Chemical measurements based on immune reactions have been used by investigators for many years to accurately and precisely determine protein in test samples. These immunochemical techniques have often been applied in situations where substances were considered extremely difficult to detect. Immunoassay systems are often quick, easy, and sensitive, with detection limits usually within the nanogram to picogram range. Using enzymes in immunoassays has helped them to become even more economical, very reliable, safe, simple, and quite versatile. Enzyme immunoassay (EIA) has resulted from a long line of advances in immunology, protein chemistry, and enzymology. EIA techniques generally involve labeling an antibody or antigen with an enzyme such as peroxidase and then measuring enzyme activity inhibition with immunochemical reactions. Fluorescein labeling of antibodies by Coons in 1942 was the starting point in the history of EIA. Improvements in protein isolation and purification, together with the availability of protein-coupling reagents, helped to advance EIA development. In 1960 Singer and Schick were among the first investigators to successfully couple two protein molecules without causing disruption of their biological and chemical activity. Enzyme labeling actually became practical when glutaraldehyde was used by Avrameas in 1969 for the coupling of peroxidase to antibodies. Once this method became available, antibodies conjugated with enzymes could easily he used for locating and identifying specific tissue antigens. Around the same time. Miles and 920A
Hales showed how enzymes could be used instead of isotopes in an immunometric assay. The great need for early and accurate diagnosis of disease also prompted further development of immunodiagnostic methods. Radioimmunoassay (RIA) and fluorescent immunoassay (FIA) have long been of great value in measuring extremely low levels of hormones in body fluids and for identification of certain infectious diseases. Both methods, however, have disadvantages that prompted investigators to seek alternative labels (such as enzymes) in immunoassays. In 1971 Engvall and Perlmann (I, 2) first described an EIA technique in which the analyte to be detected binds either to an antigen or to an antibody coating a solid surface. Unbound molecules are then readily washed free from the solid surface to allow measurement of adsorbed enzyme conjugate. They coined the term enzyme-linked immunosorbent assay (ELISA) to describe an immunoassay using enzyme-labeled antigens, antibodies, or haptens. EIA requiring several washings and steps to separate the bound from the free enzyme label is referred to as heterogeneous immunoassay. When such separation steps are not required after mixing reagents, the EIA is a homogeneous assay. In 1972 Rubenstein (3) developed a new homogeneous EIA in which competitive binding occurs between the analyte and enzyme-conjugated antigen. This method, called the
ANALYTICAL CHEMISTRY, VOL. 56. NO. 8. JULY 1984
enzyme-multiplied immunoassay technique (EMIT) has gained widespread use for rapidly assaying small molecules such as hormones or drugs in body fluids. Although still in its developmental stage, EIA has been found to be a simple, economical alternative to standard immunochemical methods (Table I). Both EMIT and ELISA are versatile methodologies designed to measure microamounts of substances in test samples. A comparison of EMIT and ELISA is shown in Table 11.Combining the specificity and sensitivity of immunoassay with the convenience, speed, and reproducibility of enzyme measurements, both EMIT and ELISA offer efficient technologies that are extremely useful in routine analytical determinations. Many industrial, agricultural, environmental, and medical EIA applications are now available. Basic Concepts EIAs offer an important method of detecting antigens or antibodies in samples tested. Antibodies are proteins called immunoglobulins produced in the animal body to neutralize and help destroy invading foreign substances known as antigens. Each antibody molecule has specific binding sites for certain antigenic determi-
0003-2700/84/0351-920A$01S O / O @ 1984 American Chemical Society
-
Report Dan Monroe
Department of Medicine InfectiousDiseases and Connective Tissues Sections University of Tennessee Center for the Health Sciences Memphis, Tenn. 38163
1
Table 1. Immunoassay Comparison f IA
RIA
Sensitivity Spcilicily
High (4-W) High
Speed
Days Shwt shelf life Scintillation or gamma cwnter Skilled with license $7/test
Reagents Equipment required Personnel cost
EM
High (ng-w) High (ng-pg) High High Days Hours Reasonable shelf life Long shelf life
w monitor Skilled $5/test
.
SpectrophOtometer Minimal training 02ltest
Table II. EIA Comparison EUSA
Heterogeneous assay Reagent separation required (centriluaation or filtration) Reagent washings required Slower than EMIT Sensitivity greater than EMIT Macromolecules measured (antigens, antibodies) For diagnosing lnlectious diseases; immunoglobulins Solid phase assay
EYll
Homogeneous assay Reagent separation not required Step washings not required Faster than ELlSA Sensitivity less man ELlSA M e w e s small molecules (haptens) For drug, hormone, metabolite determinations Liquid phase assay
nants found on the surface of an antigen. The unique molecular configurations of these antigenic determinants and their corresponding type-specific antibodies are the basis for the specificity of immunoassays. Invasive antigens include such materials as viruses, bacteria, proteins, or any high-molecular-weight substance considered foreign to the animal body. All immunoassays are directly dependent on the immune complex bond formation that enables an antigen and antibody to fit together hand-in-glove. Low-molecular-weight compounds referred to as haptens (partial antigens) can also react with type-specific antibodies once these are produced. However, haptens by themselves are too small in molecular size to elicit antibody production. Only when haptens are attached to a larger molecule or a macromolecular carrier such as bovine serum albumin can they stimulate antibody formation. EMIT: Bask Principles EMIT has been widely used for rapidly assaying microamounts of drugs and substances in human biological fluids. The key elements in an EMIT reaction are the compound to he measured, enzyme-laheled molecules of that compound, a specific antibody that binds the compound, and a spe-
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
921 A
cific enzyme chromogenic substrate that is initially colorless, but that forms color when converted bv the enzyme conjugate.
a F l
Compound to be Antibody specific measured (hapten) for compound being measured
fl E
Enzyme-labeled compound
0 Enzyme-specifc chromogenic substrate
T w o basic test principles are involved in the EMIT assay: (1)The enzyme must retain enzyme activity after hapten or compound conjugation.
E
time is determined by the amount of free enzyme left in the mixture. This results in a color change that is easy to measure spectrophotometrically. The specimen drug concentration is quickly measured by comparing the sample’s rate of change of absorbance to that of a set of known standards.A high drug concentration in the patient sample causes many antibody sites to he covered, leaving more enzyme-laheled drug unbound and able to convert more substrate for higher absorbance readings. Less drug in the patient sample allows more enzyme-labeled drug to bind the antibody, resulting in leas enzyme activity and consequently lower absorbance readings. Inactivation of the enzyme label when the hapten-enzyme complex is antibodybound makes the EMIT asnay a unique system, enabling the test to be performed without separation of bound from unbound compounds, as is necessary with other immunoassay methods.
bound antibody, and consequently antigen present, in the speximen tested. Another widely used type of ELISA is the competitive assay for antigen detection. The test specimen containing the antigen to he determined is mixed with a precise amount of enzyme-labeled antigen and both compete equally for a limited number of binding sites on an antibody adsorbed
-+A=d
Enzyme
Hapten Hapten-enzyme conjugate (Actiue enzyme)
(2) The enzyme activity of the hap-
ten-enzyme conjugate is reduced or inhibited when the hapten rea& with ita specific antibody. (Active enzyme)
9P 3 = E
+
Hapten-enzyme conjugate
Antibody
El
Antibody-bound hapten-enzyme conjugate (Inactive enzyme)
A typical therapeutic EMIT drug w a y hegins by adding to a patient specimen an excess of specific antibodies that will bind to the drug being measured. If drug molecules are present, they immediately hind to antibody sites. The enzyme-labeled drug is then added to the mixture. Antibody binding sites not occupied by molecules of the drug in the specimen are immediately filled with molecules of the added enzyme-labeled drug. Enzyme activity is then reduced because only free enzyme-labeled drug can act on the substrate. The amount of substrate converted from a colorless 9221
to a colored form in a given period of
ELSA Baslc Principles ELISA links soluble antigens to insoluble antibodies or soluble antibodies to solid phase antigens in a manner that allows both immunological and enzymatic activity to be retained. Both a competitive and a double antibody sandwich ELISA technique are available for performing antigen measurements, while antibodies can be quantitated by an indirect ELISA method. The “sandwich” technique is so called because the antigen being assayed is held between two different antibodies, one containingthe enzyme tag (Figure 1). In thii method, the inner surface of a polystyrene tube is first coated with a solid phase antibody. The test specimen containing the antigen to be measured is then added and allowed to react with the bound antibody. Any unbound antigen is washed away. A known amount of enzyme-labeled antibody (produced in an animal species different from that of the bound antibody) is next allowed to react with the bound antigen. A different set of exposed antigenic determinanta, which are not covered by the solid phase antibody bond, are involved in this enzyme-conjugated antibody reaction. Any exceas unbound enzyme-linked antibody is washed away after the reaction. Unlike EMIT, bound enzyme conjugate remains enzymatically active in the ELISA system. When substrate is added, it is quickly acted upon by the enzyme, resulting in a color change, the intensity of which is determined in a fued time period. The amount of visual color change is a direct measurement of specific enzyme-conjugated
ANALYTCM CHEMISTRY. VOL. 58. NO. 8, JULY 1984
(2) Enzyme conjugate added to well with bound antigen-antibody or
immune complex.
(3) Substrateadd$ to enzyme conjugate bound to immune complex.
t [4) Positii test or color change
denoting changed substrate due to presence of enzyme conjugate hound
lo immune complex.
,
rwn 1. wwie antibody “sandwich ELISA for antigen measurement
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to the inner surface of a polystyrene tube (Figure 2). Excess free enzymelabeled antigen is separated from that bound by washing before the substrate is added. The key element in this competitive situation is the amount of color intensity resulting from substrate addition. The ELISA antibody assay is very similar to the sandwich antigen technique except that antigen, instead of antibody, is adsorbed to the inner well or tube surface (Figure 3). The test specimen containing the antibody to be measured is then added and allowed to bind to the attached antigen. Unbound antibody is removed by several washings, after which enzymelabeled antiglobulin is added. An enzyme substrate is then added after any
unbound conjugate has been washed away. The bound enzyme conjugate causes a color change to take place by acting upon the substrate. This reaction can be stopped, and the amount of color change is then an indirect measure of specific antibody present in the test specimen. Conjugation The ease with which enzymes can he chemically bound to antibodies and antigens bas made possible protein conjugates that possess high enzymatic and immunological activity. Many mono-, bi-, or multifunctional reagents have been used for coupling enzymes to proteins ( 4 ) . However, protein conjugation using glutaraldehyde as the cross-linking reagent is proba-
bly the most widely used and best understood method. Conjugates are easily prepared by irreversibly cross-linking the e-amino group of lysine present in proteins (antigens or antibodies) with an enzyme (5).Purification of labeled ligands is usually unnecessary since very little, if any, free protein is left unbound. Bovine serum albumin (BSA), for
(1) Antigen bound to polystyrene well
plus antibody to be measured.
20
Antigen midure
(2) Antigen-enzyr.., conjugate added to
(2)
Enzyme conjugate added to well
with bound immune complex.
test sample.
Competitive inhibition occurs between antigen-enzyme conjugate and unlabeled antigen. Binding to specificantibody depends on which antigen type is in ex-.
(3) Antigen-antibody binding to form immune
I-(3) Substrate added.
complex.
(4) Substrate added
to immune complex., (4) Positivetest or color change
denoting chemically changed substrate due to presence of enzyme conjugate bound to immune complex. 1
Color change denotes cnem'caliy cnanaed sbbstrate due 6 enzyme conjugate bound to immune complex. (5)
Antibcdy lo be measured
3
Little Or No Color Change
re+ sample contains antigen) type-speciflc antibody-
,Change (unie if any antigen present in original test sample)
E
Enzyme Conjugate
Substrate Chemically changed submale
Flgure 3. Indirect ELSA for antibody Figure 2. Antigen competitive inhibition assay 924A
ANALYTICAL CHEMISTRY, VOL. 56. NO. 8. JULY I984
measurement
1
I
I
p i ... ..:
,
Fisher's Computer-Aided Titrimeter@combines enormous analytical capability with astonishing simplicity of operation. Easy-to-understand display messages lead you step-by-step through memory-stored methods, or through simple keyboard setup and storage of your own customized methods. If you need more detailed instructions at any point. just I touch the HELP key and you get a printed explanation. Help
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Performs titrations to fixed endpoint targets, titiations with derivative detection of equivalence points, computational methods such as equilibrium and incremental titrations.
8
example, can be conjugated to peroxidase in a simple, one-step procedure. Avrameas gives an excellent description of this method (5). Peroxidase -N
/H
+
\H Enzyme H
\
G-(CHJSC
/H
+
‘\O
OH
Glutaraldehyde
H *-A )
-+
Bovine serum albumin
Peroxidase -N=CHICHJ,-=N-BSA Peroxidaae-conjugated BSA Heterohifunctionsl reagents such as 4,4‘-difluoro-3,3’-dinitrophenyl sulfone (6)and N-succinimidyl-3-(2-pyridy1dithio)propionate (Pharmacia Fine Chemicals, Piscataway, N.J.) may be used instead of glutaraldehyde for production of intermolecular conjugates. Extremely sensitive EIAs have been developed based on the high affinity
uf the glycoprotein avidin for biotin
(vitamin H) and the ease with which biotin may be conjugated Lo other proteins. Several biotin molecules can easily be conjugated to a protein molecule such as an enzyme without altering immunological or enzymatic activity. Avidin, a basic glycoprotein found in egg white, has an exceptionally high affinity constant (lO-’VM/M) for biotin, about a million times greater than the assmiation constants of most antibody-antigen complexes (10-VM). A recently developed method (7,8) of indirectly coupling an enzyme like horseradish peroxidase via biotin-avi. din linking has significantly increased both EMIT and ELSA assay sensitivity and lowered the level of nonspecific label binding. The N-hydroxysuccinimide (NHS) ester of biotin is covalently bound Lo the peroxidase through free amine groups, resulting in an amide linkage. The horseradish peroxidase-biotin conjugate is then mixed with avidin in a precise ratio so that a three-dimensional complex is formed containing many enzyme molecules held together by avidin (Figure 4). At least one biotin binding site in the complex is available to bind with avidin, which in turn binds with biotin-hound antibody or antigen. Conse. quently, a single antigen, antibody, or protein molecule will be surrounded by many enzyme molecules complexed
together with biotin-avidin bonds and the EIA w a y sensitivity will be greatly amplified. When this complex is conjugated to the antigen or antibody, the complex itself surrounds the antigen or antibody protein via biotin-avidin bonds. EIA test sensitivitv can be increased by at least one or two orders of magnitude when avidin-biotin-enzyme conjugates become part of the immunoassay system. Commercial kits containing avidinenzyme conjugates and NHS-biotin eaters for biotin-protein bonding are available from Tago, Inc. (Burlingame, Calif.) and Isolab. Inc. (Akron, Ohio).
Automallon Mechanization of EIAs is available on a large-scale basis and has been used primarily for making serodiagnosis of disease (9).Practically all steps of the EMIT and ELISA systems can be mechanized, and instruments for automation are commercially available. EMIT instrumentation (Figure 5)
Wn-Enzyme Cmwnd
Wi-boHn-enryme m p l e x (C) bound to biotin-pmtein mpound. Many avMinmplexes bind to rnultlple biotins attached to a single protein (antigen ? % w b -, amplifyingtest sensiuvn)l Figure 4. Avidin-biotin system for sensitivity amplification 926 A
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8. JULY 1984
Flgure 5. EMIT instrumentation
I
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Figure 7. Instrumental block design of the Gilford (Oberlin, Ohio) Automatic Analyzer
Figure b. CLIW of diseases OZBA
steps IWserwiagnosis
includes a spectrophotometer for making absorbance measurements, a timer-printer for data handling, and a pipetter-diluter for sample handling. A semiautomatic pipetter-iiluter capable of delivering microvolumes of samples and buffers feeds test specimens to the spectrophotometer. Data can easily be processed with a clinical processor, which times and records sample absorbance measurements and automatically calculates a standard curve and test sample concentrations. Printed absorbance values are converted to sample concentration units by using a manually plotted standard m e and a timer-printer. A high-speed automated ELISA system (Figure 6 ) has been developed for large-scale screening of sera for diagnosis and treatment of infectious diseases. Disposable polystyrene microtiter plates or cuvettes serve as antigen carriers and as liquid test reaction holders. The Gilford Automatic Analyzer (Figure 7) is an instrument that simplifies the preliminary ELISA steps, such as washing and coating of cuvettes, thereby expediting the testing procedure. ELISA tests performed manually can easily be read directly in the same microtiter plate used for doing the test. The Dynatech (Alexandria,Va.) MicroELISA Autoreader is a photom-
ANALYTICAL CHEMISTRY. VOL. 56, NO. 8. JULY 1984
eter designed to measure light absorbance of test samples in clear polystyrene microtiter plates. This instrument consists of a tungsten-halogen lamp that projects a light beam horizontally through a lens and aperture. The beam passes through a 90' prism, and is then projected vertically through individual microtiter wells and a rotating Fiter disk (Figure 8). Energy from this light beam is measured on a silicon photodetector and an absorbance value is obtained. A dual-wavelength mode with ratio absorbances minimizes background interference from such factors as dirt, smudges, scratches, moisture, occlusions, and other plastic artifacts in the microtiter plate being read. Applications
Unlike most other types of immunoassays, EIAs have found immediate use in many disciplines. They have proven to be versatile chemical tools (10)with new applications being reported almost on a weekly basis. EIAs have many clinical, industrial, agricultural, and environmental uses. Very rapid detection of viruses, toxins, hormones, drugs, insecticides, antigens, and antibodies have been made possible by use of either the EMIT or the ELISA system.
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the new DL40RC MemoTitrator. 0 I’d like a demonstration Name Company Address
State Phone
ZIP (Area Code)
(Number)
Catalytic Materials Relationship Between Structure and Reactivitv
I
Tungsten Halogen
Lens
.
Aperture
rn
~
lalph A. Cataiytica Associates, inc. Eric G. Demuane, Editor Mobii TBchnicalCenter R.T.K. Baker, Editor Exxon Research & Engineering Company Reports state-of-the-art instrumentation and techniques for characterizlng the structure and reactivity of catalysts. Looks at alumina distribution In zeolites, the effectsof metal-support interactions, particle-size effects, and parameterscritical to zeolite synthesis and crystallization. CONTENTS
Prism
