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 be used for locating and identifying specific tissue antigens. Around the same time, Miles and
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 (1, 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
920 A · 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 II. 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 determi0003-2700/84/0351 -920A$01.50/0 © 1984 American Chemical Society
Report Dan Monroe Department of Medicine Infectious Diseases and Connective Tissues Sections University of Tennessee Center for the Health Sciences Memphis, Tenn. 38163
S ^ "VlMUNO&S Table I. mmunoassay Comparison Sensitivity Specificity Speed Reagents Equipment required Personnel Cost
RIA
FIA
High (ng-pg) High Days Short shelf life Scintillation or gamma counter Skilled with license $7/test
High (ng-pg) High Days Reasonable shelf life UV monitor
EIA
High (ng-pg) High Hours Long shelf life Spectrophotometer
• Skilled $5/test
Minimal training $2/test
Table II. EIA Comparison ELISA
Heterogeneous assay Reagent separation required (centrifugation or filtration) Reagent washings required Slower than EMIT Sensitivity greater than EMIT Macromolecules measured (antigens, antibodies) For diagnosing infectious diseases; immunoglobulins Solid phase assay
EMIT
Homogeneous assay Reagent separation not required Step washings not required Faster than ELISA Sensitivity less than ELISA Measures 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: Basic 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 be measured, enzyme-labeled 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 by the en zyme conjugate.
ΓΖ
/~Λ Compound to be measured (hapten)
^Λ Ε Enzyme-labeled compound
Antibody specific for compound being measured
Ο Enzyme-specific chromogenic substrate
Two basic test principles are involved in the EMIT assay: (1) The enzyme must retain enzyme activity after hap ten or compound conjugation.
+/
Ε
\ - Λ7Λ Ε
Enzyme
Hapten Hapten-enzyme conjugate (Active enzyme)
(2) The enzyme activity of the hap ten-enzyme conjugate is reduced or inhibited when the hapten reacts with its specific antibody. (Active
enzyme)
, Γ7 VI
r~\ Ε Hapten-enzyme conjugate
Antibody
^ Antibody-bound hapten-enzyme conjugate (Inactive
enzyme)
A typical therapeutic EMIT drug assay begins by adding to a patient specimen an excess of specific anti bodies that will bind to the drug being measured. If drug molecules are present, they immediately bind 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 speci men are immediately filled with mole cules 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
to a colored form in a given period of 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 be covered, leaving more enzyme-la beled drug unbound and able to con vert more substrate for higher absorb ance readings. Less drug in the patient sample allows more enzyme-labeled drug to bind the antibody, resulting in less enzyme activity and consequently lower absorbance readings. Inactivation of the enzyme label when the hapten-enzyme complex is antibodybound makes the EMIT assay a unique system, enabling the test to be performed without separation of bound from unbound compounds, as is necessary with other immunoassay methods. ELISA: Basic Principles ELISA links soluble antigens to in soluble antibodies or soluble anti bodies to solid phase antigens in a manner that allows both immunologi cal and enzymatic activity to be re tained. Both a competitive and a dou ble antibody sandwich ELISA tech nique are available for performing an tigen measurements, while antibodies can be quantitated by an indirect ELISA method. The "sandwich" technique is so called because the antigen being as sayed is held between two different antibodies, one containing the enzyme tag (Figure 1). In this method, the inner surface of a polystyrene tube is first coated with a solid phase anti body. The test specimen containing the antigen to be measured is then added and allowed to react with the bound antibody. Any unbound anti gen is washed away. A known amount of enzyme-labeled antibody (produced in an animal species different from that of the bound antibody) is next al lowed to react with the bound antigen. A different set of exposed antigenic determinants, which are not covered by the solid phase antibody bond, are involved in this enzyme-conjugated antibody reaction. Any excess un bound enzyme-linked antibody is washed away after the reaction. Un like 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 fixed time period. The amount of vi sual color change is a direct measure ment of specific enzyme-conjugated
922 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
bound antibody, and consequently an tigen present, in the specimen tested. Another widely used type of ELISA is the competitive assay for antigen detection. The test specimen contain ing the antigen to be determined is mixed with a precise amount of en zyme-labeled antigen and both com pete equally for a limited number of binding sites on an antibody adsorbed
(1) Antibody bound to polystyrene well plus antigen to be measured.
(2) Enzyme conjugate added to well with bound antigen-antibody or immune complex.
• - Ε
Ε
(3) Substrate added to enzyme conjugate bound to immune complex.
(4) Positive test or color change denoting changed substrate due to presence of enzyme conjugate bound to immune complex. Antigen to be measured Antibody
Enzyme conjugate Substrate Chemically changed substrate
Figure 1. Double antibody "sandwich' ELISA for antigen measurement
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 be chemically bound to antibodies and antigens has 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-
(1) Antibody bound to polystyrene well plus test sample containing antigen mixture.
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.
Antigen to be measured
Antigen mixture
Antigen-enzyme conjugate
(2) Antigen-enzyme conjugate added to test sample.
Substrate
(2) Enzyme conjugate added to well with bound immune complex.
Chemically changed substrate Antibody
Competitive inhibition occurs between antigen-enzyme conjugate and unlabeled antigen. Binding to specific antibody depends on which antigen type is in excess.
(3) Antigen-antibody binding to form immune complex.
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(3) Substrate added.
Unlabeled Antigen in Excess of Antigen-Enzyme Conjugate
Antigen-Enzyme Conjugate in Excess Wash (4) Substrate added to immune complex.'
Antigen-EnzymeVUnlabeled Conjugate / Antigen
Antigen-Enzyme/Unlabeled Conjugate \ Antigen
(4) Positive test or color change denoting chemically changed substrate due to presence of enzyme conjugate bound to immune complex. Antigen
A
(5) Color change denotes chemically changed substrate due to enzyme conjugate bound to immune complex.
Little Or No Color Change (Test sample contains antibody- type- specific antigen) Color Change (Little if any antigen present in original test sample)
Figure 2 . Antigen competitive inhibition a s s a y 924 A · ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
tf
Antibody to be measured
Enzyme conjugate
•
Substrate
•
Chemically changed substrate
Figure 3. Indirect ELISA for antibody measurement
example, can be conjugated to peroxi dase in a simple, one-step procedure. Avrameas gives an excellent descrip tion of this method (5). Peroxidase
Ν
+ Η
Enzyme Η
Η
χ
C — (CrU—C Ο
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\> Glutaraldehyde H
\
Ν—BSA — HT Bovine serum albumin Peroxidase
N=CH (CH2)3 CH=N Peroxidase-conjugated BSA
BSA
Heterobifunctional reagents such as 4,4 / -difluoro-3,3'-dinitrophenyl sulfone (6) and 2V-succinimidyl-3-(2-pyridyldithio)propionate (Pharmacia Fine Chemicals, Piscataway, N.J.) may be used instead of glutaraldehyde for production of intermolecular con jugates. Extremely sensitive EIAs have been developed based on the high affinity
of the glycoprotein avidin for biotin (vitamin H) and the ease with which biotin may be conjugated to other pro teins. Several biotin molecules can easily be conjugated to a protein mole cule such as an enzyme without al tering immunological or enzymatic ac tivity. Avidin, a basic glycoprotein found in egg white, has an exception ally high affinity constant (10~ l5 /M) for biotin, about a million times greater than the association constants of most antibody-antigen complexes (10-8/M). A recently developed method (7,8) of indirectly coupling an enzyme like horseradish peroxidase via biotin-avidin linking has significantly increased both EMIT and ELISA assay sensitiv ity and lowered the level of nonspecif ic label binding. The iV-hydroxysuccinimide (NHS) ester of biotin is covalently bound to 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 mol ecules 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-bound 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 assay sensitivity will be great ly amplified. When this complex is conjugated to the antigen or antibody, the complex itself surrounds the anti gen or antibody protein via biotin-avi din bonds. EIA test sensitivity can be increased by at least one or two orders of magni tude when avidin-biotin-enzyme con jugates become part of the immunoas say system. Commercial kits contain ing avidin-enzyme conjugates and NHS-biotin esters for biotin-protein bonding are available from Tago, Inc. (Burlingame, Calif.) and Isolab, Inc. (Akron, Ohio). Automation 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 sys tems can be mechanized, and instru ments for automation are commercial ly available. EMIT instrumentation (Figure 5)
I !
A—Avidin B—Biotin C—Avidin- biotin-enzyme Ε—Enzyme Ρ—Protein (antigen or antibody)
Β
Β
Β
Β
Sample
Buffer/ Reagents
Diluter
Pipetter
Test Specimen
•φ. Β
Biotin-Enzyme Compound
Β
Β
Β
Avidin-biotin-enzyme complex (C) formed when avidin incubated with biotin-enzyme compound.
