1928
Anal. Chem. 1985, 57, 1928-1930
(2) ArchlbaM, R. M. I n “Standard Methods of Clinical Chemistry”; Seieg son, D.,Ed.; Academic Press: New York, 1958;Vol. 2,p 91. (3) Doumas, 8. T.; Bayse, D. D.; Carter, R. J.; Peters, T., Jr.; Schaffer, R. Clln. Chem. (Winston-Salem, N.C.) 1981, 27, 1642-1645. (4) Variey, H. “Practical Clinical Biochemistry”, 4th ed.; Heinemann Medicai Books, Ltd.: London, and Interscience: New York, 1967. (5) Rubln, M. E.; Wolf, A. V. J. Biol. Chem. 1957, 225, 869-873. (6) Alexander, M.; Rechnitz, G. A. Anal. Chem. 1974, 46, 250-254,
860-865. (7) Gullbault, G. G.; Shu, F. R. Anal. Chem. 1972, 44, 2161-2165. (8) Berjonneau, A.-M.; Broun, T. D. Pafhol. Blol. 1974, 22, 497-502. (9) Calvot, C.; Berjonneau, A.-M.; Gellf, G.; Thomas, D. FEBS Left. 1975, 59,258-263. (IO) Havas, J.; Gullbault, G. G. Anal. Chem. 1982, 5 4 , 1991-1995.
(11) DiPaoiantomo, C. L.; Rachnitz. G. A. Anal. Chim. Acta 1982, 141, 1-10. (12) Anbrose, J. A.; Becker, R.; Blake, E.; Sldeman, L; Wainer, S. Clln. Chem. (Winston-Salem, N.C.) 1974, 20. 505-510. (13) Kalmar, A. D. M.S. Thesis, University of New Orleans, 1980. (14) Duekworth, H. W.; Coteman, J. E. J . Biol. Chem. 1970, ,245, 1613-1617. (15) Knox, W. E. “The Metabolic Basis of Inherited Disease”, 3rd ed.; Stanbury, J. E., Wyngaarden, J. E., Fredrickson, D. S., Eds.; McGrawHIII: New York, 1972;p 266.
RECEIVED for review January
30, 1985. Accepted April 11,
1985.
Solvent Perturbation Fluorescence Immunoassay Technique Clarke J. Halfman,* Franklin C. L. Wong, and Dennis W. Jay Department of Pathology, University of Health ScienceslThe Chicago Medical School, North Chicago, Illinois 60064
The use of fluorescent dyes to label analyte In llgand blndlng assays affords the posslbllty of convenlent, homogeneous assays. The homogeneous response depends upon a slgnlflcant difference In a fluorescent property of bound compared to free labeled analyte. We have found that dodecyl sulfate quenches the emlsslon lntenslty of free fluoresceln labeled gentamycln wlthout lnfluenclng the emlsslon lntenslty of labeled gentamlcln bound to gentamlcln antlbody. Thls preferentlal quenchlng by detergent Is demonstrated to serve as the basis for a homogeneous fluorescence Immunoassay lor gentamlcln requiring only slmple lntenstty measurements. The method may be used to measure other analytes when It can be demonstrated that the perturblng agent (In this case, detergent) preierentlally Influences the lntenslty of free labeled analyte. Thls preferentlal perturbation may be assured by judicious choke of perturblng agent and labellng fluor so that the lnteractlon between labeled analyte and the perturblng agent occurs with the analyte molety and not wlth the fluor molety.
Fluorescent dyes have proved practically useful as labels in ligand binding assays and provide a homogeneous response when a fluorescent property of bound labeled analyte is sufficiently different from that of free. Fluorescence intensity is the simplest property to measure, but significant intensity differences between bound and free labeled analyte do not often occur. Only for the case of fluorescein labeled thyroxine (I) has the bound/free intensity ratio ( 2.7) been reported to be sufficiently great so that measurement of intensity provided a useful, homogeneous response variable. Although a boundlfree intensity ratio of -0.8 has been reported (2) for fluorescein-labeled gentamicin, the degree of intensity difference does not seem sufficiently great to be practicably useful. A significantly greater fluorescencepolarization for bound labeled analyte compared to that of free should, in principle, be a general phenomenon for analytes of sufficiently small molecular size (3), and fluorescence polarization has been demonstrated to provide a practicable, homogeneous response variable (4). Polarization, however, is a calculated parameter determined from measurements of both the vertical and horizontal emission components with polarizers in the exciN
0003-2700/85/0357-1~28$01.50/0
tation and emission beams. One of the polarizers must be rotated before each measurement, and the presence of each polarizer in the light beam reduces light intensity and signal strength by a factor of rI. Potential sensitivity is thus reduced by up to a factor of about 10. Because of the greater convenience and potential sensitivity of intensity measurements, efforts have been expended to cause a preferential alteration of intensity from bound or free labeled analyte. The principle of excitation energy transfer was exploited (5) by additionally labeling analyte-antibody with a nonfluorescent dye, the absorption spectrum of which overlaps the emission spectrum of the dye used to label analyte. The excitation energy of the analyte label (donor dye) is transferred to the antibody label (acceptor dye) when the two dyes are sufficiently proximate, resulting in decreased emission from donor dye. Energy transfer, and consequent quenching of donor dye emission, occurs only when labeled analyte is bound to antibody (labeled with acceptor dye). Emission from bound labeled analyte is thus preferentially quenched, and the assay response is increased intensity with increasing concentration of analyte from standards or specimen. One difficulty which this method presents results from the necessity to conjugate antibody with a relatively high mole ratio (-10-20) of acceptor dye to assure that a t least one acceptor dye molecule becomes conjugated sufficiently close to each hapten binding site. On the other hand, too high a degree of conjugation may diminish antibody affinity, so that an optimum degree of conjugation must be established for each antibody preparation. Another difficulty with this method is that the acceptor dye must have minimal fluorescence since it is present a t far higher concentrations in the assay tube than is the donor dye because of the high degree of conjugation and also because when antibody is labeled with acceptor dye, so are all the other serum proteins present in the antiserum unless the specific immunoglobulinis isolated. Another means to preferentially quench emission from free-labeled analyte employs an additional antibody to the dye label (6)and is based upon the observation that fluorescein emission is quenched when the dye is bound to its antibody (8). Steric hindrance prevents dye binding by the dye antibody when labeled analyte is bound to analyte antibody. Assay response is thus a decrease in intensity, with increasing concentrations of analyte from standards or specimen. A disadvantage of this method is the requirement for a second antibody to the label which must consistently be of high 0 1985 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
1929
120
100
80
I 60
40 20
40
SDS (mg/dl) Flgure 1. Effect of dodecyl sulfate (SDS) on the emission intensity (0) and polarization (0)of fluorescein-labeled gentamicln. The emission intensity and polarization were determined for a series of tubes containing 10 nM fluorescein-labeled gentamicin and the indicated concentration of dodecyl sulfate (SDS).
affinity and cause significant quenching. Both of these characteristics are, however, variable from one antibody preparation to another (9). A simpler means of selectively altering the emission intensity of bound or free-labeled analyte would be desirable and provide for a less costly assay system.
EXPERIMENTAL SECTION Fluorescence intensity and polarization were measured with a two-channel SLM Model 4000 polarization spectrofluorometer, equipped with an excitation grating monochromator, excitation and emission Glann-Thompson polarizers, Hamamatsu R928 red-sensitive phototubes, and a water-jacketed cuvette compartment maintained at 30 aewith a Lauda RC-BOT circulating water bath. Fluorescein emission was measured with an excitation wavelength of 492 nm (8 nm band-pass) and matched Turner 12 A2 filters (transmitting at wavelengths above 500 nm) in the emission beams. Rhodamine B emission was measured with an excitation wavelength of 545 nm and matched Turner 23A filters (transmittingat wavelengths above 570 nm) in the emission beams. Polarization values (P)were determined from measurements with vertically polarized excitation and with the emission polarizers oriented in the vertical (V) or horizontal (H) positions and calculated from P = (V - H)/(V + H).Appropriate buffer-antiserum blank values (< 10% of maximum signal strength) were subtracted from all measurements. The reagents employed for the gentamicin assay, fluoresceinlabeled gentamicin, gentamicin antiserum, gentamicin standards and controls in normal human serum, and dilution buffer, were as supplied from an Abbott Diagnostics TDX gentamicin kit. Assays were conducted according to the manufacturer’sdirections. Sodium dodecyl sulfate (95%) was a Sigma product and used without further purification; working solutions were prepared in 0.1 M sodium phosphate adjusted to a pH of 7.5. A 40 mg/dL solution of dodecyl sulfate was substituted for the commercial dilution buffer for the gentamicin assay conducted in the presence of dodecyl sulfate. Fluorescein and rhodamine B were uKromexn grade products from J. T. Baker Chemical Co. (Phillipsburg,NJ). The thiocyanate derivative of each fluor was obtained from Research Organics (Cleveland, OH). Thyroxine was obtained from Sigma Chemical Co. (Kansas City, MO). The rhodamine Bthiocarbamyl-thyroxine conjugate was prepared by reacting rhodamine B isothiocyanate with thyroxine in pyridine and isolating and purifying the product by washing with ethanokwater (6040). The antibody to thyroxine was generously supplied by Abbott Diagnostics (North Chicago, IL).
RESULTS AND DISCUSSION The addition of dodecyl sulfate to aqueous-buffered solutions of 10 nM fluorescein labeled gentamicin causes quenching of fluorescein emission as shown in Figure 1. The dodecyl sulfate concentration range in which quenching occurs is in the vicinity of published estimates of the critical micelle
2c
Figure 2. Gentamicin FIA responses: curve a, intensity as the assay response without added dodecyl sulfate; curve b, intensity in the presence of 40 mg/dL dodecyl sulfate; curve c, polarization as the assay response without added dodecyl sulfate. Assay tubes were prepared by adding 20 /.&L of standards or specimens, 40 pL of fluorescein labeled gentamicin (-0.1 pM), 2.4 mL of buffer (with or without 40 mgJdL SDS), and 40 pL of gentamicin antiserum. Intensity and/or polarization was measured 5-15 mln after addition of antiserum.
‘Table I. Measurement by Solvent Perturbation FIA of Serum Specimens with Known Additions of Gentamicin specimen
gentamicin concn, pg/mL added measured
1
1.0
2
4.0
3
8.0
1.1 3.7 7.6
concentration (IO). That quenching of emission is due to an association of fluorescein-gentamicin with micelles is evidenced by the corresponding increase in polarization. Dodecyl sulfate had little effect on the emission of fluorescein-gentamicin bound to gentamicin antibody. The differential effect of dodecyl sulfate on the emission of free and bound fluorescein-labeled gentamicin constituted the basis for a homogeneous immunoassay. A typical assay response curve is shown in Figure 2. The response in terms of intensity in the presence of 40 mg f dL dodecyl sulfate (curve b) is compared to the response in terms of intensity (curve a) and polarization (curve c) in the absence of extra added dodecyl sulfate. The percentage intensity change in the absence of dodecyl sulfate is about 20%, similar to that reported by Shaw et al. (2). The polarization response is similar to that reported by Jolley et al. (4). The intensity response in the presence of dodecyl sulfate is about 2.5-fold greater than that in the absence of dodecyl sulfate and is nearly of the same magnitude as the polarization response. To evaluate the method for measuring gentamicin in serum specimens, normal human serum, to which were added accurately weighed amounts of gentamicin, was assayed. The results shown in Table I demonstrate excellent agreement between measured and expected values at low, therapeutic, and high concentrations. The phenomenon does not appear to be entirely general; Le., detergent micelles do not always preferentially interact only with free labeled analyte. For example, dodecyl sulfate affects the emission intensity of rhodamine B labeled thyroxine whether the conjugate is free or bound to thyroxine antibody, as shown in Figure 3. It appears that the interaction
1930
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985 Rhodcmine B
12
0.20
0 0
0
Intensity
I N
A 3.15 N i
T 8
E
S
N S
0 3.10 T R 0
I I
T 4
Y
Fluorescein
3.05
P
SDS ( m g l d l )
10
2
4
6
SDS ( m g / d l )
Figure 3. Effect of dodecyi sulfate on the emission intensity of rhodamine B labeled thyroxine in the presence (0)and absence ( 0 )of excess thyroxine antibody. Tubes were prepared containing 10 nM rhodamine B labeled thyroxine, with or without excess antiserum, and the indicated concentration of SDS. The dashed curve connecting the data points in the absence of antiserum has no physical significance. of detergent micelles with rhodamine B is not hindered by binding of the conjugate to antibody, as is the case for fluorescein labeled gentamicin. The factors responsible for the association of the conjugate with micelles are being investigated further so that the interaction may be controlled in a desired manner. The interaction of fluorescent dyes with micelles has been the subject of much study; and it has been demonstrated that anionic, cationic, and uncharged dyes all interact with micelles regardless of the ionic nature of the detergent ( I I , 1 2 ) . The major goal of such studies has been to characterize various properties of micelles using the fluorescent dyes as “probes” in the micromole per liter to millimole per liter concentration range. At these concentrations, there does not seem to be a preferential interaction between dye and micelle that is dependent upon the respective charge of either reactant. However, a strong influence of charge on the dye-micelle interaction, a t dye concentrations in the nanomole per liter concentration range employed in this investigation, is suggested by the follgwing additional observations. The emission intensity and polarization of free fluorescein are not affected by dodecyl sulfate a t concentrations below or above the critical micelle concentration, as shown in Figure 4,whereas the aminoglycoside conjugate does interact (Figure 1). Rhodamine B (a positively charged amino analogue of fluorescein) exhibits increased intensity and polarization in the presence of dodecyl sulfate micelles (Figure 4) as does the rhodamine B-thiocarbamyl-thyroxine conjugate, whether in free solution or bound to a thyroxine antibody (Figure 3). These results are consistent with a conjugate-micelle interaction which is dependent upon ionic charge at the low concentration of fluor and fluor anal@ conjugate employed. Like charge prevents interaction, and opposite charge favors interaction. In the case of the fluorescein-labeled gentamicin conjugate, it is apparently the cationic gentamicin moiety, rather than the anionic fluorescein moiety, which directly interacts with negatively charged micelles. When the gentamicin moiety is enveloped within the immunoglobulin combining site, micelles do not interact with the fluorescein moiety, even though it is exposed to the aqueous solvent. In the case of the rhodamine 3 labeled thyroxine conjugate, the
30
50
Figure 4. Effectof dodecyi sulfateon the intensity and anisotropy of rhodamine B and of fluorescein. Tubes were prepared containing 10 nM rhodamine B or fluoresceinand the indicated concentration of SDS. cationic dye moiety, rather than the anionic thyroxine moiety, apparently interacts directly with the negatively charged micelles and does so whether the conjugate is free or is bound to antibody via the thyroxine moiety. The desired preferential association of the free conjugate with micelles thus appears to occur when interaction with the anal@ moiety is favored and interaction with the fluor moiety is prevented. Thus, the ionic relationships required for preferential interaction of micelles with free conjugate are that (1) the detergent and fluor moiety have the same ionic charge and that (2) the detergent and analyte moiety are not of the same charge. The fluorescein-gentamicin conjugate satisfies the above requirements for the desired interaction with dodecyl sulfate, whereas the rhodamine B-thyroxine conjugate does not. However, negatively charged analyte moieties (such as thyroxin) conjugated to cationic dyes should interact in the desired manner with positively charged micelles. The requirement for nonionic analytes, which interact strongly with micelles regardless of charge (12),would simply be that the fluor used as label and the detergent be of the same charge. An additional requirement for the fluor is that emission intensity be substantially different when the adduct interacts with detergent compared to when interacting with analyte antibody. Registry No. SDS, 151-21-3; gentamycin, 1403-66-3; fluorescein, 2321-07-5;rhodamine B-thiocarbamyl-thyroxineconjugate, 96503-28-5.
LITERATURE CITED (1) Smith, D. S. f€BS Let? 1078, 77, 25-27. (2) Shaw, E. J.; Watson, R. A.; Landon, J.: Smith, D. S. J , Clin. Pathol. 1977, 30. 526-531. (3) Dandliker, W. B.; Keliey, R. J.; Dandliker, J. Immunochemistry 1973, 10, 219-227. (4) Jolley, M. E.; Stroupe, S. D.; Wang, C. J.; Panas, H. N.; Keegan, C. L. Clin. Chem. (Winston-Salem, N.C.) 1981, 27, 1190-1197. (5) Ullman, E. F.; Schwartzberg, M.; Rubenstein, K. E. J . Biol. Chem. 1976, 251, 4172-4178. (6) Zuk, R. F.; Rowley, G. L.; Ullman, E. F. Clh. Chem. (Wlnston-Salem. N.C.) 1079, 25, 1554-1560. (7) Dandliker, W. B.; Schapiro, H. C.; Meduskl, J. W.; Alonso, R.; Feigen, 0. A.; Hambrick, J. R. Immunochemistry 1964, 1 , 165-191. (8) Lopatln, D. E.; Voss, E. W. Immunochemistry 1971, 10, 208-213. (9) Voss, E. W.; Eschenfeldt, W.; Root, R. T. Immunochemistry 1076, f 3 , 447-453. (IO) Mukerjee, P.; Mysels, K. J. “Critical Micelle Concentratlons in Aqueous Surfactant Systems”; National Bureau of Standards: Washlngton, DC, 1971; NSRDS-NBS 38. (11) DeVendMls, E.; PaIumbo, G.; Parlotto, G.; Bocchini, V. Anal. Blochem. 1981, 115. 270-286. (12) Law, K. W. Photochem. Photobiol. 1981, 33, 799-806.
RECEIVED for review November 12, 1984. Accepted April 12, 1985.
