Anal. Chem. 1993, 65,1152-1157
1152
Noncompetitive Flow Injection Immunoassay for a Hapten, a=( Dif Iuoromethy I)ornithine P. Chandrani Gunaratnat and George S. Wilson'+* Department of Pharmaceutical Chemistry and Department of Chemistry, University of Kansas, Lawrence, Kansas 66045
A noncompetitive flow injection immunoassay method Bas been developed to assay small haptens. In this assay the sample containing the hapten is incubated with excess enzyme-labeledmonovalent antibody for a brief period. The excess antibody is then separated from the bound antibody by eluting through an antigen-immobilized immunoaffinity column. The enzyme label of theeluting antibody-hapten complex is fluorometrically detected. The applicability of the method is demonstrated by assaying a-(difluoromethy1)ornithine (DFMO), an anticancer drug in human plasma samples. The assay is sensitive enough to detect 200 amol of DFMO. Interferences from other similar endogenous amines have been eliminated by selective immunoaffinity purification of the antibodies. INTRODUCTION Over the past few years enzyme immunoassays have become increasingly favored as the assay choice for clinical and pharmaceutical analysis as well as for environmental analysis.l-4 Among the various immunoassay formats, the most common is the enzyme-linked immunosorbent assay (ELISA),which involves a separation step to remove the free antibody from the bound antibody. Several assay configurations can be used in ELISA, depending on the analyte and the availability of the antibodies and the corresponding enzyme label^.^ In the popular 'sandwich" ELISA, one antibody is usually immobilized on a solid surface and the analyte is allowed to bind to the antibody. After a washing step a second labeled antibody is added, and this ternary complex is then detected. Since the sandwich assay is noncompetitive and does not depend on the antibody affinity, very low detection limits are attainable by optimizing the SIN ratio of the label. The sensitivity of the noncompetitive assay depends on the sensitivity of the enzyme label, the extent of nonspecific interactions, and the method of detection. However, the sandwich assay requires multiple antigenic sites on the analyte and, therefore, excludessmall analytes (haptens)such as small peptides and most drugs. The competitive ELISA is the method available for small haptens and it requires that labeled and unlabeled analyte * T o whom correspondence should be addressed. Department of Pharmaceutical Chemistry. Department of Chemistry. (1) Dhar, T . K.; Ali, e. J . Irnrnunoi. Methods, 1992, 247, 167-172. ( 2 ) Bouve, J.; De Boever, J.; Leyseele, D.; Vandekerckhove, D. Anal. Chirn. Acta 1991, 255, 417-422. (3) Kachab, E. H.; Wu, W.-Y.; Chapman. C. B. J . Irnrnunoi. M e t h o d s 1992, 147, 33-41. ( 4 ) Harrison, R. 0.;Goodrow, M. H.; Hammock, B. D. J . Agric. Food Chem. 1991, 39, 122-128. ( 5 ) Gosling, J . P. Clin. Chem. 1990, 36, 1406-1427.
compete for the immobilized antibody. The competitive assay has a narrow linear range and is less sensitive than the sandwich assay6 because no reactant can be in large excess. The detection limit now depends upon antibody affinity. Another problem in the competitive ELISA is the incubation of the enzyme-labeled antigen with the test sample, which may contain agents such as proteolytic enzymes that could affect the activity of the enzyme label.; Currently ELISAs are performed in microtiter plates. ELISAs are sensitive and can analyze multiple samples simultaneously. However, their precision is poor and the technique is semiquantitative because it is difficult to control the amount of immobilized antibody and other assay conditions. In contrast, flow injection analysis (FIA)offers precise control of reaction times and reagent addition and improved precision required for many analytical applications. Also the FIA format can easily be automated for high sample throughput. The noncompetitive flow injection immunoassay we describe here has combined the ELISA with FIA to overcome the limitations discussed previously. In this assay configuration, the analyte-containing sample is mixed with excess labeled antibody. The unreacted antibody is removed by exposing the sample to an antigen-immobilized column in a flow system. The enzyme label of the eluting complexed antibody is directly detected. In a similar method reported by Freytag et al. for digoxin,8 instead of the actual antigen, an analog of the antigen, ouabain, has been immobilized to prepare the support used to remove excess antibody. We have found that it is not necessary to use an analyte analog which has lower affinity for the labeled antibody. The present assay provides higher sensitivity and a wider linear range than the competitive assay. The assay is much faster than a conventional ELISA and can be fully automated. Most importantly, the method is applicable to any small hapten if antibodies against the hapten can be produced. The noncompetitive assay discussed above was developed for the antineoplastic agent w(difluoromethy1)ornithine (DFMO). DFMO is currently being investigated as a potential anticancer drug because of its ability to irreversibly inhibit the enzyme ornithine decarboxylase (ODC),Ywhich is the initial rate-controlling enzyme in the polyamine synthetic pathway.l0 ODC decarboxylates its substrate, ornithine, to produce putrescine, which is the precursor for polyamines spermidine and spermine essential for cell growth.'' DFMO is present in plasma at nanomole per milliliter levels. The only analytical methods available so far for the detection of DFMO involve separation of DFMO by reversed-phase
+
0003-2700/93/0365-1152$04.00/0
( 6 ) Jackson, T . M.; Ekins, R. P. J . [rnrnunol. M e t h o d s 1986,87,13-20. ( 7 ) Maggio, E. T.,Ed. Entyrnelrnrnunoassay; CRC Press: Boca Raton, 1980; Chapter 6 . ( 6 )Freytag, J. W.; Dickinson, J. C.; Tseng, S. Y.C h n . Chem. 1984,30,
417-420. (9) Pegg, A. E. Cancer Res. 1988, 48, 759-774. (10) Metcalf,B.W.;Bey,P.;Danzin,C.;Jung,M. J.;Casara,P.;Vevert, J. P. J . Am. Chem. So?. 1978. 100, 2551-2553. (11) Tabor, C. W.; Tabor. H. Annu. Rec. Biochem. 1984.53,744-790. 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1093 DumD A I\
.
mixina
1153
I I uorescence
+p I
1
back pressure regulator
11
H2N-CH2-CH2-CH2-C-COOH
I
"2
a 0 1 M phosphate buffer
HZN-CH2-CH2-CH2-C-COOH
I
waste
"2
b
Flguro 2. Structures of ornithine (a) and a-(difluoromethyl)ornithine (b).
HPPA
H202
Flguro 1. Schematic diagram of the flow injection system.
HPLC or employment of a commercial amino acid analyzer followed by postcolumn derivatization with o-phthalaldehyde (OPA).'zJ3 These methods are tedious and time-consuming and require lengthy sample preparation. Furthermore, they are less sensitive and the detection limits are high. Procedures employing extraction and subsequent concentration of DFMO are not applicable since DFMO is extremely water soluble. Hence development of simple, sensitive assay methods for DFMO in biological fluids is highly desirable to carry out metabolic and pharmacological studies.
EXPERIMENTAL SECTION Materials. Bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH),3-@-hydroxyphenyl)propionic acid (HPPA), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochloride (EDC),and 2-mercaptoethylaminehydrochloride(2-MEA)were purchased from Sigma Chemical Co. (St. Louis, MO). Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), N-hydroxysulfosuccinimide (sulfo-NHS), and CDI-activatedReacti-gel(HW-65F)were all obtained from Pierce Chemical Co. (Rockford, IL). ELISA-grade horseradish peroxidase (HRP)was obtained from Biozyme Laboratories (San Diego, CA). The gels, Sephadex G-25 and Sephacryl S-200 HR, for the gel filtration and the ion-exchange gel DEAE-Sephacel were purchased from Pharmacia LKB (Piscataway, NJ). DFMO and the plasma DFMO samples obtained from cancer patients participating in a Phase I clinical trial were a generous donation from Dr. Milan Slavik of the VA Medical Center, Wichita, KS. All other reagents were of analytical grade. Solutions were prepared in water obtained from a Barnstead Nanopure I1system. The buffers and the reagents used in the flow injection analysis (FIA) system were additionally filtered through a 0.22-pm filter. Apparatus. A Shimadzu LC-6B liquid chromatographic system consisting of a SCL-6B controller, SIL-6B autoinjector, two LC-6A pumps, FCV-3AL valve, CR-4A integrator, and RF535 fluorescenceHPLC monitor (all from Shimadzu Corp., Kyoto, Japan) was used. The complete FIA system is shown in Figure 1. The column (0.31 x 4 cm), back-pressure regulator (100 psi), and static mixing tee were purchased from Upchurch Scientific, Inc. (Oak Harbor, WA). The gel filtration and affinity purifications were carried out with homemade columns using a Rainin Rabbit I1 peristaltic pump. The absorbance was monitored at 280 nm with a Pharmacia HR-10 single path monitor. A Shimadzu UV-160spectrophotometer (ShimadzuCorp.) was used for all the absorbance measurements. Methods. Preparation of DFMO-Protein Conjugates. Since DFMO is a small molecule (Figure 2), it was necessary to conjugate DFMO to a carrier protein such as KLH for immunization.DFMO was conjugated to KLH through the COOH group by the enhanced carbodiimide method14 by adding 100 pL of EDC (0.1 M in the final volume) in 0.1 M phosphate buffer (PB; pH 7.4) to a mixture containing DFMO (3 mM), sulfo-NHS ( 5 mM), and KLH at a 3 mg/mL concentration in PB (pH 7.4) in a total volume of 12.4 mL. The mixture was allowed to react overnight with (12)Grove, J.;Fozard, J. R.;Mamont, P. S. J. Chromatogr. 1981,223, 409-416. (13)Osei, A. A.; Kilkenny, M. F.; Riley, C. M.; Stobaugh, J. F.; Slavik, M., unpublished results, 1992. (14)Staros, J. V.; Wright, R. W.; Swingle, D. M. Anal. Biochem. 1986, 156,22C-222.
stirring at room temperature. The reaction mixture was then extensively dialyzed against 0.01 M phosphate-buffered saline (PBS) to remove excess reagents. The conjugate (KDE) was stored frozen in aliquots until use. For the screening of antisera, DFMO was conjugated to BSA through the a-NH2 group using glutaraldehyde and a dialysis method.I5 In this conjugation, 2 mL of 10 mg/mL BSA in PBS was dialyzed against 500 mL of 0.2% glutaraldehyde in PBS overnight at 4 "C. The activated BSA was then dialyzed against PBS to remove excess glutaraldehyde. After the dialysis, 100 pL of DFMO (14 mg/mL) was added to the activated BSA and allowed to react overnight at 4 "C. The remainingactivated sites of BSA were blocked by adding lOOpL of 0.1 M lysine and allowing reaction at room temperature for 2 h. The BSA-DFMO conjugate (BDG) was then dialyzed thoroughly against PBS and stored frozen in aliquots. A BSAornithine conjugate (BOG)was also prepared by the same dialysis method. Preparation of Polyclonal Antibodies. Polyclonal antibodies were made in rabbits according to a standard protocol.16 Two female New Zealand white rabbits were immunized intradermally at five sites, each site with 100 pL of KDE in Freund's complete adjuvant (1:l).At 2-week intervals, three booster immunizations with 200 pL of KDE in Freund's incomplete adjuvant or saline were given intramuscularly. Two weeks after the last immunization, the rabbits were bled from their ear artery and the antibody titer was determined. Screening of Antibodies. The antisera were screened by an ELISA. A microtiter plate was coated with 10 pg/mL BDG and incubated 90 min at 37 "C. After being washed with PBS containing 0.2% Tween 20, 100 pL of 20-fold diluted sera was added to the wells and incubated for 1 h at 37°C. Goat antirabbit-HRP was used as the reporter antibody. Preparation of Affinity Supports. BDG and BOG conjugates were coupled to Reacti-Gel HW-65F for use in the affinity purification of the antibodies. Similarly prepared BDGReactiGel support was used as the immunosorbent in the FIA assay. A solution of the conjugate (5 mg/mL) in 0.1 M carbonate buffer (pH 9.35) was mixed with 2 mL of prewashed gel for 30 h at 4 "C by mechanical inversion. The coupled gel was first washed with one volume of 2 M tris buffer (pH 8),then with one volume of 0.1 M PB (pH 2.2), and finally with 0.1 M PB (pH 7.4). The washed gel was then stored in the same buffer until further use. Purification ofdntibodies. The immunoglobulinfraction from the antisera was separated by ammonium sulfate precipitation, and the IgG fraction was obtained by anion-exchange chromatography using DEAE-Sephacel with a 0-300 mM NaCl linear gradient in 10 mM tris buffer (pH 8.0).17 The IgG fraction was then collected, concentrated, and dialyzed against 0.1 M PB (pH 7.4). This IgG fraction was next loaded on an affinity column packed with BDG-Reacti-Gel support at a flow rate of 0.2 mL/min. The loading buffer was 0.1 M PB (pH 7.4). The bound fraction which contained specific antibodies to DFMO was then eluted with 0.1 M PB (pH 2.2) and collected in a tube containing 0.2 M PB (pH 7.4) to avoid the denaturation of the antibody by pH 2.2 buffer. After the elution, if necessary, the pH was adjusted to 7.4 by adding 0.1 N NaOH dropwise. The eluate was then concentrated and dialyzed against 0.1 M PB (pH 7.4) or PBS for storage. The antibody solution thus purified was then applied to a second (15)Zegers, N.; Gerritse, K.; Deen, C.; Boersma, W.; Claassen, E. J. Immunol. Methods 1990,130,195-200. (16)Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,Struhl, K., Eds. Current ProtocolsinMolecularBiology; John Wiley & Sons: New York, 1990;Vol. 2, Chapter 11. (17)Goding, J. W. Monoclonal Antibodies: Principles and Practice; Academic Press: New York, 1983;pp 100-115.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993
affinity column containing BOG-coupled support. Only antibodies that are specific to the moiety containing the difluoromethyl group of DFMO will then be eluted, and antibodies crossreacting withornithine will be retained on the column. Therefore, the free fraction was collected, concentrated, and dialyzed against 0.1 M PB (pH 7.4) or PBS. The concentration of the DFMOspecific antibodies was determined by the absorbance at 280 nm. The antibodies were always stored in PBS. Characterization of Antibodies. The affinity-purified antibody was then screened as described before against BSA and BOG to determine the cross-reactivity to BSA and ornithine. The apparent affinity constant was determined from the same ELISA by serially diluting the antibody.la The affinity of the antibody for DFMO in solution was studied by an inhibition ELISA. This ELISA was carried out by incubating different concentrations of the affinity-purified antibody (6.25 X 10-;-3.75 x 10 l 1 M) with varying concentrations of DFMO (from 0.02 WM to 2.0 mM) in solution. After a 2-h incubation period at 37 "C, the samples were applied to a BDG-coated microtiter plate and the normal ELISA procedure was performed. Preparation of Antibody-Enzyme Conjugate. The antibodyenzyme conjugate was prepared by reducing the interchain disulfide bonds to produce a half-molecule followed by coupling to the SMCC-activated HRP according to the Pierce protocol for SMCCIYwith some modifications. The antibody (4 mg) in 0.45 mL of 0.1 M PB (pH 6.0) was reduced by adding 50 pL of 0.1 M 2-MEA in 0.1 M PB (pH 6.0) containing 5 mM EDTA. The mixture was incubated with stirring at 37 OC for 90 min. The excess 2-MEA was removed by applying the sample to a column containing Sephadex G-25 equilibrated with the same buffer. The eluate was collected and concentrated to a final volume of -0.5 mL using Millipore Ultrafree-CL 20 filters (Millipore, Bedford, MA). The protein concentration of the reduced IgG was measured from the absorbance at 280 nm. The thiol content of the reduced IgG was determined as described in the Pierce protocol. To HRP [5.0 mg in 1.0 mL of 10 mM tris buffer (pH 7.0)l was added 1 mg of solid sulfo-SMCC and the resultant mixture incubated with stirring at 30 "C for 1h. The excess sulfo-SMCC was removed by applying the sample to a Sephadex '2-25 column equilibrated with 10 mM tris buffer (pH 7.0). The first peak eluted was collected and concentrated to a final volume of -0.5 mL using same Millipore filters. The reduced antibody was then added to the SMCC-activated enzyme and allowed to react overnight (20 h) at 4 OC with stirring. To block the unreacted maleimide activated sites, 10 pL of 0.1 M 2-MEA was added and the resultant mixture incubated at room temperature for 20 min. After this step, the sample was passed through a Sephacryl S-200 HR column equilibrated with 10 mM tris buffer (pH 7.0) containing 0.1 M NaCl at a flow rate of 0.28mLt'minto remove excess 2-MEA and purify the conjugate. The absorbance was monitored at 280 nm. The fraction containing conjugate was collected, concentrated, dialyzed against PBS, aliquoted, and frozen until ready to use. The activity of the conjugate was confirmed by an ELISA. S a m p l e Preparation. All the samples were prepared in 0.1 M PB (pH 7.4). Standard samples for calibrations were prepared by adding a known excess amount of antibody-enzyme conjugate to various DFMO concentrations ranging from 50 fmol/mL to 5.0 pmolimL. The samples were incubated for 15 min at room temperature before analysis. Series of standard samples were made with different amounts of excess conjugate in order to optimize the molar excess of antibody-enzyme conjugate over analyte. Different incubation times were studied to determine the time required for maximum complexation of antibodyconjugate to analyte. Control samples were prepared by adding 0.5 pmolimL excess of antibody-HRP conjugate to known DFMO concentrations. These samples contained the same amount of diluted (200-fold) normal pooled plasma obtained from the Red Cross (Kansas City, KS) as the DFMO-containing plasma samples. The DFMO concentrations of these plasma samples (18) Beatty, J. D.; Beatty, B. G.; Vlahos. R.G. J . Irnrnunol. Methods. 1987, 100, 173-179. (19) Pierce Chemical Co. Protocoifor Sulfo-SMCC;Protocol No. 22322
and 22422, 1989.
bound
free
mmunosorbent
Figure 3.
Fluorescerce
Schematic representation of the assay format.
were predetermined by a chromatographic method using a commercial amino acid analyzer at the VA Medical Center, Wichita, KS. Plasma samples containing DFMO were diluted in 0.1 M PB (pH 7.4) 200-fold initially before assaying. Depending on the signal, some plasma samples were diluted either 400- or 1000fold. DFMO A s s a y Protocol. The samples were assayed using the FIA system shown in Figure 1. Before injecting the samples, the column was saturated with BSA to block the nonspecific sites by injecting (20 X 10 pL) a 5.0 mg/mL solution of BSA. The sample (5 FL) was injected into a flowing stream of 0.1 M PB (pH 7.4) through the immunosorbent column. The substrate for the HRP was 10 mM HPPA and 5 mM H202 in 0.1 M PB (pH 8.5 buffer). The concentration of HPPA and H202, pH of the substrate buffer, and flow rates of both streams were optimized to minimize the noise and to yield maximum sensitivity. The fluorescence generated by the HRP reaction was measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Each sample was analyzed in triplicate. The assay time not including the sample preparation is 6 min. Peak area obtained from the CR-4A integrator was used as a measure of the fluorescence signal intensity.
RESULTS AND DISCUSSION In this assay, excess labeled antibody is added to the analyte in solution. After a brief incubation period, the free antibody is separated from the bound antibody by passing through an antigen-immobilized column. Only the antibody bound to the antigen will be eluted and directly determined. The assay schematics are shown in Figure 3. Since the analyte is small, the antibody must be monovalent in order to obt,ain 1:l stoichiometry of antibody and antigen. Use of divalent antibodies can produce erroneous results since the divalent antibody is capable of binding to both the immobilized antigen and the antigen in the sample because of the small size of the antigen, although Freytag et al. have not observed any difference in assay performance with Fab' and F ( a b ) 2 conjugates.20 It is very important to preserve the enzymatic and immunological activities of the antibody-enzyme conjugate during the conjugate preparation. In the conjugation method used here, the interchain disulfide chain in the antibody was first reduced to obtain the half-molecule and the enzyme label was conjugated to the antibody through the thiol group a t the hinge region. One could also use Fab' fragments, but the preparation of the half-molecule is much simpler. Sincerabbit antibodies contain only one disulfide bridge at the hinge region,21 this assures that the antibodyienzyme ratio is 1. Therefore, measurement of the enzyme label is a direct measure of the analyte. The antibody-enzyme conjugate was purified to remove the excess enzyme, which could reduce the sensitivity of the assay by nonspecific substrate conversion. (20) Freytag, J
W ; Lau. H P , Wadsleq, J J Cltn Chem
1984, 30,
1494-1498
(21) Palmer, J. L.; Nisonoff, A.; Van Holde, K. E. Biochem. J . 1963, 150. 314-321.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993
It has been shown that purification of the enzyme conjugate yields significant enhancement in sensitivity.22 The antisera obtained by immunizing the rabbits with DFMO coupled to KLH through the COOH group were screened against DFMO conjugated to BSA through the amine group. This screening protocol eliminates the antibodies specific to KLH and to the link. The antisera had a very high titer (-40 OOO) against the BSA-DFMO conjugate. The antibodies purified by ion exchange did not show any crossreactivity toward BSA. Since BSA contains lysine residues and lysine and DFMO share similar c-NH2 chains, namely, the (CH&NHz part of the DFMO molecule, it can be concluded that the antibodies are not specific to that epitope. The affinity-purified antibody showed very little crossreactivity to ornithine (2-4%), indicating that the antibody involves mainly the epitope containing the difluoromethyl group of the molecule. The apparent affinity constant for DFMO was found to be 2.0 X lo9M-l. The inhibition ELISA showed that DFMO in solution binds to the antibody with equal affinity. High affinity of the antibody is a very important factor since the complete removal of the free antibody from the sample depends on the antibody affinity. When the affinity for the immobilized antigen (BDG) is high, the immunosorbent will be very efficient in capturing the free antibody as it passea through the column. On the other hand, the antibody must have higher or equal affinity toward the soluble antigen so that the complexed antibody will not be associated in the presence of the immobilized antigen. The affinity-purified antibody used in our assay fulfills these requirements. The antigen-immobilized immunosorbent must contain sufficient antigenic sites to capture all the free antibody from the flowing sample. In addition, the residence time of the sample in the column must be sufficient to permit complete reaction (free antibody capture). During the short time the sample spends in the column the following reactions occur:
where Agi is the immobilized antigen, Age is the soluble antigen, and Ab* is the labeled antibody. If the affinity constant is high, the dissociation of Ag,-Ab* will be negligible during the brief residence time of the sample in the column. Antibodies with affinity constants greater than lo8 Llmol satisfy this condition.23 The forward reaction between antibody and antigen is slower at a solid phase than in solution.24 Hence the displacement of soluble antigen by the immobilized antigen as shown below is a kinetically unfavorable process. Only the free Ab* will be bound by the immobilized antigen, Agi + Ag,-Ab*
Agi-Ab*
+ Ag,
Since the dissociation of hapten-antibody complex at the solid interface is much slower than in s0lution,2~it is highly unlikely that the free Ab* could be dissociated from the immunosorbent once it is bound by the immobilized antigen and thus contribute to the response. This assay takes advantage of differences in rates of immunological reactions in a manner not possible when microtiter plates are used. Contrary to the procedure suggested by Freytag et al.,20 the immunosorbent used in this assay was prepared by (22) Ngo, T. T.,hnhoff, H. M., Eds. Enzyme-Mediated Immunomsay; Plenum Press: New York, 1985; pp 203-222. (23) Ishikawa, E.; Kato, K. Scand. J . Immunol. 1978,8,43-55. (24) Nygren, H.; Kaartinen, K.; Stenberg, M. J. Immunol. Methods 1986,92, 219-225. (25) Stenberg, M.; Nygren, H. J. Immunol. Methods 1988,113,3-15.
1115
immobilizing the antigen (DFMO conjugated to BSA), not an analog of the antigen. Although Freytag's method was successful for the detection of digoxin, it is not a general case. This method requires the immobilization of an antigen analog with lower affinity. In our assay we have shown that the lower affinity constant is not a requirement for the immunosorbent, and therefore, development of similar assays is not limited to analytes which have structurally similar and more weakly binding analogs. Usually, the immobilized antigen has a lower affinity than the soluble antigen. Therefore, it is possible for the immobilized antigen-antibody complex to dissociate during the short residence time and contribute to the response. The time for the antigen-antibody complex to dissociate depends on the dissociation rate constant according to the equation t1/2 = In 2/Kd.26 Hence, when an andog of the antigen with a lower affinity constant (higher dissociation rate constant) is immobilized,the dissociation is faster and more free labeled antibody is available to produce an increased background response. An affinity column prepared with the actual antigen can more efficiently capture and retain the free antibody than a column prepared with an analog of lower affinity. Since the actual antigen is used to prepare the immunosorbent, the cross-reactivity to other molecules similar to the analyte in structure can be eliminated by choosing highly selective monoclonal or polyclonal antibodies with high affinity. The antigen-immobilized immunosorbent in this assay performed efficiently as expected. The capacity of the immunosorbent was determined by injecting small aliquots of the unlabeled antibody and determining the amount of bound and free fractions by the absorbance measurements a t 280 nm. The capacity of the gel was found to be 57.5 ng (0.74 pmol) of the labeled antibody per milliliter of gel. Performance of the gel was assessed by assaying several controls before each sample run. After several samples were assayed, the immunosorbent was regenerated by eluting the antibody from the column with 0.1 M P B (pH 2.2). The gel was repeatedly regenerated as necessary without any loss of performance. The fluorometric assay using HPPA as the substrate to detect HRP was shown to be rapid and highly sensiti~e.~'Since HPPA shows some background fluorescence, HPPA concentration needs to be optimized even though high concentrations yield high signal intensity. The HPPA concentration was kept at 10 mM. It was found that HPPA has a maximum fluorescence at pH -7.8 in 0.1 M PB. The resulting stream from the mixing of two buffer streams (0.1 M PB, pH 7.4, and 0.1 M PB, pH 8.5, at equal flow rates) provided a pH of -7.75. As the hydrogen peroxide concentration increased above 5 mM, a decrease in the signal intensity was observed. This may be due to the inactivation of the enzyme by excess hydrogen peroxide.28 In order to obtain maximum assay sensitivity, variation of the slope of the assay in the range 5 X 10-"-2.5 X lC9M was studied as a function of the amount of the monovalent antibody-enzyme conjugate. Figure 4 shows that optimum sensitivity could be achieved at 0.5 pmol/mL excess of the conjugate. Further studies and sample analysis were carried out with this amount. The time required for the binding of DFMO by the monovalent antibody-HRP conjuate was studied to determine the incubation time. A sample containing 1.0 pmoUrL DFMO was incubated with the conjugate for different time intervals and analyzed. Figure 5 shows that most of the binding ~~~~~~~
~
(28) Loceecio-Brown, L.; Plant, A. L.; Horvath, V.; Durst, R. A. Anal. Chem. 1990,62, 2587-2593. (27) Zaitau, K.; Ohkura, Y. Anal. Biochem. 1980, 109, 109-113. (28) Haldane, J. B. S. Enzymes; The M.I.T. Press: Cambridge, MA, 1965; pp 59-60.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993
1156
Table I. Recovery of Control Samples at Different Flow
600.00
Rates
recovery ( % ) 550.00
(pmol/mL)
flow rate 0.3 mL/min
0.4 mL/min
0.05 0.17 0.42 0.84
104.0 106.5 113.8 119.6
116.0 110.0 105.8 123.3
DFMO concn
.-.Z>r500.00 Y
.-n U
C
0
flow rate
v, ,450.00
2 5 ferntomc
O
I
n
1
P 400.00
Flgure 4. Variation of the assay sensltivtty wRh the amount of monovalent antibody-enzyme conjugate in excess. Standard DFMO samples in the range 5 X 10-"-2.5 X M were incubated with varying amounts of antibody-HRP conjugate in excess (0.1-1 .Opmol/ mL) for 15 min and assayed in triplicate. The slope of the calibration curve is plotted against the amount of antibody-HRP in excess. 800.00
1
I
I
600.00 n
0 c
1 2
I
X
0
0 400.00
e
I
20
I
40 Time (rnin)
I
60
0
Flgure 6. A typical flow injectiondiagram for standard DFMO samples.
U
Y
-
0
u 200.00
I I I I . I . .
I I 1 1 1 I . . .
2o.bo
40
' . " " ' ' . I " . " ' . "
.bo
60.00
8O.bO
Time (min) Figure 5. Dependency of antibody-antigen binding on time. A sample containing 1.0 pmol/mL DFMO with 1.5 pmol/mL antibody-HRP conjugate was incubatedat roomtemperature for different tlme intervals (0-60 mln) and assayed in triplicate. Peak areas are proportional to the signal intensity.
occurs within 15 min at room temperature; thus an incubation time of 15 rnin was chosen. The column capacity is another important factor in the assay. The column must have sufficient capacity to remove the free antibody-HRP conjugate from the sample efficiently. The columns used in this assay have at least 100-fold more immobilized antigen than the amount of free labeled antibody in the sample. The residence time of the sample in the column has to be optimized to achieve maximum removal without excessive broadening of the peaks. Short residence time will result in partial removal of the free antibody and cause the signal to be higher. However, if the residence time is too long, the free antibody captured by the immobilized antigen could be dissociated during the analysis. The residence time depends on the flow rate and the dimensions of the column. Therefore, the flow rate through the column should be optimized so that the residence time would be long enough for the free antibody to be captured by the immobilized antigen. Higher flow rates and narrow columns will have shorter residence times.
Three columns of different dimensions were used. A narrow, long column (0.21 X 4.0 cm, 140-pL capacity) gave sharp peaks a t flow rates of 0.2 mL/min on both streams. Both streams had equal flow rates to minimize the baseline noise. The average error of the assay using this column was about 32.8%. However, the capacity of this column was not sufficient to assay more than 12-15 samples in one run. The column must be regenerated by eluting the free antibodyH R P conjugate with 0.1 M P B (pH 2.2) for -20 min. After each regeneration, the column was coated with BSA again as described previously. A wider, short column (0.38 X 3.0 cm) of -340-pL capacity was used at higher flow rates. At flow rates of 0.2 mLimin or below on both streams, the peak broadening was a problem when the peak areas were evaluated at low concentrations, and hence the detection limit was high. At higher flow rates, the recoveries of control samples were higher, indicating the partial removal of free antibody-HRP conjugate (Table I). A longer column (0.38 x 6.0 cm) having -680-pL capacity at flow rates of 0.3 mL/min on both streams was used finally. This column gave reasonably sharp peaks (Figure 6)at these flow rates and was capable of assaying -50 samples before regeneration. It has been shown that the first-order dissociation rate constant of antibody-hapten complexes is about 10-3-10-5 s-l when the hapten is i m m o b i l i ~ e d .Therefore, ~~ the dissociation is relatively slow compared to the separation step at this flow rate. The assay was linear in the range 5 X 10-1'-2.5 X le9M DFMO. The detection limit of the assay (defined as the concentration which gives a signal with SIN > 2 and noise is considered as the signal obtained when no analyte is present (29) Karush, F. In Immunoglobulins; Litman, G. W., Good, R.A., Eds.; Plenum: New York, 1978; p 85.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993
Table 11. Precision and Accuracy Data for the Assay
DFMO concn within run ( n = 6) mean
SD
cv ( % ) between run ( n = 5) mean
SD
cv ( % )
(pmol/mL)
0.210
0.840
1.70
0.211 0.005 2.6
0.845 0.004 0.470
1.648 0.040 2.4
0.216 0.014 6.3
0.851 0.019 2.2
1.666 0.072 4.3
31m . 0 0 E
>-
8 0 800.00
i C
0 C
600.00
2 E r
in the sample) is 2 X 10-11 M DFMO or 200 am01 in a 10-pL sample. This detection limit is 1 order of magnitude lower than what has been reported for a digoxin assay.20 The precision and accuracy data for the control samples are shown in Table 11. The assay showed no interference from other structurally similar endogenous amines, namely, ornithine and putrescine. Cross-reactivity, which is the main interference, has been eliminated by selective affinity purification of the antibodies. The affinity-purified antibodies showed minimal crossreactivity to ornithine (-2-495) and no reactivity to put r e ~ c i n e .Standards, ~~ samples, and controls were all prepared in 0.1 M PB (pH 7.4) to minimize any matrix effects. The plasma samples containing DFMO were analyzed in triplicate using this large column. As shown in Figure 7, the DFMO values obtained from this assay correlated well with the values predetermined by the chromatographic method using a commercial amino acid analyzer.
CONCLUSION The flow injection analysis in conjuction with enzyme immunoassay provides a highly sensitive and rapid analytical method for small molecules. The method we have described here could be applied to any small analyte against which a high-affinity antibody can be produced. The fluorometric detection method coupled with the enzyme amplification is known to offer greater signal enhancement.31 The immunoaasay is much superior to the chromatographic methods available for this analyte mainly because no sample preparation is needed except dilution steps. Also, the derivatizing agents used in the chromatographic analysis are not selective, and hence, complex separation techniques are required. The accuracy and the precision of the method is (30)Wilson, G.S.;Gunaratna, P. C., unpublished data. (31)Maggio, E. T., Ed. Enzyme Immunoassay;CRC Press: Boca Raton, 1980;Chapter 3.
1157
400.00
.
Y
C
8
0
s
0
0.00
Concentrotion from IEC (nonomolcs/mL) Flgurr 7. Correlation between plasma DFMO concentrations measured by the flow injection immunoassay and by the chromatographymethod using a commercialamino acid analyzer Y = 1.03X+ 11.go, r = 0.94 (n = 76).
comparable to the values reported in the literature for chromatographic methods.12J3 DFMO is essentially an amino acid derivative. Making monoclonal antibodies to such a molecule is difficult and time consuming. We have been able to prepare a polyclonal antibody and have been able to fractionate the antibody population by use of immunoaffinity columns. In this way cross-reactivity has been eliminated. Since it has been shown that the antibodies to DFMO show no cross-reactivity to similar amines, this FIA assay is free from the interferences from these amines. The immunosorbent can be used repeatedly without any loss of antibody binding capability for 1month. We have not studied the capacity of the column beyond this period. The antigen-immobilized gel can be stored at 4 OC for months with no loss in performance. In conclusion, we have developed a highly sensitive, simple analytical method to detect small molecules in biological fluids.
-
ACKNOWLEDGMENT Support for this work by National Institutes of Health Grant GM40038 and a NCI traineeship (CA 09242) is gratefully acknowledged. Received for review October 5, 1992. Accepted January 11, 1993.
