Flow Immunoassay Using Solid-Phase Entrapment - ACS Publications

ment of phenytoin in a 2-min assay time. The reliable detection limit for the assay was 5 nmol L-1 of phenytoin in serum. The columns were regenerated...
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Anal. Chem. 1996, 68, 1665-1670

Flow Immunoassay Using Solid-Phase Entrapment Laurie Locascio-Brown,* Larissa Martynova,† Richard G. Christensen, and George Horvai‡

National Institute of Standards and Technology, Gaithersburg, Maryland 20899

A flow injection immunoassay was performed using a column packed with reversed-phase sorbents to effect separation of the immunoreacted species by entrapping free analyte and allowing antibody-conjugated analyte to pass unretained. Fluorescein-labeled analyte was measured in a competitive assay for the anticonvulsant drug phenytoin. The simplicity of the assay was the greatest advantage of the technique, which allowed for measurement of phenytoin in a 2-min assay time. The reliable detection limit for the assay was 5 nmol L-1 of phenytoin in serum. The columns were regenerated with periodic injections of ethanol solutions to remove the entrapped analyte and prepare the column for subsequent analyses. In a typical flow-based heterogeneous immunoassay, an antibody or antigen is immobilized onto a solid phase placed in the flow path to achieve specific separation to identify or isolate the antigen-antibody complex. Many successful flow immunoassays1-8 have been performed using this approach; however, immobilization of the immunospecies can be time-consuming if it is to be reusable, and the resultant solid phase is not generically useful for many applications. Gubitz and Shellum have provided a thorough review of solid-phase flow injection immunoassays in a recent publication.9 A generic solid phase can be produced via the attachment of binding proteins such as protein A or protein G. These proteins attach to the Fc portion of an antibody and can be used to affix all antibodies to a solid phase.10-15 Others have utilized immobilized species-specific antibodies, such as anti-human or anti†

Present address: Russian Academy of Sciences, Moscow, Russia. Present address: Technical University of Budapest, Budapest, Hungary. (1) Locascio-Brown, L.; Chesler, R.; Kroll; M., Plant, A. L.; Durst, R. A. Clin. Chem. 1993, 39, 386-391. (2) Locascio-Brown, L.; Choquette, S. J. Talanta 1993, 40, 1899-1904. (3) Locascio-Brown, L.; Plant, A. L.; Horvath, V.; Durst, R. A. Anal. Chem. 1990, 62, 2587-2593. (4) Siebert, T. A.; Reeves, S. G.; Durst, R. A. Anal. Chim. Acta 1993, 282, 297-305. (5) de Alwis, W. U.; Wilson, G. S. Anal. Chem. 1985, 57, 2754-2756. (6) Tang, H. T.; Halsall, H. B.; Heineman, W. R. Clin. Chem. 1991, 37, 245248. (7) Nilsson, M.; Mattiasson, G.; Mattiasson, B. J. Biotechnol. 1993, 31, 381394. (8) Hage, D. S.; Thomas, D. H.; Beck, M. S. Anal. Chem. 1993, 65, 16221630. (9) Gubitz, G.; Shellum, C. Anal. Chim. Acta 1993, 283, 421-428. (10) Khokhar, Y.; Miller, J. N.; Seare, N. J. Anal. Chim. Acta 1994, 290, 154158. (11) de Frutos, M.; Paliwal, S. K.; Regnier, F. E. Anal. Chem. 1993, 65, 21592163. (12) Palmer, D. A.; Xuezhen, R.; Fernandez-Hernando, P.; Miller, J. N. Anal. Lett. 1993, 26, 2543-2553. (13) Cassidy, S. A.; Janis, L. J.; Regnier, F. E. Anal. Chem. 1992, 64, 19731977. (14) Janis, L. J.; Regnier, F. E. Anal. Chem. 1989, 61, 1901-1906. (15) Evans, M.; Palmer, D. A.; Miller, J. N.; French, M. T. Anal. Proc. 1994, 31, 7-8. ‡

0003-2700/96/0368-1665$12.00/0

© 1996 American Chemical Society

mouse antibodies, to capture antibodies onto a solid phase.16 An alternative is the immobilization of avidin or streptavidin, which can be used to capture any species, antigen or antibody, that is linked to biotin. These immobilized materials may then be used for immunoassay applications without discretion based on the analyte. A still broader approach, based on gel exclusion, was recently reported by Nakamura.17 The method utilizes a gel column placed in the flow path to separate enzyme-antibody from enzymeantibody/antigen complex based on size. In practice, the method requires the use of a second antibody to allow for resolution of the peaks in flow. This method is most applicable to larger analytes that have multiple antigenic sites and can bind to two antibodies simultaneously. Many investigators are interested in the combination of liquid chromatography (LC) and immunoassay techniques to provide greater separation power and enhanced detection limits. An extensive review of this literature was recently published.18 Most of these methods use immobilized antibody to capture antigen prior to elution into an LC system for further resolution.19-21 Other methods involve LC analysis followed by postcolumn detection by immunoassay.22 Among other advantages, this preliminary cleanup step allows for sample concentration and elimination of matrix effects. This paper describes a new approach to the performance of flow immunoassays and makes use of reversed-phase LC stationary phases to separate species from an immunochemical reaction. The technique embodies all of the advantages of a generic solid phase and is applicable to the measurement of hydrophobic analytes in complex matrices such as serum or plasma. In this protocol, a competitive immunoassay is performed in solution using a fluorescein-labeled antigen as the competing species. In general, there will be several resulting species including antigen (AG), labeled antigen (AG*), and the antigenantibody complexes (AbAG, AbAG*). When the reaction mixture is injected into the flow path, the uncomplexed species are retained on a hydrophobic LC stationary phase. The complexed labeled antigen will pass through the column unretained, and then through the fluorometric detector. The results are interpreted as in any other competitive immunoassay. The desired properties of the column packing material include the following: (1) completely retains the tracer; (2) does not retain (16) Lu, B.; Xie, J.; Lu, C; Wu, C.; Wei, Y. Anal. Chem. 1995, 67, 83-87. (17) Nakamura, K.; Satomura, S.; Matsuura, S. Anal. Chem. 1993, 65, 613616. (18) de Frutos, M; Regnier, F. E. Anal. Chem. 1993, 65, 17A-25A. (19) Flurer, C. L.; Novotny, M. Anal. Chem. 1993, 65, 817-821. (20) Farjan, A.; Brugman, A. E.; Lingeman, H.; Brinkman, U. A. Th. Analyst 1991, 116, 891-896. (21) Rule, G. S.; Mordehai, A. V.; Henion, J. Anal. Chem. 1994, 66, 230-235. (22) Oosterkamp, A. J.; Irth, H.; Beth, M.; Unger, K. K.; Tjaden, U. R.; van de Greef, J. J. Chromatogr. B 1994, 653, 55-61.

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Table 1. Description of Column Packings

no.

packing

column length (mm)

1 2 3 4 5 6 7 8

Sep-Pak C18 Sep-Pak CN C4 XAD-2 Pinkerton ISRP Sep-Pak Diol Vydac TP PRP-1

10 50 50 250 10 50 30 30

a

active surface

particle size (µm)

retention of tracer (%)

octadecyl propionitrile n-butyl polystyrene oligopeptide diol octadecyl polystyrene

200 200 200 250 10 200 5 10

100 100 100 100 100 80 100 100

protein absorption without with SBBa (%) SBB (%) 15 23 40 95 1 nt 100 100

5b 5 20 5 ntc nt nt nt

source Waters, Milford, MA Waters synthesized in-house Rohm & Haas, Philadelphia, PA Regis Chemical Co., Morton Grove, IL Waters Separations Group, Hesperia, CA Hamilton, Reno, NV

SBB, SuperBlock Buffer as described in the text. b Blotto Buffer used in this case. c nt, not tested.

Figure 1. (A) Block diagram of flow injection analysis system. The main components are an LC pump, a refrigerated autosampler, a column packed with the solid-phase material, and a fluorescence detector. (B) The reaction mixture consists of tracer (AG*), serum containing analyte (AG), and antiserum containing phenytoin antibody (Ab). The mixture is passed through a column that selectively traps the unbound tracer and analyte, but elutes antibody bound to the analyte. The eluted species pass through the fluorescence detector, and the fluorescence peak associated with the tracer-antibody complex is monitored.

proteins; and (3) does not contribute significantly to peak broadening. Column materials containing different hydrophobic moieties were evaluated on the basis of their capability to bind the analyte (antigen) and to elute protein with and without the use of various commercial protein blocking buffers. Regeneration of the column was performed by injecting ethanol solutions after several sequential immunoassays were performed on the columns. Performance parameters of various column packings will be discussed in detail. The therapeutic drug phenytoin was chosen as a convenient model analyte for the development of this new assay format. EXPERIMENTAL SECTION Reagents. All immunoassay reagents including antiserum containing phenytoin antibodies (Ab), fluorescein-labeled phenytoin tracer (AG*), and serum calibrators containing phenytoin (AG) were purchased from Sigma Chemical Co. (St. Louis, MO) as part of the Abott fluorescence polarization immunoassay (FPIA) kit. The serum calibrators contained 0, 10, 20, 40, 80, and 160 µmol L-1 phenytoin. NIST Standard Reference Material (SRM 900, NIST, Gaithersburg, MD) containing phenytoin was obtained as lyophilized sera and was reconstituted in phosphate-buffered saline (PBS). Three levels of phenytoin in SRM 900 were used 1666

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which were labeled as “toxic”, “therapeutic”, and “subtherapeutic”. All dilutions of tracer, antiserum, and calibrators were prepared in a PBS solution (pH 7.5) containing 0.14 mol L-1 NaCl, 2.7 mmol L-1 KCl, 8.1 mmol L-1 Na2HPO4, and 1.5 mmol L-1 KH2PO4. The carrier solution in the flow system was also PBS. The column regeneration solutions consisted of either pure ethanol or 10% ethanol solution in water unless otherwise specified. Commercially available blocking buffers were used in some cases to reduce adsorption of Ab on the columns. Superblock Buffer (SBB) and Blotto Buffer (BB) were purchased from Pierce Chemical Co. (St. Louis, MO). Fluorescein-conjugated human immunoglobulins, IgG whole molecule, were obtained from Pierce Chemical Co. Various column packing materials were used in the system to function as analyte-entrapment zones. These are listed in Table 1 with some characteristic features of each column packing material. Safety. All solutions from the Abott FPIA kit contained sodium azide (0.1%) as a preservative. This chemical is toxic if injested and can react with lead and copper pipes to form explosive compounds (metal azides). With serum samples, use normal precautionary measures associated with the handling of biological materials. Experimental Methods. The flow injection system is shown in Figure 1A. Components of the system are a refrigerated autosampler (Bio-Rad, Hercules, CA), an HPLC pump (Isco, Lincoln NE), and a variable-wavelength fluorescence detector. All connecting tubing in the flow system consisted of 0.5-mm-i.d. PTFE tubing. PBS was pumped through the system at a flow rate of 1.0 mL min-1. Excitation of the fluorescein tracer was performed at 490 nm, and emission was detected through a 515nm cutoff filter. Fluorescence polarization experiments were conducted using the Model K-2 fluorometer (ISS, Champaign, IL) to evaluate the stability of the antibody-antigen complex in the presence of ethanol solutions used for column regeneration. Six samples were prepared containing 100 µL of antiserum and 100 µL of the stock tracer solution. The samples were mixed and then incubated at 37 °C for 1 h. Following incubation, the solutions were diluted with ethanol and PBS. Control solutions contained only tracer with no antiserum. These solutions were placed in a cuvette, and polarization was determined using a 490-nm excitation wavelength and measuring emission at 516 nm on two channels (90° and -90°). Prior to use, serum calibrators (Sigma Chemical Co.) were diluted 50-, 100-, or 200-fold in PBS. The reconstituted standards

(SRM 900) were also diluted 200-fold in PBS before use. A 300µL aliquot of each calibration solution, as well as each standard, was diluted in half with the addition of 100 µL of tracer, 30 µL of antiserum, and 170 µL of PBS. These calibrators and standards were prepared and diluted in glass tubes because polypropylene tubes adsorbed the drug. The resulting solutions were incubated for 2 h at 37 °C and subsequently placed in the refrigerated autosampler (4 °C) for analysis. Aliquots (50 µL) of these solutions were injected into the system every 3.3 min for analysis. When blocking buffers were used to reduce nonspecific adsorption, either a single 500-µL injection of a 20% solution of blocking buffer in PBS was made prior to sample injection or a 5% solution in PBS was used as the carrier solution. When the blocking buffer was used as the carrier solution, the same buffer was also added to the sample in the same concentration. Following the reaction, the column packing was regenerated with solutions of pure ethanol or buffer containing ethanol. Data Manipulation. The calibration curves for the immunoassays were generated using the four-parameter logistic model.23 The model utilizes the following equation:

f(x) ) β2 +

(β1 - β2) 1 + (x/β3)β4

where x is the concentration, β1 is the asymptote as x goes to zero when β4 > 0, β2 is the asymptote as x goes to infinity, β3 is the predicted concentration halfway between the two asymptotes, and β4 is related to the slope. Because the value of the concentration was plotted on a logarithmic scale, the following equation applies:

f(x) ) β2 +

(β1 - β2) 1 + (exp(β4(log(x) - β3)))

The estimated lower and upper confidence limits, the minimal detectable concentration, the reliable detection limit, and the dynamic range have also been derived using the four-parameter logistic function. RESULTS AND DISCUSSION The assay protocol for this approach is outlined in Figure 1B. Phenytoin and fluorescently labeled phenytoin were incubated in solution with phenytoin antibodies allowing the two phenytoin derivatives to compete for a limited number of antibody binding sites. This reaction mixture was then passed through the column following some initial incubation time. As depicted in the figure, ideally only antibody-tracer complex should be detectable in the postcolumn solution. The resulting signal was then used to generate calibration curves. The amount of antibody-tracer complex was inversely related to the amount of free analyte in the serum. In a separate experiment, it was determined that the amount of tracer bound to the column could be eluted with a solvent to produce calibration curves. In this case, the amount of bound tracer was directly related to the amount of analyte in the serum. Selective trapping of the analyte was accomplished using several column packing materials. All column materials were (23) O’Connell, M. A.; Belanger, B. A.; Haaland, P. D. Chemom. Intell. Lab. Syst. 1993, 20, 97-114.

tested for their ability to trap and retain tracer. Injections of diluted tracer were made with and without the columns in place, and the percent retention is calculated from the ratio of the two resulting peak areas. Eight column packings were compared, and the results are shown in Table 1. All column materials except 6 strongly retained the labeled drug and were then tested for protein adsorption characteristics. Samples containing 100 µg mL-1 fluorescein-labeled human IgG in PBS were injected through the system with and without the column in place, and the eluted peaks were monitored. The ratio of the area of these peaks was used to evaluate nonspecific adsorption. All columns exhibited some retention of the fluorescein-labeled protein although, with the Pinkerton column (5 in Table 1), only 1% of the injected protein remained on the solid phase. The Sep-Pak C18 and Sep-Pak CN packing materials (1, 2) were comparable to one another, with moderate adsorption, while the remainder (4, 7, 8) demonstrated the greatest amount of protein adsorption. In cases where protein adsorption was greater than 1%, blocking buffers were used to discourage this effect. A carrier solution containing 5% SBB was very effective at reducing the nonspecific adsorption on most of the columns. This buffer, however, had no effect on reducing the nonspecific adsorption on column 1, therefore, an alternative blocking buffer (5% BB) was tried as the carrier. The high protein content of this buffer resulted in precipitation and column blockage during regeneration with ethanol solutions. Therefore, a single 500-µL injection of concentrated blocking buffer (20% BB) prior to the sample injection was used to minimize protein adsorption on the C18 column. The C18 and Pinkerton columns were used in subsequent experiments to demonstrate the immunoassay protocol. The C18 packing was chosen since it was somewhat superior to materials 2 and 4 with respect to tracer affinity and nonspecific protein adsorption, and it is an inexpensive material that is readily available in Sep-Pak columns (Waters, Milford, MA). Experiments were performed to determine the ratio of tracer to antiserum necessary to optimize immunoassay performance with regard to sensitivity and slope of the resulting calibration plot. Various amounts of antiserum containing phenytoin antibodies were added to 100 µL of tracer. The fluorescence intensity was measured initially by injecting the undiluted tracer directly into the detector, bypassing the column. All subsequent injections were passed through a Pinkerton column to capture unbound tracer. The intensity is proportional to the amount of tracer bound to antibody since this fraction elutes while the free tracer is retained on the column. Figure 2 shows the relationship between amount of antiserum and intensity of the fluorescence of the eluted peak. The maximum intensity is achieved when all tracer passes through the column bound to antibody. As shown in the figure, the curve plateaus with the addition of approximately 30 µL of antiserum; therefore, all samples were prepared with an antiserum to tracer ratio of 0.3. Solvent injections were used as a simple means of regenerating the column for continuous usage. Injections of pure ethanol successfully removed tracer and renewed the column capacity for subsequent analyses; however, column blockage occurred frequently as a result of precipitated protein or precipitated salts. Dilute solutions of ethanol in buffer were also tested as the carrier solution in place of PBS for two purposes: (1) to discourage antibody adsorption and (2) to continuously elute the tracer at a slow rate to obviate the need for periodic regeneration. Figure 3 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

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Figure 2. Calibration curve for optimization of the immunoassay sensitivity. Several aliquots of antiserum (from 20 to 80 µL) were incubated with the same volume of tracer (100 µL) and then injected into the Pinkerton column. The fluorescence intensity of the eluted peak area is plotted versus the amount of antiserum added to determine the minimal amount of antibody necessary to bind all tracer.

shows the fluorescence peaks observed when tracer was injected into the flow system in the presence of varying concentrations of ethanol in the carrier buffer. At 0% ethanol (pure PBS), all tracer was retained on the column and a subsequent 200-µL injection of pure ethanol produced a peak associated with elution of the tracer. With increasing concentrations of ethanol, there is more evidence of column bleed and the subsequent injection of pure ethanol produces a smaller fluorescence peak. At lower ethanol levels, this tracer bleed can be subtracted out as background and therefore the inclusion of ethanol in the carrier buffer can serve to regenerate the column continuously. Fluorescence polarization experiments were performed to determine the stability of the antigen-antibody complex in the presence of ethanol solutions. In fluorescence polarization studies, all tracer bound to the antibody should emit polarized light, and free tracer should emit nonpolarized light. If the presence of ethanol disturbed the complex, the amount of polarized light should decrease as shown in Figure 4. Between 20 and 30% ethanol, there was a significant change in the stability of the antibody-tracer complex. Buffer containing 20% ethanol had little effect on the stability of the complex. At 10% ethanol, the complex was completely intact, and this solution was used as the regeneration buffer in several subsequent experiments. The protocol initially included a 2-h incubation step followed by injection from the autosampler at room temperature. The reproducibility of this method was very poor as sequential injections of the same sample produced antibody-tracer peaks that continually increased in area. The reproducibility of the method was significantly improved by storing the reacted samples at lower temperatures (4 °C) in the refrigerated autosampler to minimize further reaction of the species in solution so that the time between injections did not effect the reproducibility of the assay. All subsequent immunoassays were performed with samples at 4 °C. For the immunoassay to be successful, the affinity of the drug, phenytoin, for the hydrophobic packing must be greater than the 1668 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

Figure 3. Peaks obtained by injecting tracer (peak 1) onto the Pinkerton column in the presence of varying concentrations of ethanol in the carrier buffer. Injections of tracer were followed by a single 200-µL injection of pure ethanol to remove tracer from the column (which produced peak 2). The carrier buffer contained the following concentrations of ethanol: (A) 0, (B) 10, and (C) 15%.

association with the serum carrier proteins. Otherwise, the drug would pass through the column since the proteins are not adsorbed under the conditions used for these analyses. The SepPak C18 and Pinkerton columns were found to be very efficient at extracting the drug from serum proteins. This fact enabled us to inject serum onto the column without pretreatment, e.g., incubation with surfactant to remove the drug from the carrier proteins. A 10% ethanol solution used as the carrier buffer allowed for analysis of many sequential samples on the C18 column without a separate column regeneration step. After approximately 18 analyses, however, the column reproducibility decreased and a regeneration step was performed with infusion of the column with 10% ethanol in PBS for 15 min at a flow rate of 2 mL min-1. A broad peak was observed resulting from the removal of tracer from the column. The signal returned to baseline after 15 min, and the column was rinsed with PBS prior to the next sample injection. The area of the peak resulting from unretained antibody-tracer complex was plotted versus the concentration of phenytoin in the sample to generate a calibration curve, together with a 95% confidence envelope, shown in Figure 5. Data in this plot were generated by injecting calibrators (lowest to highest concentration) and then performing repeat injections in the same order. All data were produced on a single day with no additional regeneration step. The relative standard deviation of the fluorescence measurement was 1.1% for n ) 36. The reliable detection

Figure 4. Effect of the different concentrations of ethanol on the stability of the Ab-AG* complex. The stability of the complex was monitored using fluorescence polarization. In this experiment, tracer (AG*) that is bound to Ab emits polarized light, and when the tracer is uncomplexed, the polarization decreases. Fluorescence polarization of samples (9) is plotted versus the amount of ethanol in PBS. Fluorescence polarization of control solutions (b) are also shown as described in the text.

Figure 6. Standard calibration curve of the flow injection immunoassay for phenytoin using Pinkerton column entrapment. The concentration of phenytoin in serum is plotted versus the peak area using a log-linear scale. The calibration plot is based on 64 experimental runs for three dilutions of standard samples: 1:50, 1:100, and 1:200 in PBS. Each point is the average of 6-10 replicates. The antiserum to tracer ratio was 0.3 in this experiment. The envelope lines represent the 95% confidence limits. Table 2. Determination of Phenytoin in SRM 900a levels

av value (nmol L-1)

SD (nmol L-1)

n

certified value (nmol L-1)

toxic therapeutic subtherapeutic

1240 380 100

100 20 13.5

6 5 5

1200 330 83.5

a

Figure 5. Standard calibration curve of the flow injection immunoassay for phenytoin using C18 column entrapment. The concentration of phenytoin in serum is plotted versus the peak area using a log-linear scale. The calibration plot is based on 18 experimental measurements for three dilutions of standard samples: 1:50, 1:100, and 1:200 in PBS. The antiserum to tracer ratio for these measurements was 0.5. The envelope lines represent 95% confidence limits.

limit for the assay at the 95% confidence level was found to be 35 nmol L-1, and the minimal detectable concentration was calculated to be 18 nmol L-1.23 Using the 95% confidence interval, the relative uncertainty in the determination of concentration at the inflection point on the log-linear calibration plot was found to be approximately 11%. The dynamic range was found to be 170-1750 nmol L-1 (10% CV) and 70-2600 nmol L-1 (20% CV) although more concentrated phenytoin samples can easily be measured following dilution with buffer. Baseline-to-baseline resolution of the fluorescence peaks was accomplished in 1.8 min; therefore, with this column it is possible to process 33 samples per hour. The Pinkerton column packing is an HPLC column material designed for serum analyses. A short 1-cm column, intended for use as a guard column, was incorporated into the flow system. Since this material exhibited very little adsorption capacity for

All serum samples diluted by a factor of 200 with PBS.

human IgG, the assay was performed without blocking buffers. The relative standard deviation of the fluoresence measurement was 1.1% for n ) 48. Baseline-to-baseline resolution of the peaks was accomplished in 2.2 min; therefore, 27 samples could be analyzed per hour. The calibration curves shown in Figure 6 extended over the range from 70 to 1800 nmol L-1 (10% CV) and 20 to 3400 nmol L-1 (20%). Again, the relative uncertainty in the determination of concentration at the inflection point on the loglinear calibration plot was found to be approximately 11%. The reliable detection limit was found to be 5 nmol L-1 at the 95% confidence level, and the minimal detectable concentration was determined to be 2 nmol L-1. The performance of the immunoassay was validated by analyzing three different standards (SRM 900) that contained phenytoin at toxic, therapeutic, and subtherapeutic levels in serum. The samples were analyzed on two different days, and the results are shown in Table 2. The “toxic” sample was cloudy upon reconstitution in buffer; however, the results indicate that filtering is unecessary even when the serum contains precipitated material. The column was regenerated every 30 samples by injecting a solution containing 10% ethanol into the system at a flow rate of 2 mL min-1. After an 8-min flush with the regeneration buffer, the signal returned to baseline. Inclusion of dilute ethanol in the carrier for continuous regeneration was unnecessary, since the packing demonstrated an extremely high affinity for the drug. CONCLUSIONS We have demonstrated the utility of a new approach to flow immunoassays that uses the hydrophobic retention mechanism Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

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of a reversed-phase LC column packing material to separate complexed from uncomplexed species in an immunoassay. The system was tested using phenytoin in a serum matrix, and the adsorption of other serum components did not adversely effect the assay since these column packing materials have a very high capacity. The assay had a demonstrated throughput of approximately 30 samples per hour with excellent reproducibility, and the sample throughput could be easily increased by increasing the carrier flow rate. The assay was found to have a reliable detection limit of 5 nmol L-1 when the Pinkerton packing was used. These data compare favorably to the sensitivity of the fluorescence polarization immunoassay method using the same reagents which was reported to be 1.8 µmol L-1. Other advantages of the solid-phase entrapment flow immunoassay include rapid column regeneration and long column lifetime. The simplic-

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ity of the approach should make it suitable for use in clinical as well as environmental applications. ACKNOWLEDGMENT L.L.-B. and G.H. acknowledge a grant from the U.S.-Hungarian Joint Fund, which supported this collaborative work. Certain commercial products are identified in order to adequately specify the experimental procedure. This does not imply endorsement or recommendation by the National Institute of Standards and Technology. Received for review December 4, 1995. February 16, 1996.

Accepted

AC951173N X

Abstract published in Advance ACS Abstracts, March 15, 1996.