Report
Fluorescence Immunoassays C. Michael O'Donnell Clinical Chemistry Laboratory Department of Pathology University of California Los Angeles. Calif. 90024
Stephen C. Suffin Department of Pathology University of California Los Angeles. Calif. 90024
The technique of radioimmunoas say, RIA (see Appendix for definitions of selected terms used in this paper), has revolutionized the analysis of many biologically important sub stances. Examples of the impact of this method are found throughout the areas of biology and medicine with new applications continually being de veloped. The impact of this technique results from its sensitivity (10 - 1 2 10 -15 M), selectivity, and batch sam pling capabilities. However, the sensi tivity of this method depends on the counting of trace quantities of radio activity which places several restric tions on RIA. These restraints are reg ulation of production, shipping, han dling, and disposal of radioactive ma terial; the decay of radioactivity is a destructive process that prevents long-term assay standardization; and expensive counting equipment that requires significant maintenance. A new generation of immunological tests that do not require the use of ra diolabeled materials is currently being developed. Substitution of the radiolabel by an enzyme or fluorescent mole cule may be accomplished without de stroying the specificity of the ligand— antibody reaction, where ligand repre sents both immunogen and antigen. These new immunochemical ap proaches are currently competing with RIA in a number of areas, but the ouccess of any method will depend on the 0003-2700/78/0351-033 A$01.00/0 © 1978 American Chemical Society
attainment of sensitivity and specifici ty equivalent to that already achieved by the use of radiolabeled material. The use of fluorescence labels in im munoassays has the potential to re place many, if not all, RIA procedures. We will assess the current status of fluorescence immunoassays (FIA) and indicate which directions this impor tant new technology appears to be taking. Labeled immunoassays generally trace their origins to studies per formed by Yalow and Berson (7) and reported in 1959. Although each pro cedure differs in specifics depending on the type of analysis or the type of label selected, the various constituents of each assay are all characterized by similar kinetics and have been exten sively reviewed (2). The mathematics of these reactions are based on two as sumptions. One is that the reactants obey the law of mass action, and the second is that the antibody is unable to distinguish between labeled ligand and unlabeled (native) ligand. Subsequent to mixing and incuba tion, an equilibrium is established: [L—Ab] = K([Ab]0 - [L-Ab]) [L] where [Ab]o = initial concentration of antibody, [L] = ligand concentration, [L—Ab] = concentration of ligand bound to antibody, and Κ = equilibri um constant. This simplified descrip tion of a labeled immunoassay serves as an adequate predictive model when antibodies are of a single chemical species and react in a univalent fash ion. Unfortunately, neither of these conditions holds in a real immunoas say because almost all antisera contain
several antibodies of similar reactivity but of differing binding strengths, and almost all antibodies have some cross reactivity with similar chemical species present in the biological millieu. We will ignore most of these sub tleties to concentrate on the fluores cence aspects of immunoassays. The reaction between a ligand (L), a ligand labeled with a fluorescent mole cule (LO, and an antibody [Ab] specif ic toward both L and Lf may be writ ten: [L] + [LT + [ A b ] ^ k-l
[L—Ab] + [Lf—
Ab]
where k] » k_i. L f may be considered to exist in two separate microenvironments, i.e., the environment of the so lution and the environment of the antibody binding site. Both local envi ronments are different as evidenced by the high binding affinity of the Ab for L and Lf. These differences result from solvation effects, hydrophobic binding, hydrogen binding, and ionic interactions with concomitant changes in enthalpy and entropy. The degree of environmental influences will be re flected in perturbations (or lack of perturbations) in the electronic and vibrational structure with parallel changes in fluorescence properties of V. Quantitative analysis of L may be accomplished in one of two ways. First, a change in the fluorescence properties of Lf may occur upon bind ing to Ab. If this change in bound Lf fluorescence is sufficiently different from unbound Lf fluorescence, it may be used to quantitate L without a sep-
ANALYTICAL CHEMISTRY. VOL. 51. NO. 1, JANUARY 1979 · 33 A
Table 1. Homogeneous FIA Principle
Fluorescence excitation transfer Fluorescence polarization
Enzymatic
Chemiluminescence Enhancement of fluorescence Quenching of fluorescence
Analyte
Analytical data 9
Codeine IgG Morphine 2-Aminobenzimidazole Gentamicin
Insulin Trypsin Biotin Gentamicin
2,4-Dinitrofluorobenzerie Biotin Thyroxine Thyroxine Gentamicin
Ret
1 Χ Ι Ο " Μ detectable
5
1 X 1 0 - ' ° Af detectable 1 X 1 0 - 1 0 W detectable 1 X 1 0 " 8 M detectable 2 μρ/mL concentration detectable with a sample of 1.25 μ ι and 2 min incubation 1 Χ 1(Γ β Μ detectable Activity measured 1 X 1CT8 M detectable 1 Mg/mL detectable with 1 μί. sample and 2-3 min incubation 1 Χ Ι Ο - 8 Μ detectable 1 X 1 0 _ " M detectable 1 X 1 0 - 1 0 M detectable 3 X 1 0 - 9 M detectable
5 5 8 7
1 Mg/mL concentration with sample of 10 μ ί and 5 min incubation
10
6 6 11, 13 12
11. 13 14 15 9
aration of Lf bound from Lf unbound. This type of assay is called homoge neous. A second type of FIA has been developed based on the separation of Lf bound and Lf unbound and is called a heterogeneous assay. The molecular properties important in FIA such as fluorescence quantum yield are de pendent on pH, the presence of heavy atoms, the linkage between ligand and label, the details of ligand—antibody binding, and the presence of endoge nous quenchers. This sharp depen dence of Lf on its environment may be contrasted to a radiolabeled ligand which has far fewer restraints. Success of any FIA will ultimately depend on the minimization of potential fluores cence interferences and rigid control of the basic fluorescence property being measured.
upon irradiation with light of an appropriate wavelength which is equivalent to the condition that fluorescence bleaching is negligible for the duration of the measurement. All of the above fluorescence labels have been demonstrated to fulfill this condition. Longterm stability is more difficult to define but implies that fluorescence characteristics of the label are constant during long-term storage, i.e., several years. Only fluorescein labels
Fluorescence Labels The choice of a label in FIA is criti cal and must satisfy several require ments. A fluorescence label should possess stability; have high absorptivi ty and quantum yield; absorb and fluoresce at appropriate wavelengths; and fulfill specific structural criteria. The labels most frequently used to date in FIA are fluorescein, tétramethylrhodamine, umbelliferone, isoluminol, and nicotinamide derivatives. Refer to Tables I and II for label applications. The first requirement implies both short- and long-term stability. Short-term stability consists of achieving stable fluorescence readings
igM
have been adequately demonstrated to have long-term stability, but tetramethylrhodamine and other labels are probably stable when stored lyophilized in the dark. The second condition is desirable in order to have the maximum absorption of light on a molar basis and a significant portion of this absorbed light reemitted in the form of fluorescence without appreciable losses due to radiationless decay processes. Although not a strict requirement, it is certainly a convenience that the label absorb in the visible region of the spectrum to avoid the use of ultraviolet light sources as a source of instrumental instability and to decrease interferences from impurities. This restraint is consistent with the practical finding in biological systems that as the wavelength of excitation is decreased the number of fluorescence interferences increase. Fluorescence from the label should occur in a region of the spectrum where multiplier phototubes are sensitive. At present, a useful lower limit is found around 300 nm, and an upper limit around 600 nm. The last condition necessitates that the label not interfere with the ligand—antibody reaction, and that the label meet the above criteria after conjugation and under appropriate assay conditions. Conjugation may or may not significantly perturb the electronic structure of a label and cause shifts in absorption and fluorescence maxima and decrease or increase in molar absorptivity and/or fluorescence quantum yield. Such effects are observed when fluorescein ieothiocyanate is coupled with thyroxine presumably due to some heavy atom mechanism via the iodine atoms. Additionally, the presence of ionizable functional groups on the ligand neces-
Table II. Heterogeneous FIA Analyte
Principle
Bivalent ligand, fluorescentlabeled antibody
IgG IgA
igE
c3 c4 Alpha-fetoprotein Internal reflectance
Morphine
Antigen adsorbed on solid phase fluorescent-labeled antibody
Toxoplasma gondii antigen Entamoeba histolytica antigen
34 A · ANALYTICAL CHEMISTRY, VOL. 5 1 , NO. 1, JANUARY 1979
Analytical data
6 mg/dL detectable 60 μg/dL detectable 40 μg/dL detectable 200 ppb detectable 125 ng/mL detectable 150 ng/mL detectable 25 ng/mL detectable 2 X 10-8 M detectable Screening test Screening test
Rat
17, 18 18 18 20 21 22 23 19 24 25
sitates strict control of parameters such as pH. T h e recent introduction of rareearth based fluorochromes for labeling (3) represents a significant departure from the labels previously discussed. Most labels possess wide absorption and emission bands. This reduces their specificity. However, the rareearth fluorochromes emit well-de fined, spectrally distinct peaks that would permit the simultaneous mea surement of several labels without in terference. This work is indicative of the type of flexibility possible with properly designed label systems. A re port on fluorescence studies of xanthene dyes (4) points out the deficien cies t h a t exist in our knowledge of the electronic structure of the labels, let alone their conjugates to various ligands.
Approaches to FIA (See Table I) Homogeneous FIA. These assays are based on changes in fluorescence properties upon ligand—antibody binding. T h e properties t h a t have been studied to date are fluorescence excitation transfer (5), polarization (6-8), enhancement (9), and quench ing (10). In addition, conjugates have been used as labels which depend on additional enzymatic reactions (7715). Fluorescence excitation transfer (PET) requires a fluorescent molecule (donor) which fluoresces in the same spectral region in which a second fluo rescent molecule (acceptor) absorbs. T h e efficiency of transfer will depend on the overlap of donor emission with acceptor absorption and the spatial separation of the two molecules. In the application of F E T immunoassay to the analysis of morphine, two variants were examined. In the first approach, fluorescein-labeled ligand was used as the donor and tetramethylrhodaminelabeled antibody as the acceptor. This reaction may be written: [L] + [L
