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H K321 91 068 ANALYTICAL ALTERNATIVES TO RADIOIMMUNOASSAY: A REV IE6V A. Castro, N. Monji, W. Voigt and Ii. Malkus Department of Pathology Hormone Research Laboratory University of Miami, School of Medicine Miami, Florida 71I3STRACT • Radioimmunoassay has been used increasingly in recent years as an analytical technique for the detection of minute.quantities of substances in numerous body fluids.' It is high ly sensitive and specific. This procedure, however, has several disadvantages, such as radioactive hazard, waste disposal, licensing, FDA regulations over instruments and reagent production, and the short shelf life of reagents. Thus we have reviewed analytical alternatives to radioimmunoassay to consider the possibility for the development of methods capable of performing quantitative assays which will. detect level of substances with less than 10-9g/ml. In all immunoassay techniques reviewed, the use of radioactive material is avoided. CTR MN C0\FIpENTIAL- MINNE-93TA TOBACCO LITIGATION

H K021 91 069 INTROI)UC'TION During the past few years, radioimmunoassay has become one of the most rapidly growing analytical techniques used in the clinical laboratory for the detection of minute quantities of steroids, hormones, peptides, drugs, and other substances 'in body fluids (Anderson and Breillatt 1971; Belanger et al, 1976; Castro, 1974; Castro 1975; Castro et al, 1974a; Castro et al, 1974b; Castro et al, 1973a; Castro et al 1973bs Castro et al, 1974c; Castro and Prieto, 1975; Castro et al, 1977). This technique has proven to be highly sensitive and specific due mainly to the homogeneous antibody. Also, the use of gamma-emitting isotopes for labeling (i.e., 125 I) renders detection relatively simple. FIowever, these labeled reagents have several disadvantages, such as short shelf life, radioactive hazard, expensive equipment, and inefficiency in the total number of disintegrations measured. Analytical alternatives to radioimmunoassay -tacr detection method, light scattering and slide-based techniques and isodensity analyses - have been thus studied to determine the feasibility of developing new approaches for the measurement of minute quantities of substances;which could be free from the above mentioned restric- tions. Such.methods urine. cerebrospinal -9 of 10 g/mh,-:Plasma significant trace -:~.: vitamins, tllerapeutic Castro, 1973a=;^ Castro et at. 1972) could perform quantitative assays on serum, fluid and/or saliva for substances at levels and serum antigens considered to be clinically levels include endocrine hormones, steroids, drugs; and drugs of abuse (Castro, 1974; and Prieto, 1975; Cheng et al, 1973; Chung 2 CTR M CDNFTDENTIAL ~ MZNhiE-MTA TC1Z3ACCQ LITxGATION

N K321 91 070 Most chemical analyzers presently in use are based on spectro- photometry (colorimetry or fluorometry) and have a measurement range of 10-6 to 10-9g/ml in concentrations. Several automated immunoassays have been developed for the measurement of various proteins in which the antigen-antibody complexes are determined by their light scattering ability. Many non-radioactive labels, such as enzymes (Castro et al, 1977), coenzymes, proteins, particles, bacteriophages, and fluorogenic compounds, are also used. These labels have'a longer shelf-life and less complex end point detector than the isotope labels. Also, some of these assays do not require separation of antibody bound and free fractions. Other direct measurement techniques (i.e., gas chromatography, liquid chromatography, differential pulse polarography, etc.) lack specificity for the detection of trace quantities of substances (steroids, hormones, etc.). On the other hand, indirect measurement procedures using the antibody-antigen (tag) reaction are highly specific and it now appears that any type of molecule (hapten) can be attached or tagged to a protein carrier or synthetic polypeptide (inununogen) and used to induce in animals antibodies specific for the antigen determinant group. These determinants include sugars, peptides, steroids, drugs, purines, pyrimidines, etc. In the present review on immunoassays all the techniques employing radioactive material are avoided. This advantage eliminates not only the radioactive hazard to laboratory personnel, the psychological implication involved and the problems of waste disposal, but also eliminates licensing, strict F.D.A. regulations on the manufacture of. the instruments and reagents, and the short shelf life of reagents. - G.T_R IrtN CDNF4TXNTI'a. : M14ESCtTA MfiACC0.L.I'LT.GAT11CN

A. Tag Detection W32 19 1O71 A substance or substances other than that being measured sometimes interferes and reduces the limits of detection of a given method. Thus, only new immunoassay methods which are highly specific are outlined herein. i. Electrochemistry - Although there are many variations of the polarographic chemistry technique (Schneider and Sehon, 1961), it essentially involves electrolysis of an electrolyte solution in a fine bore glass capillary cell using a dropping mercury electrode and a non-polarizable electrode. From the resulting current-voltage characteristic curves, or polarograms, the identity and concentration of the electro-oxidizable or -reducible substances present are determined. As many as six substances can be identified from a single polarogram with detection limits in the ppb range. However, sensitivity is low, about 10-sM." Oxygen interferes with polarographic analysis; however, if the quantity of 02 in the sample is known, the characteristic half-voltage parameter can be correlated to a well-defined substance concentration. Since the antibody bound hapten is polarographically inactive, free concentration of a polarographically active hapten can be determined. With this technique, separation is eliminated in the characteri- zation of the antibody-hapten interaction. Good agreement between polarographic determination and conventional quantitative precipitation has been shown (Zikan, 1966). Cost of equipment is low although advanced methodology is necessary to lower the limits of detection. 4 LTR M a2wFIDENrLQt :r PITNNESQTA. TLI6'ACCQ LITI+CATILiN

N K3 21 91 072 ii. Spin hlagnet.ic Resonancn •- Spectrometers, or spin resonance instruments are capable of measuring several parameters related to the precision of magnetic subatomic particles about an applied steady magnetic field. "Free" electrons, protons and nuclei have not only an intrinsic spin magnetic moment but mechanical spin angular momentum around the magnetic axis. Electron spin resonance (ESR) has been applied to drug screening. In this method, known as FRAT (free radical assay technique), replacement of spin-labeled analogues of hapten bound to an antibody produced against the hapten with unlabeled (free) hapten results in immediate increase of characteristic ESR triplet signal to yield the free hapten concentration in the sample (Leute et al, 1972). Reducing detection limits by ESR, however, mentation costs. will increase instru- iii. blagnetome-'try -*In this method, either the magnetic moment (magnetization/uhit sample volume) or the magnetic susceptibility (magnetization/magnetic field) is measured. Iron or any molecule with a permanent magnetic dipole moment could be used as the tag. With magnetometry, antigens or antibodies are tagged with a magnetic label. After separation of bound and free fractions the concentration of antibody bound..magnetic tags is determined in a magnetometer through a calibration curve of concentration vs. magnetization (Williams and Chase, 1971). iv. P'luorometry - There are several variations of this method: a. An.antigen is labeled with a fluorochrome and a procedure similar to radioimmunoassay (competition, separation, measurement) is followed. A fluorometer is used for measurement. 5 CTR W .CONF'...MEh1T1'FL' MTNNESOTA TOEAGGO LITTMTIDN

H K321 91 073 an<1 sample level is calibrated ng:iinst fluorescence intensity. Intensity measurements, however, are unreliable (Cukor et al, 197G). b. When the antibody is bound, fluorescence quenching of a fluorophored antigen occurs. This method also can be undependable in its intensity measurements due to•quenching reactions (Ullman et al, 1976). c. A method of fluorescent polarization is used. The assay principle is similar to the FRAT. When fluorochrome labeled antigen or hapten is attached to a large antibody molecule, polarization of fluorescence results. Replacement of labeled antigen or hapten with unlabeled one in antibody bindinc, sites results in inhibition of polarization (Dandliker and Saussure. 1970). The sensitivity of this assay so far is similar to other currently available fluorometric technique. As the change in molecular tumbling is dependent upon the difference in molecular weight between the bound and unbound fluorophore the method is limited to compound of molecular weight of 20,000 or less. v. Enzyme Immunoassay Techniques - Assay procedures using antigens, haptens or antibodies labeled with an enzyme have heen applied to the measurement of substances in biological'fluids. These enzyme immunoassays (EIAs) are specific and highly sensitive for the identification and measurement of various substances. The assays are relatively simple and inexpensive. Extensive reviews on this subject have been reported (Wisdom et al, 1976; Schuurs and van Weemen, 1977). Although, EIAs are as sensitive comparable radioimmunoassay, labeling and conjugation are complicated. They are more susceptible to interference than 6 as CTR M CDh1F".3DWMq...= -MIhlNESOTA TOBACCO LITIGATILIN

H i(321 91 074 other immunoassays. On the othoi- hand, all clinical chemistry laboratories have the equipment necessary for enzyme determinations; no special radioactive materials and handling procedures are required and the photometric detection can be easily automated. There are several types of EIA. a. Competitive EIA for antigen. Labeled and unlabeled antigens compete for binding sites on a limited number of antibodies. Solid phase or specific second antibody is used for separation. Enzyme activity is determined and related to the concentration of the unlabeled antigen (Engvall et al, 1971). This procedure is similar to the classical Yalow and Berson (1960) radioinununo- assay. In the sequential saturation variation the labeled •antigen is added after antigen-antibody binding takes place (Castro, 1975). This is analogous to the sequential radio- immunoassay (Hales and Ran31e, 1963). b. "Imrr.unoenzymometric" assay for antigen. In this method, antigen reacts with excess labeled antibody. Excess solid phase antigen is added after incubation. The solid phase antigen and the free labeled antibody remaining react. After separation of the solid phase, enzyme activity associated with soluble antigen is measured and related to antigen concentration (Maiolini et al, 1975). c. "Sandwich" EIA for antigen. In this method the antigen has at least two binding sites. Antigen reacts first with excess solid phase antibody. Fllowing incubation and washing, the bound antigei, is treated with excess labeled antibody. 7 CT!? Mh1_LCtWICEPtTIAL. MINh1E'SOTA TCOACC:O LITICATICN

N K321 91J75 After another washing the bound l abel is assayed providing a direcL quantitati.ve measure of the antigen present (Maiolini and Masseyeff, i975)'. d. EIA for antibody. Antibody to be quantitated is first bound with excess solid phase antigen. After incubation and washing, labeled second antibody specific for the first antibody is added. Following further washing the bound label is assayed. This provides a direct measurement of the amount of specific antibody present (Kato et al, 1976). e. Homogeneous EIA for haptens. In this EIA, separation of bound and free label is not required as the technique depends on inhibition or activation of the enzyme label by antibody binding. In the first of this type of assay, a morphine der.ivative was attached to lysozyme (Rubenstein et al, 1972). In a later assay, phenobarbital derivative was attached to glucose 6-phosphate dehydrogenase (Rowley et al, 1975): f. Homogeneous reactant-labeled fluorescent immunoassay. In this system, no conjugation of hapten to enzyme is needed. Instead the hapten is covalently linked to fluorescent dye/enzyme substrate conjugate. This hapten/fluorescent dye conjugate is non-fluorescent under assay conditions and react with the enzyme to yield fluorescent products. When the hapten/dye conjugate. reacts with an antibody produced against the hapten, a complex forms that prevents interaction of the bound conjugates with the enzyme. In competitive binding reactions, hapten/dye conjugate displaced by non-conjugated hapten in the sample reacts with the 8 CTR MN CDh1UMNUMV.. MINN~SOTA TOBAC,CO LITTGATIOh!

N K321 91 076 enzyme to give fluorescence which yield estimation of the hapten concentration in the sample (Watson et al, 1976; Burd et al, 1971). g. Other. EIAs. Three more EIAs for antibodies have been r.eported. (Schuurs and van Weemen, 1977). In one of these methods, labeled and unlabeled antibodies compete for solid phase antigen. In the second method, labeled antigen reacts with it. In both methods the quantity of antibody is determined by measuring enzyme activity in the free.and bound fractions after centrifugation. In the third method, homogeneous EIA technique is employed using phospholipase.C as a labeled enzyme and erythrocytes as substrates (Wei and Riebe, 1977). vi. Viroimmunoassay - The principle of this assay uses the phenomena that bacteriophage-anti-phage complexes cannot adsorb onto bacteria or inject their nucleic acid to produce plaques on a layer of agar seeded with bacteria. In the assay, various antigens or haptens were attached chemically to bacteriophage; "anti-antigen" sera to neutralize the infectivity of the coupled phages before subjecting to the plaque assay. The inhibition of the neutralii"ation of the antigen-bacteriophage conjugate with free antigens;was used as an antigen quantitation assay (Iiaimovich et al, 19704Mray et al, 1975). This ass=a.y system was shown to be very sensitive but both the use of microb;iological assay systems and a long incubation period make this approach of immunoassay rather unattractive to many immunoassayists. CTR MN COhlFIaENTTAL- MINNESt?TA TC1BACCLC.LIT7GATI0hJ

I3. Light Scattering Technique_; Nlt02191077 In this.section, several optical. techniques which depend on the operational principles of electromagnetic scattering theory are discussed. These procedures monitor particle size or steric changes which accompany reactive association of antigens and antibodies rather than utilizing labels. i. Ellipsometry - It involves the measurement of the effect of reflection on polarized light and is used to determine the thickness and refractive index of thin films with several angstrom in thickness on solid surfaces and to measure optical constants of reflecting surfaces. The ellipsometer has been used to detect immunochemical reactions at liquid-solid interfaces (Mathot and Rothen, 1969). It was found that if a properly polarized electric current (ti300ljamp),was applied during the antigen and antibody adsorption periods, a clear differentiation was obtained between homologous and heterologous sera. This current served to induce a substantial concentration of antigens and antibodies near the charged surface, at the same time reducing or preventing nonspecific material adsorption of charge opposite that of the antibody molecules. Research with this technique has strongly suggested that it is capable of detecting levels much lower than any other techniques (Rothen and Plathot, 1971). ii. "Dip Slide" Assay - in this procedure a discontinuous surface of.indium is coated onto a glass s.lide to which a monomolecular layer of antigen is adsorbed. The presence of homologous antibody is confirmed if, when the slide is dipped 10 , CTR MN GOWS4ENU6,,.:_,.WNAIE'SC1TA TL]EACt:O LITICATIC?N