Anal. Chem. 1996,67, 606-612
Capillary Electrophoresis-Based Immunoassay for Cortisol in Serum Dieter Schmaldng, Wassim Nashabeh, Xian-Wei Yao, Rohin Mhatre, Fred E. Regnier, Noubar 6. Afeyan, and Martin Fuchs* PerSeptive Biosystems, Cambridge, Massachusetts 02139
A competitive immunoassayfor cortisol based on capillary electrophoresis (CE) and laser-induced fluorescence is described. The work involved the production of assay reagents and the development of separation conditions allowing for routine analysis of serum samples. Fluorescein-labeled cortisol was synthesized and purified. Fab fragments were produced from mouse monoclonal anticortisol antibody and puri6ed using a POROS cation exchange chromatography column. After incubation of these reagents with serum, free and bound labeled antigen were separated by CE with high reproducibility. No prior sample cleanup of the serum samples was necessary. Serum calibration curves were established and used for the quantitation of cortisol in serum. The results demonstrate feasibility for a cortisol assay based on CE operating directly on serum samples. Immunoassays permit the highly selective and sensitive detection of macromolecular substances (e.g., proteins and polysaccharides) and of smaller molecules (e.g., peptides, hormones, and drugs) in complex biological matrices.' Their selectivity is based on the high specificity of the antibody/antigen recognition, whereas the sensitivity results from the use of labeling techniques such as radioactivity, fluorescence,chemiluminescence,or enzyme amplification. Many immunoassay strategies rely on the separation of bound and free forms of antibody or antigen. Capillary electrophoresis (CE) has proven to be a powerful separation tool and is therefore being examined as a useful separation method for rapid and efficient immunoassays.2 Indeed, in a 1991 report, Nielsen3et al. demonstrated the ability of CE to separate antigen/ antibody complex from antibody and free antigen. More recently, initial results on CE-based immunoassays for the measurement of insulin4and human growth hormone5 have been reported. In related work, a latex particle agglutination immunoassay using a capillary flow system and laser-based particle counting has been applied to the measurement of glucose &phosphate dehydrogenase in single erythrocytes6 In principle, CE should have a number of advantages for performing immunoassays. First, CE requires only a few micro(1) Masseyeff, R F. In Methods of Immunological Analysis; Masseyeff, R F., Albert, W. H., Staines, N. A, Eds.; VCH: New York, 1993; Vol. 1,pp 115130. (2) Afeyan, N. B.; Regnier, F. E. US. Patent 5,376,249, 1992. (3) Nielsen, R G.; Rickard, E. C.; Santa, P. F.; Sharknas, D. A; Sittampalam, G. S. J. J. Chromatogr. 1991, 539, 177-185. (4) Schultz, N. M.; Kennedy, R T. Anal. Chem. 1993, 65, 3161-3165. (5) Shimura, IC; Karger, B. L. Anal. Chem. 1994, 669-15. (6) Rosenmeig, 2.; Yeung, E. S. Anal. Chem. 1994, 66, 1771-1776.
606 Analytical Chemistry, Vol. 67,No. 3, February 7, 7995
liters of sample and hence offers very low reagent consumption. Second, the overall assay methodology can be greatly simplified since many of the steps required in conventional immunoassays, including multiple wash steps followed by measurement steps, can be eliminated and replaced by a single CE separation. As such, the CE approach will be straightforwardto automate. Third, the time necessary for incubation might be reduced compared to that for solid-phase assay systems7,*since there is no longer a need for reactants to diffuse to a surface. Fourth, the high resolving power of CE can, in principle, allow the simultaneous determination of multiple analytes in a single capillary. Fifth, an array of capillaries operated in parallel could be used to obtain even higher sample throughput. Finally, CE, when combined with appropriate labeling techniques and laser-induced fluorescence &IF) detection, for which detection limits as low as M have been reported?-" can provide high sensitivity. Nevertheless, there are certain challenges unique to the performance of immunoassays in the CE format. To achieve the sensitivity needed in many assays and to prevent interferences with matrix components, fluorescent labeling is desirable. Hence, labeled antibody (antibody fragment) or antigen must be synthesized and purified. Moreover, to make the assay work reproducibly and quantitatively in biological matrices such as urine or serum, adsorption of sample components to the capillary wall must be prevented. For multiple analyte systems, where there will be many components to be separated and quantitated, it may be advantageous to work with monovalent antibody fragments rather than with the intact antibody, necessitating a digestion and purification step. In this paper, we present results from our work on developing a competitive immunoassay for the determination of cortisol in serum based on CE and LIF detection. Cortisol is the major glucocorticoid steroid secreted by the adrenal gland. It shows antiiammatory activity and influences blood pressure and metabolism of proteins and carbohydrates. The determination of its concentration in serum (typically in the range of 1-60 pg/ dL or 30-1700 nM) provides diagnostic information for adrenal malfunctions, e.g., Addison's disease (chronical adrenal insufficiency) and Cushing's syndrome (adrenal overproduction). (7) Chaiken, I.; Rose, S.; Karlsson, R Anal. Biochem. 1992,201, 197-210. (8)Wimalasena, R L.; Wilson, G. S.J. Chmmatogr. 1991, 572, 85-102. (9) Cheng, Y. F.; Dovichi, N. J. Science (Washington, D.C.)1988, 242, 562564. (10) Sweedler, J. V.; Shear, J. B.; Fishman, H. A; Zare, R N.; Scheller, R H. Anal. Chem. 1991, 64, 496-502. (11) Yeung, E. S.; Wang, P.; Li, W.; Giese, R W. J. Chromatogr. 1992, 608, 73-77.
0003-2700/95/0367-0606$9.00/0 0 1995 American Chemical Society
EXPERIMENTAL SECTION Instrumentation. A CE system constructed in-house was used for all separations using W detection, consisting of a Spellman CZE lOOOR power supply (Spellman, Plainview, NY) and a PerSeptive Biosystems UVIS205 absorbance detector (PerSep tive Biosystems, Cambridge, MA). Detection was done at 214 nm. BioCAD software from PerSeptive Biosystems was used for data acquisition. The LIF system consisted of a P/ACE instrument Model 5500 (Beckman, Fullerton, CA) fitted with an argon laser source. Excitation was at 488 nm and detection at 520 nm. Capillary isoelectric focusing (cIEF) separations were performed on fused silica columns coated with C18/methylcellulose prepared according to Yao and Regnier,12and the other CE separations were carried out with capillaries having a modified form of the siloxanediol/poly(acrylamide) coating of Schmalzing et al.13 Mass spectrometry was done on Laseflec (PerSeptive Biosystems, Vestec Mass Spectrometry Products, Cambridge, MA). The HPLC puriiication was carried out on a BioCAD instrument also from PerSeptive Biosystems. Materials. Polyimide-coated fused silica capillary columns of 50 pm i.d., 360 pm 0.d. were from Polymicro Technologies (Phoenix, AZ). Cortisol 3-(O-carboxymethyl)oxime, cortisol, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride, N-hydroxysuccinimide, 2-amin0-2-methyl-l,3propanediol (AMPD), PI markers @I range from 4.6 to 7.2), N-tris(hydroxymethy1)methyl-3-aminopropanesulfonic acid (TAPS),and Pharmalytes 3-10 were supplied by Sigma (St. Louis, MO). 5((5Aminopentyl) thioureidyl) fluorescein (fluorescein cadaverin) was from Molecular Probes (Eugene, OR). Mouse monoclonal anticortisol antibody (subclass IgG2b) was purchased from Fitzgerald (Concord, MA), and the Fab preparation kit was from Pierce (Rockford, IL), The POROS S/M cation exchange column (4.6 mm d x 100 mm I, 20 pm) was from PerSeptive Biosystems. Cortisol serum standards were obtained from Incstar (Stillwater, MN) . &Anilino1-naphthalenesulfonic acid ammonium salt (ANS) was from Aldrich (Milwaukee, wr). Labeling of Cortisol and Fab Production. The labeling of cortisol with fluorescein was performed according to Pourfarzaneh14et al. The purity and integrity of the labeled compound was assessed by TLC, CE, and mass spectrometry. Fab fragments were produced as described in the ImmunoPure Fab preparation kit from Pierce and purified by HPLC on a POROS cation exchange column. The Fab fragments were characterized by CE, cIEF, and mass spectrometry. Assay Protocols. (i) In Water. Into small vials were pipetted 10 pL aliquots of cortisol standard solutions prepared in water in the range from to lo-* M and 10 p L of fluorescein-labeled M). To each was added 10 p L of Fab (2.3 x antigen M). After 15 min of incubation at room temperature, the samples were analyzed by CE. (ii) In Serum. Into small vials were pipetted 10 pL aliquots of undiluted serum standards with 0, 1,3,10, and 60 pg/dL cortisol and 10 p L of a solution containing M labeled antigen and 6 mM ANS.l5 To each was added 10 p L of Fab (2.3 x M). (12)Yao, X.-W.; Regnier, F. E.J. Chromatogr. 1993,632,185-193. (13) Schmalzing, D.;Foret, F.; F'iggee, C.; Carrilho, E.; Karger, B. L. J. Chromatogr. 1993,652,149-159. (14)Pourfarzaneh, M.;White, G. W.; Landon, J.; Smith, D. S. Clin. Chem. 1980, 26,730-733. (15)Brock, P.;Eldred, E. W.; Woiszwillo, J. E.; Doran, M.; Schoemaker, H. J. Clin. Chem. 1978,24,1595-1598.
M e r 1 h of incubation, the samples were measured by CE. (iii) CE Separation. The coated 50 pm columns had a total length of 27 cm and an effective length (to detector) of 20 cm. The samples were pressure injected for 8 s at the negative electrode. The applied field strength was 30 kV, and 20 mM TAPS/AMPD (PH 8.8) was used as the background electrolyte. The current was 14 pA RESULTS AND DISCUSSION
Competitive versus Direct CE Immunoassay. In the C E based competitiveimmunoassay, fluorescently labeled tracer and antibody are added in specific amounts to the sample to be analyzed. The labeled tracer competes with the antigen present for the limited number of antibody binding sites. After equilibrium is established in the free solution, a small volume of the incubate is injected into the capillary, whereupon free and bound labeled tracer are separated by CE and quantitated by measurement of fluorescence intensity. The amount of labeled tracer displaced by the antigen is a measure of the antigen concentration in serum: a high amount of antigen will give a large signal for the free labeled antigen and a small signal for the complex. In principle, both signals can be used for quantitation. The competitive format was chosen since exploratory work showed that a direct immunoassayfor cortisol based on CE would be less feasible. In direct assays, the complex formed by the antigen with the labeled antibody must be separated from excess labeled antibody. Separation in free zone capillary electrophoresis is due to differences in electrophoretic mobility. Thus the separation of free and bound forms of the antibody in a direct assay requires a change in charge to mass ratio for the antibody resulting from the complexation with the antigen. However, cortisol is a neutral molecule of low molecular mass (362 Da) . In our experiments, its binding to the much heavier antibody (150 kD)or antibody fragment (50 kD)did not lead to any observable mobility difference between free and complexed antibody or antibody fragment. Labeling of Antigen. To stimulate immunoresponse of an animal against small antigens (haptens), the haptens are attached to a large carrier protein before immunization.16 The antibody used in this work was produced by innoculation of mice with cortisol which had been covalently linked at the 3 position of the steroid to bovine serum albumin. The linker used for the conjugation of the hapten to the carrier protein often becomes part of the epitope, and the antibody affinity and specificity will depend not only on the hapten but also on the linker position and chemistry.17J8 We therefore decided to use a labeling strategy in which the fluorescent label is attached to the 3 position of cortisol through a spacer, seeking to avoid alteration of other portions of the antigen molecule and resulting in a labeled compound which is similar to the immunoreactive component of the hapten-BSA conjugate. Figure 1shows the reaction scheme for the labeling of cortisol with fluorescein. The carbonyl group at position 3 of cortisol was first reacted with carboxymethoxylamine to form cortisol 3-(Ocarboxymethyl)oxime. In a second step, the carboxy group of the oxime was activated by N(16)Shyer, L. Biochemktw, W. H. Freeman and Co.: New York, 1988;pp 890891. (17)Hosoda, H.; Kobayashi, N.; Ishii, N.; Nambara, T. Chem. Phann. Bull. 1986, 34,2105-2111. (18)Ogihara, T.;Miyai, IC; Nishi, IC; Ishibashi, K. J. Clin. Endocn'nol. Metab. 1977,44, 91-95.
Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
607
CHI-OH
0.004
.a ,004
0,003
0.003
0.002
0.002
0.001
> .001
0.000
1.000
I
0 0 OH
N ‘O-CH~-C-OH
OH
-+ n 0 1) carbodiimide
2) rucdnimide DMF. RT E
s
H
Y
I
CH2-OH
I
2
N
‘0
-CH2 -C
a
- NH-
(CH&
-NH-C
-NH
’0H
Figure I. Synthesis scheme of labeled antigen.
hydroxysuccinimide and finally reacted with the free amine of 0.001 -o.oor fluorescein cadaverine to form a stable amide linkage. 2.5 3.0 2.0 Fab Production and Purification. To avoid the heterogenemigration time [min] ity inherent in a polyclonal antibody preparation, a monoclonal Figure 2. CE of mouse monoclonal anticortisol antibody (0.3 mg/ instead of a polyclonal antibody was selected for the assay. Even mL). Coated fused silica column, 20/30 cm, 50 pm; pH 8.8 (20 mM so, intact monoclonal antibodies displayed strong microheteroTAPWAMPD), 30 kV, 18 pA, 21 4 nm; 10 s hydrodynamic injection. geneity when analyzed by CE, each antibody yielding a very broad peak consisting of partially resolved antibody isoforms. Figure 2 to a reduction in the antifolate antibody fine structure but had no depicts a CE separation of mouse monoclonal anticortisol IgG2b effect on the overall peak width (Figure 3). antibody performed at 30 kV and pH 8.8 on a coated fused silica In principle, the microheterogeneity should not pose a problem column in which there is essentiallyno electroosmotic flow. Five for competitive assays where the quantitation can be based on antibody isoforms were partially resolved in less than 3 min, but free labeled antigen, as long as free and bound forms are well the total peak width was almost 1min. Similar results have been separated by CE. However, one aim of our work was to illustrate reported in the literature for monoclonal antibodies studied by the applicability of CE to multiple analyte systems. In this case, c I E F , c~h~r o~ m~ a~t ~ g r a p h yand , ~ ~slab ~ ~gel ~ ~electroph~resis.~~-~~ ~~ a high peak capacity is desirable, and the wide peaks of intact Previous studies have revealed monoclonal antibody heteroantibodies are less acceptable. The broad peaks might also result geneity due to variations in g l y c o s y l a t i ~ n .For ~ ~ the ~ ~ ~antibodies in poor quantitation and loss in sensitivity. Furthermore, we we studied, treatment with neuraminidase or other glycosidases observed adsorption to the coated column surfaces for several to remove the negatively charged sialic acids and other sugarsz5 antibodies. Also, whole antibodies can form bivalent as well as did not result in a s i m c a n t decrease in heterogeneity, indicating monovalent complexes, thereby spreading the signal over several that the observed microheterogeneity is only partially caused by peaks. We therefore decided to use Fab fragments produced by differences in glycosylation. Other phenomena such as variations papain digestion for this assay. in sequence, posttranslationalmodification, improper folding, etc. The main cleavage points for papain are in the antibody hinge appear to play important roles as well. CE of an antifolate antibody region adjacent to the disulfide bonds which hold the two antibody partially separated more than eight antibody isoforms in less than heavy chains together. As such, papain digestion yields an Fc 2.5 min. Removal of sialic acid by neuraminidase treatment led portion and two Fab fragments, each with one antigen binding (19) Silverman, L.; Komar, M.; Shields, K; Diegnan, G.; Adamovics, J. /. Liq. domainz6 The digestion process was monitored in real time with Chromatogr. 1992, 15, 207-219. CE by sampling directly from the digestion mixture. The high (20) Costello, M. A; Woititz, Ch.; De Feo, J.; Stremlo, D.; Wen, L.-F. L.; Palling, D. J.; Jqbal, K; Guman, N. A /. Liq. Chromatogr. 1992, 15, 1081-1097. speed of separation and direct UV detection, combined with (21) Jungbauer, A; Tauer, C.; Wenisch, E.; Uhl, K; B m n e r , J.; Purtscher, M.; minimal sample consumption, makes CE an attractive alternative Steindl, F.; Buchacher, A /. Chromatogr. 1990,512,157-163. to SDS slab gel electrophoresis for digestion monitoring since (22) Kaltenbmnner, 0.: Tauer, C.; Bmnner, J.; Jungbauer, A /. Chromatogr. 1993, 639, 41-49. the latter requires that a small scale test digestion be run with (23) Patel, T. P.; Parekh, R B.: Moellering, B. J.; Prior, C. P. Biochem. /. 1992, periodic sampling and the use of agents such as iodoacetamide 285, 839-845. to stop papain activity in the samples. (24) Rademacher, T. W.; Parekh, R B.; Dwek, R A; Isenberg, D.; Rook, G.; Axford, J. S.; Roitt, I. Springer Semin. Immunopathol. 1988, 10,231-249. (25) Chaplin, M. F.: Kennedy, J. F., Eds. CarbohydruteAnalysis; IRLPress: Oxford 1986.
608 Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
(26) Harlow, E.; Lane, D., Eds. Antibodies; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY,1988 pp 626-628.
0.09
neuraminidase eeaaent
0.07
Ab
A
Fc
-0.07
.O.OlO
I
a
0.041
1
.0.005
Smin
Fab
I I
i
0.0:.
Ab
0.00
1.5
2.0
3.5
migration time [mi.]
Figure 3. CE monitoring of neuraminidase(Clostridiumperfringens) treatment of mouse monoclonal antifolate lgG2b antibody at 37 "C, pH 4.4 (50 mM sodium citrate). Coated fused silica column, 20/30 cm, 75 pm; pH 4.4 (20 mM EACNHAc), 24 kV, 20 pA, 214 nm.
The digestion of the antibody proceeded rapidly. The appearance of three Fab fragment peaks and a broad peak for the Fc portion was observed only minutes after the enzyme was added (peak assignments based on protein A purification) (Figure 4a). After 30 min, the digestion was essentially complete, and the Fab fragments were separated from undigested antibody and Fc portion using a protein A af6nity cdlumn. The purity was determined by CE (Figure 4b,c), The nature of the three separate peaks for the Fab fragments is not yet clear. It could be the result of microheterogeneities in the Fab fragments or due to the fact that papain cleavage is not very specific,27 particularly for mouse IgGBb, which is known to be very susceptible to secondary digestion.26 MALDI-TOFMS gave the expected molecular mass of about 50 kD for the three Fab fragments but did not reveal observable differences between the fragments. In cIEF, three bands were observed with PI values of 5.4, 5.5, and 5.8, respectively. The three Fab fragment isoforms were separated and collected by HPLC on a cation exchange chromatography column making use of the small PI dzerences observed in cIEF. The purity of the collected fractions was assessed by CE. All three fractions preserved their activity for the antigen as determined by CE binding studies, and any one of the fractions could be used for the assay. CE of Cortisol in Serum. Figure 5 shows electropherograms obtained when the CE immunoassay was performed for the determination of serum cortisol within the clinically s i g " t (27) Fersht, k Enzyme Structure and Mechanism; W. H. Freeman and Co.: New York, 1985; pp 413-416.
0
5.0
10.0
migration time [mi.]
Flgure 4. (a) CE monitoring of the papain digest of mouse monoclonal anti-cortisol lgG2b antibody. (b) Protein A fractionation of digest: undigested antibody and Fc portion. (c) Purified Fab fragments. Coated fused silica column, 30/40 cm, 50 pm; pH 8.8 (20 mM TAPS/AMPD), 30 kV, 14 PA, 214 nm.
range of 1-60 pg/dL (10-6-10-8 M). At an applied field strength of 1100 V/cm, the free labeled antigen was well separated from the complex in a run time of about 2 min. There was no noticable dissociation of the complex during the runs, as evidenced by the lack of a pedestal connecting the labeled antigen and the complex peaks. Because the speed of the separation is limited by the present experimental setup, even faster separations could be obtained by changing instrumental parameters. We estimate that a separation time of less than 1 min would be possible by shortening the effective separation length or increasing the field strength. Both free and bound labeled antigen follow the behavior expected for competitive assays: an increase in the amount of serum cortisol led to a decrease in signal for the complex and an increase in signal for the free labeled antigen. No cleanup of the serum samples prior to the CE runs was required, and the sera were injected without further dilution. As can be seen, the high salt and protein content of serum did not have an adverse effect on the separations. Most impressively, the migration time reproducibility between both individual runs and different columns was better than 0.5%. This results in part from the elimination of electroosmotic flow and its contribution to migration time variability. Furthermore, there is essentially no adsorption of the analytes to the column surface. The slight broadening of the free labeled antigen peak is likely due to the presence of two isomeric forms (syn and anti coniigurations at the oxime double bond or different attachment sides of the linker to the fluorescein) of the labeled antigen. Analytical Chemistry, Vol. 67, No. 3, February 7, 7995
609
free Ag* i.s.
I
1 Fg/dL
I
0
,
,
,
,
,
1.0
-
*
a
,
I
2.0
3.0
2.0
3.0
migration time [min]
I
0
1.0 migration time [minl
migration time [min]
Figure 5. CE-based immunoassay of cortisol in undiluted serum, 8 s pressure injection at the negative electrode. Coated fused silica column, 27/30 cm, 50 j”pH 8.8 (20 mM TAPSAMPD), 30 kV, 18 PA, LIF (excitation, 488 nm/emission, 520 nm).
An approximately l@fold decrease in fluorescence yield was observed for the FabAabeled antigen complex compared to the free form. Such a change in emission is not surprising since complexation can result in a drastic change in the local environ610 Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
ment of the f l u o r o p h ~ r e .The ~ ~ ~quantum ~~ yield of fluorescein is known to be sensitive to environmental conditions such as pH and electrostatic effects. The use of another fluorophore which is less susceptible to environmental changes should reduce the observed quenching. As previously mentioned, quantitation in competitive immunoassays can be carried out without the signal from the complex. Still, there are advantages to having both signals available: (1) the second signal can be used as a control; (2) peak ratios can be applied; and (3) the signals are inversely proportional, hence precision can be improved by using both signals. We found that the equilibrium in water was established after 10 min of incubation due to a relatively rapid complex formation in solution. In serum, however, because of the presence of endogenous cortisol binding proteins, a longer incubation time was needed to release the bound fraction of The cortisol releasing agent 8anilino-1-naphthalenesulfonic acid (ANS) was added to the serum samples to displace bound corti~0l.l~ Under these conditions, the incubation took 1 h at room temperature. Insufficient incubation resulted in broad peaks with poor reproducibility for the free labeled antigen, possibly because of interaction with binding proteins in the serum. Quantitation. The peak heights of free labeled antigen and labeled antigedantibody complex were used to establish the calibration curves for cortisol in water and serum (Figures 6 and 7). Because of the very high run-to-run reproducibility, normalization for migration time was not necessary. Slight variations in injection volume were corrected by using an internal standard (fluorescein). The calibration curves show the sigmoidal shape typical for competitive assays with the curves for the labeled antigen and complex following the characteristic opposite trend. The operating concentration range of more than 3 orders of magnitude is sufficient, given that the clinically relevant cortisol concentration in serum spans 2 orders of magnitude. The midpoint of the calibration curve (Le., the point at which 50%of the bound labeled antigen has been displaced) sets the analyte concentration range which can be measured by the assay and hence determines the assay suitability for the clinically relevant range. With the reagent concentrations shown, the assay extends over the clinically significant range but is not centered on it as would be desired. However, the calibration curve can be shifted to lower concentrations by decreasing the concentration of the labeled antigen and the antibody, provided the affinity of the antibody is sdciently high.31 In our case, we were not able to work with lower reagent concentrations. This limitation was due not to the affinity of the antibody (reported affinity constant for the intact antibody, 1.2 x 10-9 M) but to the detection limit of the LIF detector (5 x low9M fluorescein). The noise level in the detector is high compared to what others have reported for laser fluorescence detection. This is not an inherent limit but is due to the fact that our detector is not optimized. Improved detection capabilities are under development. (28) Shaw, E. J.; Watson, R A A; Landon, J.; Smith, D. S.J. Clin. Pathol. 1977, 30, 526-531. (29) Soini, E.; Hemmila, I. A Clin. Chem. 1979,25,353-361. 1869-1890. (30) Pratt, J. J. Clin. Chem. 1978,24, (31) Delaage, R; Barbet, J. In Methods of Immunological Analysis; Masseyeff, R F., Albert, W. H., Staines, N. A, Eds.; VCH: New York, 1993 Vol. 1, pp 493-497.
25
1
20
-
15
-
10
-
I
Cortisol Concentration (M)
Cortisol Concentration (M)
5
0.0
1
10.8
'
'
'
" '."
1 0 . ~
'
'
'
'
""
104
Y
10.5
104
1
Cortisol Concentration (kl)
0.1 I 10.8
10.~
10.6
Cortisol Concentration
10.5
(M)
Figure 6. Calibration curve for cortisol in water measured by CE: (a) ratio of free labeled antigen peak height to the peak height of the internal standard versus cortisol concentration in water and (b) ratio of complex peak height to the peak height of the internal standard versus cortisol concentration.
Figure 7. Calibration curves for cortisol in serum measured by CE: (a) ratio of free labeled antigen peak height to peak height of internal standard versus cortisol concentration in serum and (b) ratio of complex peak height to the peak height of the internal standard versus cortisol concentration.
Because fluorescence quenching resulted in lower signal to noise ratios for the complex, we chose to use the labeled antigen signal for quantitation. Table 1 presents the data based on the
peak height for the free labeled antigen for a set of incubations where known amounts of cortisol were added to cortisol free serum. The measured values are in reasonable agreement with Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
611
Table 1. Accuracy of Serum Cortisol Determination
cortisol added (ug/dL)
cortisol found (ug/dL)
1.0 3.0 10.0 25.0 60.0
3.0 5.0 7.5 20.0 65.0
the expected values. Better results can likely be obtained if the calibration curve is shifted to lower concentrations, thereby centering it more closely on the clinically relevant range. Separately, a set of serum samples with a range of cortisol concentrations was measured repetitively (n = 5) by CE (Table 2). The precision of quantitation using the free labeled antigen is satisfactory. As expected,poorer precision is obtained by using the signal for the complex. These results demonstrate the essential elements of a CEbased serum cortisol immunoassay. Further development is required to make it a clinically acceptable assay. Work in our laboratory is focused on the improvementof the laser fluorescence detection system, which we consider to be a major limiting factor with regard to accuracy and precision. Also ongoing are crossreactivity studies and comparisons with other assay methodologies. At this point, the results indicate that CEbased immunoassays allow for rapid and quantitative determination of analytes of diagnostic relevance in complex sample matrices such as serum. Furthermore, given the speed and high separating power of CE, such assays show advantages in terms of reducing reagent consumption, simplifying assay methodology by eliminating wash-
612
Analytical Chemistry, Vol. 67, No. 3, February 1, 1995
Table 2. Preclslon of Serum Cortisol Determination (n = 5)
cortisol (ug/dL) X SD cv (%) (a) Using Free Labeled AntigenAntemal Standard Peak Height Ratio 1.0 3.0 10.0 25.0 60.0 (b)
1.12 1.76 1.82 2.72 3.70
0.021 0.045 0.031 0.058 0.120
1.9 2.7 1.8 2.1 3.9
Using Complex/Intemal Standard Peak Height Ratio 1.0 3.0 10.0 25.0 60.0
0.71 0.44 0.54 0.39 0.14
0.035 0.019 0.069 0.022 0.011
4.9 4.4 13 5.6 5.9
ing steps, and potentially providing multianalyte and multilane capabilities. Based on our results, we believe that CE-based immunoassays will become an important tool in clinical diagnostics. ACKNOWLEDGMENT
The authors would l i e to thank Dr. Simin Maleknia for performing the MALDI-TOFmeasurements and Renee Piggee for running some of the CE experiments. Received for review September November 18, 1994.@
7, 1994.
Accepted
AC940891R @Abstractpublished in Advance ACS Abstructs, December 15, 1994.
