Anal. Chem. 1990, 62, 2587-2593
2587
Liposome Flow Injection Immunoassay: Implications for Sensitivity, Dynamic Range, and Antibody Regeneration Laurie Locascio-Brown,* Anne L. Plant, Viola Horvlth,' and Richard A. Durst
National Institute of Standards and Technology, Gaithersburg, Maryland 20899
We have developed a Ilposome-based flow injection Immunoassay (FI I A ) system for quantltation of a cllnical analyte, theophylline. With very minor changes In assay format, this procedure can also be used for the quantltation of anti-theophylllne. Automated sequentlal analyses were performed at room temperature with plcomole sensitlvlty and a day-to-day coefficient of variation of less than 5 % for aqueous solutions. The system components Include llposomes that contain fluorophores In thelr aquepus centers and an immobilized-antibody reactor column. The lmmunoreactor was regenerated hundreds of times over 3 months of continuous use wlth no measurable loss of antlbody actlvky. The two assay formats studied produced dlstlnct dynamlc ranges for thelr respective anaiytes. The special advantages of using flow injection analysls for Immunoassays and of uslng ilposomes In F I I A are discussed.
INTRODUCTION Recent interest in applying the advantages of immunoreagents to the field of analytical chemistry has led to the development of automated techniques using biomolecules. Currently, most solid-phase immunoassays are performed with 96-well microtiter plates in which multiple samples can be processed simultaneously. This technique has proven to be sensitive, but it is only semiquantitative (I,2) and is difficult to automate. Flow injection analysis (FIA), on the other hand, is an easily automated technique that can be adapted to accommodate many immunoassay formats. Previously developed FIA immunoassays use electrochemical detection (3) or optical detection (4,5). FIA is an attractive technique to apply to immunoassays because precise control of reagent addition and reaction times offers the potential for high analytical precision. Antigen-antibody binding is not accompanied by a readily detectable physical or chemical change; therefore, a detectable signal is usually generated by the addition of a labeled species. Labels typically used in immunoassays include radioisotopes, fluorophores, and enzymes. Enzymes are most commonly employed in solid-phase assays as labels to enhance sensitivity because of their ability to convert substrate into a detectable product (6). Very low detection limits are achievable by using long incubation times during which many enzyme turn-over events can occur (7). A major drawback of this approach is the long analysis time due to the multistep process involving antigen-antibody complexation, enzyme labeling, and substrate conversion. In the immunoassay described in this paper, the signal is provided by liposomes which entrap a fluorophor, carboxyfluorescein, as the detectable label. Liposomes are spherical structures that consist of a phospholipid bilayer which surrounds an aqueous interior. Approximately 105 water-soluble
* To whom correspondence should be addressed.
Permanent address: Institute for General and Analytical Chemistry, Technical University of Budapest, Budapest Hungary.
fluorescent molecules can be entrapped in a single liposome. Since the detection and signal amplification using liposome labels are not dependent on a secondary reaction, as is required for enzyme labels, the use of liposomes has the advantage of providing large signal amplification immediately. Previously, we have demonstrated that, in addition to allowing faster analysis, liposomes provide 2 orders or magnitude more sensitivity than enzymes in a solid-phase immunoassay (8). Other work done in our laboratory involved the examination of the hydrodynamic behavior of liposomes in FIA (9). In this report, we describe an automated flow injection system, based on the use of liposomes in competitive immunoassays. The system was developed by using theophylline, a therapeutic drug usually measured in serum, as a model compound for evaluation of the technique. Most often, clinical measurements of theophylline concentration are made by fluorescence polarization, or by the enzyme multiplied immunoassay technique (EMIT). Both of these assays are homogeneous and require the addition of new reagents for each assay. This is a potential disadvantage when the reagents are costly or difficult to produce. In addition, the fluorescence polarization technique is limited to the detection of compounds that are small (1000-10000 molecular weight). The flow injection immunoassay system described in this report has been used to measure a small analyte, theophylline (180 MW), as well as a large protein, anti-theophylline (160000 MW). This system can be calibrated and is reusable. We have used a packed-bead column containing immobilized antitheophylline on nonporous silica particles to measure both analytes with only a slight variation in the assay procedure. Both immunoassays described are competitive and heterogeneous where separation is carried out on the antibody column. The basis for the substantial differences in the dynamic range and sensitivity of these two assays, and the analytical implications, will be discussed.
EXPERIMENTAL SECTION Apparatus. A diagram of the flow injection immunoassay (FIIA) system is shown in Figure 1. The flow system was constructed with 6-V solenoid pinch valves to control the movement of samples and reagents. Valve operation was controlled by a microprocessor for the precise timing of events. The sample was aspirated into a 50-pL sample loop at a rate of 180 pL s-l. The immunoreactor column consisted of a glass tube 10 cm in length and 2 mm i.d. (6.35 mm 0.d.) packed with antibody-derivatized silica particles which were contained by nylon frits with a mesh size of 60 pm. Connecting Teflon tubing had an inner diameter of 0.5 mm. The flow rate through the system was 0.2 mL min-' and was controlled by a peristaltic pump at the outlet. Sample peaks were detected in a 4-pL flow cell using a fluorescence detector. The excitation wavelength was 480 nm, and emission was measured through a 515-nm long-pass filter. For experiments involving the use of KSCN, solutions were degassed under vacuum for 30 min each day prior to use in the system. If not degassed, the large temperature differential created when the KSCN solution was injected onto the antibody column caused bubbles to form and be trapped within the column. Reagents. Phosphate buffered saline (PBS) (8.1 mM Na2HPO4, 1.5 mM KH2P04,2.7 mM KCl, 0.14 M NaCl, and 0.01%
This article not subject to US. Copyright. Published 1990 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23,DECEMBER 1, 1990
2588
I
OG
I
I
1 I3 9
waste
U
sample
vacuum
Flgure 1. Schematic of flow injection immunoassay (FIIA) system. OG and PBS indicate the N,-pressurized reservoirs of those reagents.
Rectangular boxes are solenoid valves. Column indicates the immunoreactor column containing silica particles on which antibody is immobilized. Boxes marked D and P indicate fluorescence detector and peristaltic pump, respectively.
NaN3, pH 7.4) was used in all experiments except when otherwise noted. Distilled and deionized water was used to dilute buffers (OG) and samples. Solutions of l-O-octyl-fi-D-glucopyranoside at a concentration of 21.4 mM in buffer and potassium thiocyanate (KSCN) at a concentration of 3 M in buffer were used. Mouse monoclonal anti-theophylline IgG was purchased from American Qualex, Inc. (La Mirada, CA), as ascites fluid and was purified on Protein A agarase (Bio-Ftad Laboratories, Richmond, CA). The [8-3H]theophyllinewas purchased from Amersham Corp. (Arlington Heights, IL). Preparation of Immobilized Antibody. Fused silica particles, provided by Dr. Wolfgang Haller (Ceramics Division, NIST), ranged in size from 150 to 180 pm in diameter. The particles were treated in concentrated nitric acid for 18 h over a steam bath, washed repeatedly in distilled water, and dried for 3 h at 140 "C. The particles (20 g) were then reacted over a steam bath for 30 h in a 3% solution of (3-glycidoxypropyl)trimethoxyshe (GOPS) in toluene (30 mL). The silica was washed exhaustively in toluene and methanol, dried under a heat lamp, and cured for 3 h at 135 OC. Five grams of silanized particles was added to a solution of 0.5 g of periodic acid in 100 mL of 80% glacial acetic acid for 1.5 h to cleave the diol and oxidize the alcohol to an aldehyde. The particles were rinsed thoroughly with distilled water and used immediately or placed in a desiccator until needed. Purified anti-theophylline or y-globulin was coupled to the solid support as described previously (10). Determination of Immobilized Antibody Binding Activity. The immunospecific binding activity of immobilized anti-theophylline on silica particles was determined by use of a [3H]theophylline derivative as previously reported (10). The following controls were used to determine nonspecific adsorption of the radiolabeled antigen to the silica: (1)GOPS-derivatized particles following oxidation with periodic acid to the aldehyde, and (2) silanized particles reacted with bovine y-globulin. All measurements of binding activity were done in duplicate. Activity of the immobilized protein was determined by subtracting the value of the highest control sample from the value obtained with the immobilized anti-theophylline particles. Nonspecific binding of [3H]theophyllineto control particles was less than 0.5% of the specific binding to anti-theophylline particles. The apparent binding constant (K,)of immobilized antitheophylline was determined by varying the concentration of [3H]theophylline added to 0.2 g (0.1 mL) of particles while maintaining the specific activity of the radiolabel. The amount of bound theophylline was determined for each concentration and subtracted from the total amount added to calculate free theophylline. Concentrations were determined by using the reaction volume of 1 mL. The equilibrium constant was determined by Scatchard analysis (II), in which the ratio of bound-to-free theophylline is plotted vs bound theophylline, and the resulting slope is -K,. Liposome Preparation and Characterization. Liposomes containing 80-100 mM 5- (and 6-)carboxyfluorescein (CF) in their aqueous centers were prepared from a mixture of dimyristoylphosphatidylcholine,cholesterol, and dicetyl phosphate in a molar
ratio of 5:4:1, using the injection method described in detail in a previous paper (12). To make the liposomes immunogenically reactive to anti-theophylline, a conjugate of 8-(3-carboxypropyl)-1,3-dimethylxanthine and phosphatidylethanolamine (theophylline-PE) was prepared (13). This partially purified preparation of theophylline-PE was used in liposomes at a final molar ratio of 5% and 0.8% of total lipid. The diameter of the theophylline-PE liposomes was measured by dynamic light scattering to be 135 f 65 nm. Lipid concentration of liposome preparations was determined by phosphorus analysis according to the method of Bartlett (14). By use of these measured values, and assuming a surface area per lipid molecule in liposomes of 50 A2 (15),the number of liposomes per milliliter was estimated to be 4 x 10l2. Interaction of Liposomes with Immobilized Antibody. Apparent binding constants were determined in order to quantitatively compare theophylline-PE liposomes and theophylline as ligands for immobilized antibody. Liposomes were treated as individual macromolecular assemblies, each of which behaves as a multivalent ligand. To measure the apparent binding constant between theophylline-PE liposomes and immobilized anti-theophylline, different concentrations of liposomes in 1-mL solutions were incubated with 0.2 g (0.1 mL) of particles. Particles plus liposomes were incubated on a rocker for 1h at rwm temperature, and the particles were washed 5 times with buffer. Liposomes bound to the particles were then disrupted by addition of 1.5 mL of OG, and the associated fluorescence was quantified by fluorometry. Concentrations of bound and free liposomes were determined with respect to the initial reaction volume. Free liposomes were determined by subtraction of the intensity of the carboxyfluorescein entrapped in bound liposomes from the intensity of total liposomes added. The ratio of the molar concentration of bound-to-free liposomes was plotted against the molar concentration of bound liposomes. The slope of the Scatchard plot is -Ka. Dissociation Rate Constants. The rate constants for dissociation of [3H]theophylline and theophylline-PE liposomes from immobilized anti-theophylline were compared. Immobilized anti-theophylline particles (0.2 g) were incubated with either [3H]theophylline or theophylline-PE liposomes to achieve apM in liposomes, proximately 90% of maximal binding (1X and 5 X lo-' M in [3H]theophylline). This was followed by the addition of a buffer containing a 1000-fold higher concentration of theophylline, and incubation for varying times. Liposome assays were performed in test tubes as described above, but [3H]theophylline experiments were performed in a filtration apparatus in order to permit rapid processing of samples. For liposome experiments, following the initial incubation with immobilized antibody, the particles were washed and incubated for varying times with theophylline. At the appropriate times, supernatant solution was removed, the particles were washed once and then treated with OG to release the carboxyfluorescein from the bound liposomes, and the resulting fluorescence was measured. For the [3H]theophyllineassays, incubation of particles with [3H]theophylline occurred in a volume of 0.1 mL in filtration wells. The well contained glass fiber filters that had been pretreated with 0.3 mL of buffer containing 10% bovine serum albumin (BSA) (w/v) for 15 min to block nonspecific binding. After the initial incubation, the wells were drained and washed 10 times with a solution of buffer containing 0.3% gelatin. Unlabeled theophylline solution (0.1 mL) was then added to the wells for varying periods of time. A t the appropriate times, the wells were drained, and the filters and particles were removed so the bound [3H]theophylline could be counted. Assays performed in test tubes and assays performed by filtration, both under the same conditions, gave the same results. Comparison of Regeneration Methods. To test regeneration of active sites, immobilized antibody particles were incubated in test tubes with 0.5 mL of either KSCN or OG for 4 min. This treatment was preceded either by no preincubation or by preincubation with 8 X 1O'O liposomes in a volume of 0.5mL for 1 h. After particles were washed 3 times with buffer, antibody activity was measured with [3H]theophylline. To test long-term effects of the reagents, KSCN or OG solutions were cycled through an immunoreactor column for 4 min followed by rinsing with buffer for 5 min. This protocol was repeated for 50 cycles before the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23,DECEMBER 1, 1990
C
2589
A
Y
4
ANTI.THEOPHYLLINE
'
-
gh0t:ATED ANTIBODY. PARTICLES
y
#
I
' A
A
Figure 3. Description of assay formats. Part A depicts the format of FI I A for anti-theophylihe. Anti-theophylline in the sample binds to theophylline-PE liposomes, blocking binding of the liposomes to the immobilized antibody column. Part B represents the format of FHA
for detection of theophylline. Theophylline liposomes directly compete with free theophylline in the sample for antibody sites on the silica particles.
1.3s
6"
!mn 4min
5 min
Flgure 2. Flow injection fluorescence peaks from a single injection experiment. Peak A represents Intact liposomes that passed through the immunoreactor column without binding. Peak B results from rinsing all lines (but not the column) with 00 to remove any liposomes caught
in the tubingconnectingtees. Peak C is the analytical peak that results from lysing of the liposomes on the column with 00 and is a measurement of the amount of liposomes which interacted specifically with the immobilized antibody reactor. See text for details. immobilized antibody activity was again measured with t3H]theophylline. Flow Injection Immunoassay for Determination of Theophylline. Solutions of varying concentrations of theophylline, in buffer or in reconstituted lyophilized serum, were mixed with liposomes so that the final concentrationof liposomes in solution was 8 X 1O'O liposomes mL-'. For some experiments, caffeine, which has a cross-reactivity in immunoassays for theophylline, was substituted for the theophylline. The assay timing sequence and typical signal output are shown in Figure 2. The following describes the sequence of events in a single assay: (1) The sample (50 rL) containing the analyte (theophylline or caffeine) plus liposomes was aspirated into the sample loop. (2) Injection of the sample occurred by passing buffer solution through the sample loop. Liposomes that did not bind to the immobilized antibody in the reactor column flowed downstream and through the detector to produce peak A in Figure 2. (3) The sample loop lines were rinsed with buffer. (4)OG flowed through solution lines (not in series with the reactor column) to remove any nonspecifically adsorbed liposomes. This step produced the small second peak B. (5) OG solution flowed over the column for 4 min. OG disrupted the liposomes that were immunospecificallybound to the immobilized antibody, and their entrapped fluorophores were released and flowed downstream through the detector. This resulted in a third peak, C, which represented the analytical signal. Calibration curves were generated for theophylline and caffeine in buffer based on the measurement of the height of peak C. (6) The column was restored with buffer. During double-injection experiments, steps 1-3 were repeated before performing steps 44. During triple-injection experiments, steps 1-3 were repeated twice before performing steps 4-6. Flow Injection Immunoassay for Anti-Theophylline. Prior to analysis, samples containing anti-theophyllinewere mixed with an amount of theophylline-PE liposomes so that the final concentration of liposomes in solution was 8 X 1O1O liposomes mL-'. The mixture was allowed to react for 30 min prior to injection onto the column. The timing of events was identical with the procedure outlined above for the theophylline determinations. A calibration curve was generated by plotting maximum intensity of the peak C versus the log concentration of antibody.
Determination of Assay Sensitivity and Minimal Detectable Concentration, The calibration curves contained an apparent linear portion, which was used to simplify calculation of the concentrations of samples. Assay sensitivity is defined as the slope of the apparent linear portion of the calibration curves. The minimal detectableconcentration (MDC)represents the limit of detection for the methods and was determined by using a statistical method described by Rodbard (16)using a one-sided Student t test for three degrees of freedom. For determination of the MDC of the assays, it was assumed that the zero response (when the concentration of analyte added was zero) was equal to the highest point on the calibration curves. Stability of Liposomes in Complex Matrices. Carboxyfluorescein was encapsulated into the aqueous center of liposomes at a concentration which resulted in quenching of the fluorescence. Stability (i.e., resistance of liposomes to leakage) was measured by monitoring the increase in fluorescence intensity of a liposome solution when diluted with buffer or serum.
RESULTS AND DISCUSSION We have developed two different flow injection immunoassays that use fluorescent dye containing liposomes. One assay is for the quantitation of the therapeutic drug theophylline, and the other is for anti-theophylline. For both assays, anti-theophylline was immobilized onto nonporous silica particles, which were packed into an immunoreactor column, and both assays employed liposomes that contained theophylline-PE in their membranes. As depicted in Figure 3A, for the detection of anti-theophylline, the analyte competed with immobilized anti-theophylline for sites on the theophylline-labeled liposomes. In the detection of theophylline (Figure 3B), the analyte competed with theophylline-labeled liposomes for immobilized antibody sites. The result in both cases was an inverse relationship between the amount of analyte in the sample and the amount of liposomes that bound to the solid support. The two assays, however, differed considerably in sensitivity for their respective analytes. This difference was a function of assay format and the characteristics of the interaction of liposomes with immobilized antibody. In this report, we will begin by discussing the apparent binding constants and dissociation rate constants of liposomes for immobilized antibodies and the resulting analytical implications. This dicussion will be followed by a complete description of the automated immunoassays. The competitive assay is a convenient and popular format for immunoassays. It is appreciated that the dynamic range of competitive assays is inherently limited to analyte concentrations that center around the inverse of the binding constant (i.e., l/Ka) of the antibody for the analyte (11).
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990 30.
Table I. Characteristics of Immobilized Monoclonal Anti-Theophylline
'3
1 2e-14
i
binding reverse half-time of ligand constant, rate constant, dissociation, densit9 K,, M-' k,, min-' min theophylline theophylline-PE liposomes 0.8 mol % 5.0 mol %
1
881 5839
2.1 X 10'
0.553
5.8 X 10'O 8.0 X loxo
0.048 0.017
1.25 14.4 40.8
Ligand density refers to the approximate number of theophyllineP E molecules in the outer leaflet of the liposome bilayer.
Therefore, in general, a higher binding constant results in a more sensitive assay. As shown in Table I, our monoclonal anti-theophylline demonstrated a moderate affinity for its ligand, theophylline, a t 2 X 10' M-l. Therefore, competitive immunoassays developed by use of this antibody would be expected to have a measurement range at best around 5 X lo4 M theophylline, since this is the inverse of the antibody binding constant for theophylline. In the development of one-shot or nonreusable devices, the binding constant is by far the most relevant antibody characteristic. In these devices, formation of the antibody-antigen complex is very important; removal of the complexed antigen for regeneration of the antibody site is inconsequential. In the development of reusable devices, however, it is equally important to consider the reverse rate of the reaction and the ease of dissociation of the antigen from the antibody. For the reaction Ag
+ Ab + AgAb
K, =
[&Ab1 12, [Agl[Abl = k,
where K , is the apparent binding constant, kf is the forward rate constant, and k , is the reverse rate constant. Since the binding constant is inversely related to k,, rapid dissociation is usually associated with a lower affinity antibody. Therefore, in the design of a reusable assay system using a competitive assay format, there is a trade-off between sensitivity (high binding constant) and the ease of regeneration (fast dissociation). As seen in Table I, the apparent binding constant of liposomes for immobilized anti-theophylline is several orders of magnitude higher than for [3H]theophylline. The magnitude of the apparent binding constant is greater for liposomes containing higher surface densities of theophylline-PE, indicating that the effect is the result of the multivalency of the liposomes. Dissociation rate constants, k,, were determined by nonlinear least-squares regression analysis of fluorescence or radioactivity associated with particles as a function of time of incubation with unlabeled theophylline. Because of the presence of a high concentration of unlabeled theophylline, reassociation of liposomes or [3H]theophylline to immobilized antibody was inhibited. Decay of signal with time always fit a monoexponential function. Half-times for dissociation, tl,*, were calculated from the rate constant
Rate constants for dissociation, also shown in Table I, are inversely related to the apparent binding constants, as predicted. It is important to note that t I l zfor dissociation of univalent theophylline from immobilized anti-theophylline is only 1.25 min. This dissociation rate is a key to the gentle and efficient regeneration of the immunoreactor column.
I
m
2
Q
4
c
c
C40r-15
1 :
3
I
20
40
50
8
Time (min)
Figure 4. Dissociation of theophylline and theophylline-PE liposomes from immobilized anti-theophy". Relative fluorescence intensity from theophylline-PE liposomes and moles of theophylline determined from radioactivity are plotted as a function of time of incubation of immobilized antibody in the presence of large concentrations of unlabeled theophylline. Actual data are shown by symbols. Dotted lines are the single exponential decay functions.
Regeneration of Immobilized Antibody Activity. Two different methods of column regeneration were compared for their effect on antibody activity as well as for their effect on relative sensitivities and reproducibility of sequential immunoassays. The first method involved the use of the chaotropic agent, KSCN. Chaotropic agents generally act to disrupt antigen-antibody complexes by exposing the antibody to chemical conditions that induce a change in the antibody structure resulting in a weakening of the interaction with the antigen (17-1 9). Depending on the chaotropic reagent used, the protein may become more or less denatured and can lose activity if the denaturation is irreversible (18). An alternative approach to column regeneration is by infinite dilution allowing dissociation to occur by mass action. This method is easily performed in a continuous flow system where dissociated antigen is continuously removed and prevented from reassociating with antibody. In this case, the dissociation rate constant determines the time required for regeneration of sites. For a low or moderate affinity interaction, the antibody can be freed in a reasonable time under very mild, nondenaturing conditions. A graphic comparison of the time course of dissociation of theophylline-PE liposomes and theophylline from the immobilized antibody is shown in Figure 4. From calculations based on the data in Figure 4, in a continuously flowing stream, 90% of the theophylline is dissociated from the antibody in 7.5 min. If theophylline is the only reactive species in the sample, 99% of column activity is restored in 8.75 min. In order to perform a competitive assay, however, theophylline liposomes also react with the immobilized antibody. Liposome dissociation, because of the liposome multivalency,is very slow. However, if the liposomes are disrupted, 99% of the resulting univalent theophylline-PE can also be removed in 8.75 min from the column, and the column activity can be completely restored in a short time. OG and KSCN were compared for their effectiveness as column regeneration reagents. Three criteria were evaluated: regeneration of binding sites, long-term effect on antibody activity, and effect on fluorescence signal. A comparison of their effectiveness at disrupting liposome-antibody complexes is shown in Table IIA. OG treatment for 4 min actually increased antibody activity (an effect which is currently being studied in more detail), while KSCN reduced activity by approximately 60%. Furthermore, OG was more effective at removing liposomes from the solid phase: 60% of original binding activity was recovered following this low-volume regeneration treatment. KSCN treatment following liposome
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
Table 11. Effect of Regeneration Methods on Antibody Activity
I
2591
i
A. Stability of Immobilized Antibody Activity following Regeneration of the Antibody Binding Sites by Various Methods
treatment none KSCN (4 min) theophylline-PE liposomes; KSCN (4 min) none OG (4 min) theophylline-PE liposomes; OG (4 min)
remaining binding sites, mol/mm2 x ioi7
fraction of original activity
11 h 70 4.7 h 0.6 2.5 f 0.4
1 0.43 0.23
7.7 f 3.2 17 h 2 4.6 h 1.3
1 2.2 0.6 .I1
50 Regeneration Cycles Using Different Methods
treatment
initial activity, mol/”* x 1017
KSCN OG
3.5 f 1.2 5.8 h 1.0
a Uncertainty
remaining fraction of activity, original mol/”* x 101~ activity 0.09 f 0.006 6.2 h 2.8
.
0
B. Stability of Antibody Activity following
0.03 1.1
represents standard deviation of the measurement.
binding resulted in recovery of approximately 20% of the initial activity. To test the effect of long-term treatment with the reagents, test regenerations were performed on two packed antibody columns for 50 cycles, and the remaining antibody activity was determined. As shown in Table IIB, the column treated with OG retained all of its original activity, whereas the column treated for 50 cycles with KSCN lost 97% of its original activity. When used as a component of the FIIA system, an immobilized antibody column regenerated with OG performed 274 assays over a period of 3 months, at room temperature, with no measurable loss in antibody activity. In contrast, the application of KSCN as regeneration reagent for the anti-theophylline FIIA was not nearly as successful, as is discussed below. The third criterion was the effect of the reagents on fluorescence intensity of carboxyfluorescein. A solution of 2.5 X M carboxyfluorescein was prepared in either OG or KSCN. The fluorescence of the carboxyfluorescein solution was reduced by 93% in the presence of KSCN. Conversely, there was no signal reduction in the OG solution other than that caused by dilution. Therefore, in addition to other disadvantages, the sensitivity of assays performed by using KSCN was greatly compromised due to the large signal attenuation caused by fluorescence quenching. Flow Injection Immunoanalysis for Anti-Theophylline. The assay for anti-theophylline was performed by adding anti-theophylline aliquots directly to a solution of the theophylline-PE liposomes and then injecting this sample into the column for reaction with the antibodies on the solid support. Liposome binding to the column was reduced by the binding of anti-theophylline in the sample to theophylline in the liposomes. The assay was successfulpresumably because partial blocking of the sites resulted in a reduction in the apparent binding constant of the liposomes. The anti-theophylline assay was performed initially by using KSCN as the regeneration buffer. With a new column, the standard deviation was less than 5%. The column rapidly deteriorated after 40 measurements to a within-day relative standard deviation of >50% for N = 9. The poor reproducibility of the KSCN-regenerated assay can be explained by the loss in activity during regeneration as described previously. Degradation of antibody activity or corresponding assay reproducibility was not seen when OG was the regeneration reagent. The results of assays using the two regeneration
-153
, .10
.
.
(1)
, .9
.13 3
.
, .8
.
, .7 .I1 3
.
, .6
..5
.93
M moles
Log Amount Anti-theophylline Figure 5. Calibration curves for anti-theophylline using 00 or KSCN regeneration. The KSCN curve (1) was generated by using the triple injection method described in the text. Three curves were generated by using OG regeneration with single (2), double (3), and triple (4) injections.
reagents are shown in Figure 5. Curves 1and 4 are the results of the assays for anti-theophylline with KSCN or OG for regeneration, respectively, and were obtained by using the three-injection protocol to enhance the signal of each. Single injection experiments for anti-theophylline using KSCN were below our detection capabilities. Signal intensities obtained with the two reagents differ by approximately a factor of 20. All other assays for theophylline or anti-theophylline (described below) were performed using OG regeneration. Curves 2 , 3 , and 4 in Figure 5 are the results of the anti-theophylline assays in which the sample was injected one, two, and three times, respectively, prior to disruption of bound liposomes using OG. As shown in the figure, after a maximum on the calibration curve was attained, binding of the label to the solid phase decreased. This response is different from a typical dose-response curve where the binding reaches saturation at lower antigen concentrations. This effect occurred in our assays a t lower levels of analyte, therefore increasing the relative amounts of liposomes and liposome binding, and is similar to the “hook effect” noted by others in solid-phase immunoassays (20). The linear range in all three cases was less than 1order of magnitude. As shown in the figure, the fluorescence intensity at the minimal detectable concentration increased by a factor of 2.1 in double versus single injection experiments. Prior to liposome lysis with OG, the total time for a double injection experiment was 12 min. Due to the slow rate of dissociation of multivalent theophylline-PE liposomes, approximately 85% of the bound liposomes from the first injection remained on the column after this time. Since there was very little loss of liposomes from the second injection, the increase in signal from a single to a double injection protocol was expected. A third injection increased the signal by a fador of 1.1compared to double injection. A third injection required an additional 6 min. In 18 min, 78% of the liposomes from the first injection remained on the column. The loss of more liposomes from the column, and the fact that fewer sites remained available for binding by additional liposomes, explained the less impressive signal gain for the third injection. Since each additional injection required 6 min, the loss in time and throughput for this final 10% increase in signal was not justified. The apparent linear ranges, sensitivities (as related to the slope of the apparent linear range), and minimal detectable concentrations (MDC’s) of each of the different injection protocols are compared in Table 111. The FIIA for anti-theophylline demonstrated a concentration-dependent response between 4 X lo-’ and 6 X lo4 M.
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
Table 111. Range and Sensitivities of.Anti-Theophylline and Theophylline Assays (Characteristics of Calibration Curves) slope of linear range, fluorescence intensity
detectable concn
M-1
(MDC), M
10-8
3.1 X IO6 1.4 X IO7
10-8
1.5
6.0 X IO4 1.5 x 10-9 4.2 x 10-9
linear range, M anti-theophyllinedetermination single injection double injection triple injection theophylline determination single injection double iniection
3.5 x 10-7 to 4.7 x 3.5 x 10-7 to 5.6 x 3.5 x 10-7 to 7.4 x
3.5 x 10-5 to 3.3 x 3.5 x 10-5 to 3.3 x
0
"
I
2
M
I 12
10
8
163
113
123
6 103
4
83
63
moles
Log Amount Analyte
Figure 8. Calibration cuwes for theophylline and caffeine. The curves for theophylline represent double (1) and single (2) injection of sample and liposomes onto the column prior to measurement and determination of theophylline concentration. The caffeine curve (3) was produced by using the single injection method. The inset shows the structures of the two competing ligands.
The midpoint of this range was 4.9 X lo*, which was very close to that predicted for a competitive assay using this antibody. Typical relative standard deviations for anti-theophylline FIIA using OG as the regeneration reagent were
