylidene difluoride, which exhibits very low nonspecific adsorption. It is hydrophilic and porous and has been chemically activated to couple with a primary amine group on the protein in aqueous solutions. The manufacturer claims that the activated membrane can covalently immobilize up to 500 pmol of antibody/cm2 because of its large internal surface area (155 cm2/cm2frontal area). The effect of the emission “enhancer”, p-iodophenol, on the output signal in the flow injection system was investigated by varying the concentration of p-iodophenol with the immunoreactor containing a fixed amount of HRP immobilized on the membrane. It was observed that the peak area is directly proportional to the concentration of p-iodophenol in the carrier buffer up to about 2 x M. The peak area is about 4 times larger with 5 X M p-iodophenol present in the carrier buffer than without it. The analytical performance of the membrane-based reactor was evaluated by using the sequential injections shown (one assay cycle) at a flow rate of 0.1 mL/min. Each assay cycle consists of four injections: (1) an unknown or a standard mouse anti-bovine solution ( t = 0), (2) goat anti-mouse-HRP ( t = 2 min), (3) pH 7.4 buffer ( t = 4 min), and (4) the enzyme substrate and luminol reagents (t = 6 min). The 2-min interval between injections is a requirement of the particular autosampler employed. An autosampler with a faster cycle time would result in sampling intervals perhaps as short as 15-30 s. A calibration was effected by repeating the assay cycle with standards containing 0, 25, 50, 75, and 100 fmol of mouse anti-bovine antibody. The amount of goat anti-mouse-HRP injected was 1.5 pmol, which is more than 1 order of magnitude
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into the reactor, all of the specific sites will eventually be occupied. Figure 5 shows the plot of peak area of the signal vs total picomoles of mouse anti-bovine injected into the reactor. After about 3 pmol of mouse anti-bovine are injected, the reactor approached saturation. Thus the reactor has an operating capacity of approximately 3 pmol. The apparent maximum capacity is about 10 pmol instead of the 200 pmol suggested by the manufacturer. There are several explanations for this discrepancy. First, it is not known whether all of the attached antibody is active. The active antibody could vary between 5 and 50% of the total. Second, many if not most of the available sites are on the interior of membrane. If such sites have actually reacted with antibody, they may be inaccessible to antigen in the mobile phase. Thus the total measured capacity of the membrane may well be reasonable under the circumstances. The effect of the flow rate of mouse anti-bovine and goat anti-mouse-HRP through the reactor on the output signals was studied. The mobile phase was set at various flow rates while the samples and the conjugate were injected and was set a t a fixed rate of 0.1 mL/min while the substrate was injected. As shown in Figure 6, the output signal decreases with increasing flow rate. This indicates that the immune reaction inside the reactor does not reach completion. This occurs because the extent of the reaction is controlled by the hydrodynamic properties of the reactor. The extent of the reaction increases as the residence time of the injected pulse of mouse anti-bovine inside the reactor increases, which is calculated to be 12 s when the flow rate is 0.1 mL/min and about 2 s a t a flow rate of 0.5 mL/min. A mouse anti-bovine spiked human serum sample was assayed with the system in order to check its analytical performance in a complex matrix. The serum was diluted 1:20 with 0.1 M phosphate buffer saline (pH 7.4) and was then spiked with mouse anti-bovine IgG. A 2 5 - ~ Lsample containing 50.0 fmol of mouse anti-bovine IgG was injected into the system. From two standards of 25 and 75 fmol for calibration, a value of 51.5 f 4.5 fmol was obtained.
CONCLUSIONS Enhanced chemiluminescence of luminol was successfully demonstrated for a flow injection immunoassay system. A universal calibration curve with femtomole detection limits
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was possible with the membrane-based immunoreador with an assay time of about 10 min. The operating capacity of the system (-3 pmol) is sufficient for assaying at least 30 samples containing femtomoles of analyte, thus avoiding the necessity of system regeneration and greatly shortening the assay time. The membrane-based immunosorbent has limited surface area and this limited capacity, which is nevertheless more than sufficient for the low analyte concentrations studied. The low surface area leads to low nonspecific adsorption. Moreover, the membrane immunosorbent configuration affords the possibility of analyzing samples that are turbid or contain particulate matter, which would clog a packed reactor. Thus this new configuration provides a promising approach to solid-phase assays.
ACKNOWLEDGMENT We gratefully acknowledge Rohan Wimalasena for his generous donation of mouse monoclonal anti-bovine IgG. LITERATURE CITED Lim, C. S.;Miller, J. N.; Bridges, J. W. Anal. Chim. Acta 1980, 774, 183- 189. Kelly, T. A.; Christian, G. D. Tabnta 1982, 29, 1009-1112. Lee, I. H.; Meyerhoff, M. E. Anal. Chim. Acta 1990, 229, 47-55. de Alwis, U.; Wilson, G. S. Anal. Chem. 1985, 57, 2754-2756. de Alwis, U.; Wilson, G. S. Anal. Chem. 1987, 5 9 , 2786-2789. Mattiasson, B.; Berden, P.; Ling, T. G. I. Anal. Biochem. 1989, 787, 379-382. Thorpe, G. H. G.; Kricka, L. J. Methods Enzymoi. 1988, 733, 331-353. Van Dyke, K. Bioluminescence and Chemiluminescence: Instruments and Applications; CRC Press: Boca Raton. FL, 1985. Weeks, I.; Woodhead. J. S. Trends Anal. Chem. 7988, 7 , 55-58. Kricka; L. J.; Whitehead, T. P. J. Pharm. Biomed. Anal. 1987, 5 , 829-833. Thorpe, G. H. G.; Kricka, L. J., Moseley, S.B.; Whitehead, T. P. Clin. C h W . 1985, 3 1 , 1335-1341. Thorpe, G. H. G.; Williams, L. A.; Kricka, L. J.; Whitehead, T. P. J . Immunoi. Methods 1985, 79, 57-63. Sheppard, M. C.; Black, E. G. Clin. Chem. 1987, 33, 179-181. Leong, M. M. L.; Fox, G. R. Anal. Biochem. 1988, 772, 145-150. Hool, K.; Nieman, T. A. Anal. Chem. 1987, 59, 869-872. Hool, K.; Nieman, T. A. Anal. Chem. 1988, 6 0 , 834-837. Nieman, T. A. J. Res. Natl. Bur. Stand. 1988, 93, 501-502. Shellum, C.; Gubitz, G. Anal. Chim. Acta 1989, 227, 97-107. Liu, H.; Wilson, G. S. Unpublished data, University of Kansas, 1990. Orlovic. M.; Schowen, R. L.; Givens, R. S.;Alvarez, F.; Matuszewski, B.;Parekh, N. J. Org. Chem. 1989, 54, 3606-3610.
RECEIVED for review October 1,1990. Accepted December 27, 1990. This work was supported by National Science Foundation Grant No. CHE 8804263 (J.C.Y.), The Kansas Technology Enterprise Corp., and Oread Laboratories.
