Journal oflmmunological Methods, 150 (1992) 91-97
© 1902 ElsevierScience Publishers B.V. All rights reserved 0022-1759/92/$05.00
Homogeneous enzyme immunoassay Sarah H. Jenkins Pathology and Lab Medicine, Universityof Cincinnati, Cincinnati, Ot145267.0714, USA
(Accepted 12 February 19921 Key words: Enzyme immunoassay; Enzyme-multiplied immunoassay technique; Substrate-labeled fluorescence immunoassay;
Apoenzyme reactivation immunoassay;Cofaetor-labeled;Inhibitor-labeled
Homogeneous enzyme immunoassays
Homo~'~:icous immunoassay is defined as an immunoassay system in which both antigen-antibody reaction and the measurement of the extent of the reaction are performed in solution without separation of the free and antibody-bound components (Khanna, 1991). This allows for simple and fast assays that are easily automatable for use in clinical labs. Homogeneous immuooassays are necessarily competitive in nature and can be described by the following equation:
Enzyme-multiplied immunoassay technique
Ag+Ag* +Ab :---"Ag:Ab+ Ag* :Ab A competition is set up between the unlabeled analyte (Ag in the sample) and labeled analyte (Ag*) for antibody binding sites by utilizing a limited amount of antibody. Either free label or antibody-bound label can be measured. When the distinction between bound and free label cannot be made a separation step is required. But in the case of homogeneous assays, no separation is required because the signal generated by the label is somehow moderated by the antibody binding so that bound and free label can be distinguished. Correspondence to: 5.H. Jenkins, Pathology and Lab Medicine, University of Cincinnati, Cincinnati, OH 452670714, USA.
The first homogeneous immunoassay developed utilized an enzyme labeled in assays for low-molecular weight ligands. This type of immunoassay is known as enzyme-multiplied immunoassay technique or EMIT. A typical EMIT
. VV V SampleHapten Inactive Enzyme ~"i" ~
Fig. I. Principle of homogeneous enzyme immunoassay (enzyme-multipliedimmunoassaytechnique, EMITJ.
format is shown in Fig. 1. in this type of assay, the binding of enzyme-labeled ligand to antibody results in a change in the enzymatic activity observed. Most commonly a decrease in activity is observed. The mechanism of inhibition is thought to be due to steric hinderance, caused by the antibody physically blocking the substrate binding site of the enzyme (Chan, 1978). In some cases, the inhibition of certain enzymes can be due to conformational changes within the enzyme induced by antibody binding (Samuels, 1963). A typical dose-response curve for this type of homogcneous assay is shown in Fig. 2. The labeled ligand and unlabeled ligand compete for a limited number of antibody binding sites so that as the concentration of ligand in the samples increases the concentration of free labeled ligand left at equilibrium increases. This leads to an increase in
Fig. 2. Typical dose-response curve for an enzyme-multiplied immunoassaytechnique (EMIT). signal or enzyme activity with increasing dose as seen in Fig. 2. The dose-response curve eventually reaches a maximum when all labeled ligand is
Fluorescent Product Fig. 3. Principleof a substrate-labeled fluorescence imm~noassay(SLFIA).
in the free form (or all antibody is bound by the sample ligand) and therefore exhibits maximum enzyme activity. Detection limits are on the order of 10 -9 mol/I for haptens. There has been very limited applicability of this type of format to protein analytes and success has been achieved only with the use of macromolecular enzyme substrates (Gibbons et al., 1980; Armenta et al., 1985). It is possible to observe an increase in enzymatic activity upon antibody binding. An example of this is a thyroxine (T 4) homogeneous immunoassay. In this case, the conjugated ligand is the inhibited form of t t e enzyme, malate dehydrogenase, and the binding of antibody results in the reversal of enzyme inhibition. Thus free thyroxine in the sample prevents the inhibition reversal so that the enzymatic activity is reduced as
the concentration of thyroxine in the sample increases (Thompson, 1989).
Substrate-labeled fluorescence irnmunoassay The substrate-labeled fluorescence immunoassay (SLF1A) is based upon the principle shown in Fig. 3. The hapten is labeled with a fluorogenic enzyme substrate, [J-galactosyl umbelliferone. While bound to the hapten, /3-galactosyl umbelliferone is not fluorescent but is hydrolyzed by the enzyme, /3-ga!actosidase, to a fluorescent product, umbelliferone. Thtts in the absence of hapten in the sample, the substrate-!~d.Jeled hapten is bound by antibody, sterically restricting the enzyme's access to its substrate and no fluorescent product will be generated. As the concentration of bapten in the sample increases, the subs':ate-labeled hapten is displaced from the anti-
III SampleHapten W
A0ymej . ~
,,~ Active ' ~
Fig. 4. Principleof a prosthetic group-labeled immunoassay(PGLIA)or apoenzymc reactivationimmunoassay(ARIA;.
limits are on the order of 10 -t' mol/I for haptens and 10 -5 mol/l for i~roteins. This particular scheme has al.~o been appiied to measurement of haptens using chemiluminescent labels (Shroeder et al., 1976).
body thus freeing substrate for hydrolysis by the enzyme and fluorescent product will be generated. The enzymatic product is monitored as kinetic or endpoint measurements. This type of a scheme has a limited detection limit when compared to other enzyme immunoassays in that it lacks the amplification feature that most enzyme labels provide. The detection limit is dependent upon the detection of the fluorescent product. Despite, the not-so-low detection limit of these assays, the method has been applied to assays for aminoglycosides and anticonvulsant drugs as well as some serum proteins (Wong et al., 1979; Ngo et al., 1980). Detection
/l HAD Cofact0r-labeledHapten It/HAD
Prosthetic group-labeled immunoassay Prosthetic group-labeled immunoassays (PGLIA) are also known as apoenzyme reactivation immunoassay (ARIA). This variation is based on the reactivation of the enzyme, glucose oxidase, upon interaction with its prosthetic group, FAD ~ (Morris et al., 1981). The assay format is shown in Fig. 4. The hapten labeled with the
"1- II III SampleHapten HAD +
~+(~ u,,HAD V
Fig. 5. Principleof a cofactor-labeledimmunoassay.
prosthetic group competes with the unlabeled hapten for antibody. The remaining unbound labeled hapten will then combine with the inactive apoenzyme, forming an active enzyme, thus reactivating the en~,'matic reaction. Therefore, as the concentration of hapten in the sample increases, the enzyme activity also increases. Glucose oxidase is an excellent enzyme for this type of application because the apoenzyme is completely inactive and stable, yet can be easily reactivated upon combination with FAD. Detection limits are in the order of 10 -s -- 10 -9 mol/I for haptens, such as drugs.
Cofa~io,'.lcb."!ed immunoassay The cofactor-labeled homogeneou~ immonoassays are very similar to the ARIA. In the case
presented in Fig. 5, the NAD-labeled hapten participates in an enzyme cycling reaction when present in the free form (Carrico et ul., !976). If the NAD labeled hapten is bound by antibody it is no longer able to participate in these reactions so that enzyme-generated product, that is determined, decreases. As in ARIA the signal measured is directly proportional to the concentration of the hapten in the sample. This scheme is unique, though, in that there is significantly more amplification associated with enzyme cycling and theoretically lower detection limits could be obtained. In practice, however, there are significant and variable amounts of NAD present in serum making it necessary to limit the amount of sample serum utilized in these types of assays which consequently limits the dctecfi6n !imits obtainable.
~ Inhibit0r-labeled Hapten 7 ~
III qP SampleHapten
Active Inactive ' ~
~ l l InhibitedEnzyme
Fig. 6. Principleof an inhibitor-labeledimmunoassay.
hlhibitor-labeh'd immunt,assay Irreversible inhibitors have also been used in the development of homogeneous enzyme immunoassays. Modulation of enzyme activity, is achieved through the use of an irreversible inhibitor attached to the hapten. If the labeled hapten is bound by antibody the complex is unable to inhibit the enzyme. Conversely, if the labeled hapten is unbound by the antibody at equilibrium, it is able to bind to the e.,',zyme and inhibit enzymatic activity. As ~he concentration of the hapten anaiytc in the sample increases, more labeled hapten will be in the unbound form and thus the enzymatic activity will decrease. Detection limits are in the order of 10-7-10 -8 mol/l for haptens. The format for this type of homogeneous assay is shown in Fig. 6. Clotted enzyme donor hnmunoassay A relatively newly introduced homogeneous enzyme immunoassay is the cloned enzyme donor immunoassay (CEDIA). This is a fascinating
technology in that it merges the recombinant DNA technology and immunoassay technology (Henderson et al., 1986). It basically involves the generation of two fragments of the enzyme, /3galactosidase, neither of which alone has enzyme activity. When mixed together the two fragments recombine to form an active enzyme (a process called complementation) with the specificity and substrate turn over associated with natural /3galactosidase. The first of these enzyme fragments is termed the enzyme donor fragment (ED) which is a small polypeptide fragment of 70-90 amino acids. The second fragment, the enzyme acceptor fragment (EA), is a larger protein that contains about 97% of the native amino acid sequence. The assay format is shown in Fig. 7. The hapten is attached to the ED fragment (labeled hapten) in such a way so that the activity after complementation is virtually unchanged. If the hapten-ED component is bound by antibody, the complementation process cannot occur. As the
SampleHapten +ll I EAM. . . . . .
Fig.7. Principleof the clonedenzymedonorimmunoassay(CED1A).
c o n c e n t r a t i o n of h a p t e n in t h e s a m p l e increases, t h e h a p t e n - E D is available to c o m b i n e with t h e E A f r a g m e n t , resulting in an active e n z y m e a n d an increase in signal. T h i s is a particularly interesting assay f o r m a t in that by careful choice o f h a p t e n - E A a n d E D pairs in addition to careful s c r e e n i n g o f antibodies h a s resulted in a linear r e s p o n s e curve in contrast to t h e typical sigmoidal r e s p o n s e curves o f o t h e r h o m o g e n e o u s e n z y m e i m m u n o a s s a y s . T h e C E D I A assay is also t h e m o s t sensitive o f commercially available hom o g e n e o u s e n z y m e i m m u n o a s s a y s with limits o f d e t e c t i o n b e i n g o n t h e o r d e r of 10 - H m o l / I o f hapten.
Advantages and limitations B e c a u s e t h e e n z y m e i m m u n o a s s a y s described above do n o t r e q u i r e a s e p a r a t i o n step, they a r e mt~re rapidly a n d conveniently p e r f o r m e d as well as e:asily a d a p t a b l e to m o d e r n a u t o m a t e d analyzers. H e t e r o g e n e o u s i m m u n o a s s a y s c a n now be a u t o m a t e d b u t typically require special imm u n o a s s a y analyzers w h e r e a s h o m o g e n e o u s assays c a n be a c c o m m o d a t e d by any g e n e r a l a n a lyzer c a p a b l e o f t h e basic visible, U V , or fluorescence measurements. T h e lack of a s e p a r a t i o n step c a n also be a limitation. If n o s e p a r a t i o n is m a d e , all t h e c o m m o n i n t e r f e r e n c e s of s a m p l e m a t r i c e s will be p r e s e n t a n d c a n limit t h e sensitivity o f s u c h assays. D e t e r m i n a t i o n o f t h e e n z y m a t i c activity in a kinetic m o d e r e d u c e s t h e effect o f e n d o g e n o u s s a m p l e i n t e r f e r e n c e a n d b a c k g r o u n d noise. T h e o retically, t h e m o r e sensitive h o m o g e n e o u s imm u n o a s s a y s are t h o s e t h a t rely o n a n increase r a t h e r t h a n a d e c r e a s e in t h e m e a s u r e d signal with increasing analyte c o n c e n t r a t i o n . Currently, h o m o g e n e o u s e n z y m e i m m u n o a s says a r e also be limited to only s m a l l e r h a p t e n s b e c a u s e t h e binding o f antibody m u s t c a u s e a c h a n g e in e n z y m a t i c activity. If t h e antibody directly c a u s e s a c h a n g e in e n z y m a t i c activity by sterically h i n d e r i n g t h e s u b s t r a t e b i n d i n g site of
the e n z y m e label, t h e n t h e label m u s t be small e ~ o u g h to bind t h e antibody while also allowing it to block s u b s t r a t e access to the enzyme. T h e homogeneous enzyme immunoassay has had a major impact on t h e ability of hospital laboratories to m o n i t o r low m o l e c u l a r weight analytes such as d r u g s a n d h o r m o n e s . F u t u r e developm e n t s are ~,nticipated which will increase t h e sensitivity a n d reliat~ility a n d p e r h a p s lead to the design of an assay capable of m e a s u r i n g proteins.
References Armenia, R.. Tarnowski, T., Gibbons, I., et al. (1985) Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate. Anal. Biochem. 146, 211. Carrico, R.J., Christner, J.E. and Bogulaski, R.C. (1976) A method for monitoring specific binding reactions with cofactor ligands. Anal. Biochem. 72, 271. Chan, D.W. (Ed.) (1987) immunoassay: A Practical Guide. Academic Press, Orlando, FL, pp. 18-21. Gibbons, !., Skold, C., Rowley, G.L., et al. (1980) Homogeneous enzyme immunoassay for proteins employing flgalactosidase. Anal. Biochem. 102, 167. Henderson, D.R., Friedman, S.B., Harris, J.B., et al. (1986) 'CEDIA', a new homogeneous immunoassay system. Clin. Chem. 32, 1637. Khanna, P. (1991) In: C.P. Price and D.J. Newman (Eds.), Principles and Practice of Immunoassay. Stockton Press, New York, pp. 326-364. Morris, D.L., Ellis, P.B., Carrico, R.J., et al. (1981) Flavin adenine dinucleotide as a label in homogeneous colorimettic immunoassays. Anal. Chem. 53, 658. Ngo, T.T., Carrico, R.J., Boguslaski, R.C., et al. (1981) Homogeneous substrate-labeled fluorescent immunoassay for lgG in human serum. J. Immunol. Methods 42, 93. Samuels, A. (1963) Immunoenzymology reaction processes, kinetics and the role of conformational alteration. Ann. NY Acad. Sci. 103, 858. Shroeder, H.R., Vorelhut, P.O., Carrieo. R.J., et al. (1976) Competitive protein binding assay for biotin monitored by chemiluminescence. Anal. Chem. 48, 1933. Thompson, S.G. (1989) L.A. Kaplan alld A.J. Pesce (Eds.), Clinical Chemistry: Theory, Analysis, and Correlation, 2rid edn. Mosby, St. Louis, MO, pp. 191-206. Wong, R.C., Burd, J.F., Carrico. R.J., et al. (1979) Substratelabeled fluorescent immunoassay for phenytoin in human serum. Clin. Chem. 25, 1402.