In the Laboratory
Enzyme-Linked Antibodies: A Laboratory Introduction to the ELISA Gretchen L. Anderson and Leo A. McNellis Department of Chemistry, Indiana University South Bend, South Bend, IN 46634
ELISAs (enzyme-linked immuno-sorbent assays) have revolutionized diagnostic procedures in medicine, agriculture, and home health-care management. Enzyme-linked antibodies are used routinely to detect minute quantities of viruses (e.g., hepatitis virus), hormones (e.g., human chorionic gonadotropin as evidence of pregnancy), and other antibodies (e.g., antibodies to the AIDS virus as an indication of infection). The use of ELISAs has spread from clinical applications to layman use in home pregnancy tests, golf-course turf management, and diagnosis of crop disease. ELISAs provide an ideal laboratory exercise for elementary biochemistry students such as health science and nursing majors, students enrolled in chemistry survey courses, or high school students. The technique is current, conceptually straightforward, and rapid enough to be completed in a typical 2-hour laboratory class. It provides students with a clear example of practical, contemporary uses of basic chemical principles. It introduces them to the microscale quantities typical of ELISAs and integrates the use of enzymes, antibodies, and the specificity of protein binding. The procedure outlined below demonstrates the impressive sensitivity of the ELISA through the detection of femtomolar (10᎑15 M) quantities of biotin, a water-soluble vitamin. The advantages of this choice of antigen are the low cost ($1.00 or less per student), minimal equipment needs, moderate amount of instructor or technician preparation time, and nonpathogenicity of the samples. Background In vivo, antibiodies (or immunoglobulins) constitute a family of proteins that specifically bind to antigens recognized as foreign to the host. The antigen may be a polypeptide or a portion of a protein, a carbohydrate, or virtually any small molecule. Formation of the antigen–antibody complex is an essential step in the immune response to eliminate molecules, viruses, or cells that are not part of the host organism. Antibodies bind noncovalently to target antigens by virtue of their complementary 3-dimensional structures. A specific antibody will bind only to its target, even in complex mixtures. The ELISA takes advantage of the specificity of antibodies to detect small amounts of a particular antigen in a complex mixture such as blood or extracts of plant tissue (1–9). One way to detect the presence of a specific antibody–antigen complex is to measure the activity of an enzyme covalently attached to the antibody. The enzyme chosen (usually peroxidase or alkaline phosphatase) is one that catalyzes a color conversion in a dye when appropriate substrates are added. Since each enzyme molecule catalyzes the formation of thousands of colored dye molecules, and since the absorbance of the dye is proportional to the amount of antibody–antigen complex present in the sample, the ELISA can detect picomolar or femtomolar (10᎑12 and 10᎑15 M, respectively) concentrations of a particular antigen in a mixture.
Many antibodies used in ELISAs are commercially available as enzyme-linked antibodies (also called enzyme-linked antibody conjugates). Antibodies are produced by injecting an animal (e.g., a rabbit) with the antigen of interest. Within several weeks, the animal produces many copies of an antibody that specifically recognizes and binds the antigen. The antibodies are identified, purified, and covalently bound to an enzyme. This enzyme-linked antibody now acts as a specific probe to detect the presence of the antigen in a sample. A different antibody or enzyme-linked antibody preparation is purchased for each antigen to be tested. This laboratory exercise demonstrates the effectiveness of ELISAs by using enzyme-linked antibodies to detect biotin in the form of biotinylated albumin. This choice of antigen is advantageous in terms of availability and cost of reagents (enzyme-conjugated antibiotin antibodies are among the least expensive) and avoidance of potentially hazardous biological specimens. Because the volumes of samples are very small (usually less than 100 µL), the assay can be performed in commercially available small plastic (polyethylene) wells similar to a tiny plastic muffin tin. For ill-understood reasons, large molecules, particularly proteins, bind tightly to the inside surface of the wells, but small molecules such as biotin do not bind efficiently. The efficiency of the ELISA is therefore improved by attaching the biotin to a large protein such as albumin. Commercially available biotinylated albumin is used here to insure that biotin remains bound to the surface of the ELISA wells. Peroxidase-linked antibiotin antibody is also commercially available. It catalyzes the reduction of colorless TMB (3,3′,5,5′-tetramethylbenzidine) dye in the presence of hydrogen peroxide to yield a colored product: peroxidase
TMBox + H2O2 → TMBred + O2 + 2H+ (colorless)
(colored)
Typical ELISA Procedure A typical ELISA procedure consists of four basic steps (Figure 1 shows these steps as related to the assay for biotinylated albumin described here): 1. Bind the antigen to the ELISA well and wash away excess. Samples used in ELISA diagnostic procedures are generally complex mixtures consisting of a few molecules of the antigen of interest in a matrix of extraneous proteins, lipids, carbohydrates, etc. Some of these “contaminating” molecules will bind to the ELISA well, but will not be detected later. Any molecules that do not bind to the well are removed with a buffered rinse. 2. Block exposed sites on the wells with albumin and wash away excess. The enzyme-linked antibody (to be added in a subsequent step) will bind nonspecifically to the ELISA well if any of the well surface is exposed. To prevent this, a large excess of an inexpensive protein (such as albumin) is added to the well. Excess albumin is removed from the well with a buffered rinse.
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In the Laboratory 3. Bind enzyme-linked antibody to available antigens. When the appropriate enzyme-linked antibody is added to the well, it binds only to its specific antigen. After washing to remove unbound antibody, the amount of enzyme-linked antibody remaining in the well is proportional to the amount of antigen in the well. At this point, the enzyme-linked antibody is undetectable because it is colorless and present in minute quantities. 4. Add enzyme substrate to detect presence of antigen. The amount of enzyme bound to the ELISA well can be determined by its activity with a suitable substrate. If the enzyme-catalyzed reaction results in a color change of a dye, the presence of the enzyme can be determined visually and its concentration estimated by the intensity of the color produced or quantified by spectroscopy. Since enzymes typically catalyze the specific reaction of hundreds of molecules of substrate, the detection power of the ELISA is greatly amplified.
1:2000 dilution of antibiotin peroxidase conjugate (Sigma) or according to recommendations of vendor TMB (3,3′,5,5′-tetramethylbenzidine-HCl) tablet (Sigma) 30% hydrogen peroxide Strip of 8 polyvinyl ELISA wells (ELISA plates, available from scientific warehouses as 96-well plates, can easily be cut into 8-well strips) Humid box (a plastic sandwich box containing a damp paper towel works well) Pipettors capable of measuring 100 µL and 2 µL (drops and droppers can also be used) Pasteur pipet attached to an aspirator for removing liquid from ELISA wells Distilled or deionized water should be used to make all reagents.
Experimental Procedure
Materials
Bind the antigen to the ELISA well and wash away excess: PBS buffer (phosphate buffered saline): 8.0 g NaCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per liter, pH 7.2 0.05 M phosphate citrate buffer pH 5.0: 51 mL 0.2 M Na2HPO4 added to 49 mL 0.1 M citric acid and brought to 500 mL with water 3% BSA (bovine serum albumin) (Sigma) in water Serial dilutions of biotinylated BSA (Sigma) including 1:100, 1:500, 1:1000, 1:5000, 1:10,000 and 1:50,000 dilutions
1. Add 100 µL (or one drop from an eyedropper) of each dilution of biotinylated BSA to an ELISA well, and 100 µL of 3% BSA to one well to serve as a negative control. 2. Incubate 15 min at room temperature in a humid box. 3. Remove the contents of the wells by aspiration. This is most easily accomplished using a Pasteur pipet attached to an aspirator. To avoid contamination between wells, rinse the pipet by aspirating water before
Figure 1. General ELISA protocol. Antigens in a complex mixture bind nonspecifically to the sides of a polyethylene well. After removal of any unbound antigens, albumin is used to coat the remaining exposed surface of the well, and excess albumin is removed. An enzyme-linked antibody specific for the antigen of interest is added and the excess is removed. When the enzyme substrate is added, a color change is observed, which is proportional to the amount of antigen of interest in the original sample. Legend:
antigen of interest albumin enzyme-linked antibody
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In the Laboratory continuing to the next well. Avoid scraping the sides or bottom of the well. To wash the wells, completely fill each well with PBS; remove the wash by aspiration. Repeat twice.
Block the wells with albumin and wash away excess: 1. Completely fill each well with 3% BSA. Incubate 15 min at room temperature in a humid box. 2. Aspirate to remove the BSA solution and then wash the wells three times with PBS as before.
Bind enzyme-linked antibody to available antigens: 1. Add 100 µL of antibiotin-peroxidase conjugate solution to each well. 2. Incubate 15 min at room temperature in a humid box. 3. Meanwhile, prepare the enzyme substrate (the substrate must be made within 10 min of use). Dissolve one TMB tablet in 1.0 mL of phosphate-citrate buffer. When the tablet is dissolved, dilute the TMB with 9.0 mL of phosphate-citrate buffer and add 2 µL of 30% hydrogen peroxide. 4. Wash the ELISA wells three times with PBS.
Add enzyme substrate and observe color change: 1. Add 100 µL of prepared enzyme substrate to each well. The intensity of the blue color is proportional to the amount of biotin (and biotinylated BSA) in the sample well. Simple visualization can be used to determine the detection limits of the assay and the useful concentration range of the antigen. This can serve as a powerful demonstration of the detection power of ELISAs. 2. If a microplate reader is available, the absorbance can be obtained for each well. In this case, careful measurement of biotinylated albumin added to each well can serve as a standard curve and demonstrate the linearity of the response through Beer’s law as well as the usual concentration range of the antigen. If using a microplate reader, the enzyme reaction should be stopped after 20 min by adding 100 µL of 3 M sulfuric acid. The absorbance is read at 450 nm.
Discussion The ELISA described here is simple, fast, and relatively cost-effective. The experiment demonstrates the power of antibodies and enzymes to detect the presence of small amounts of a particular antigen in a mixture. ELISAs are often used in this way to detect the presence of antigens (e.g., human chorionic gonadotropin in urine, the “pregnancy test”) or of a particular virus in a biological sample to confirm a diagnosis. This laboratory exercise has been used successfully in our freshman chemistry classes for nursing and health science majors. Technical difficulties are generally related to adding reagents in the proper order to the proper wells. Aspiration of the excess reagents avoids cross contamination of wells and is reproducible so long as care is taken to rinse the aspiration pipet between wells and to avoid scraping the wells. Followup questions incorporated in our lab reports ask the students to calculate the detection limit of biotin in their experiment, extrapolate the ELISA concept to develop a diagnostic test
for pregnancy, and design an ELISA experiment that would detect the presence of HIV antibodies in serum. Variations of the basic ELISA are used in hospitals, clinics, and agricultural and research laboratories for many purposes. For example, the basic ELISA can be used in histochemistry to determine the presence and location of compounds within a cell using a dye that precipitates when reduced. The presence of particular antibodies, such as antibodies to the AIDS virus, can be detected and quantified using a competitive ELISA in which enzyme-linked antibodies compete with patient antibodies for HIV antigens on a solid support. Therapeutic and nontherapeutic drugs, infectious agents, hormones, and numerous biological compounds are among the types of materials detected and quantified by ELISAs. ELISAs are often used to quantify antigens by choosing appropriate dilutions and comparing absorbances in these ELISA wells to those of standards in other wells of the same ELISA plate. Because of the small volumes and large number of wells in an ELISA plate, absorbances are usually measured directly in the well via a microplate reader. Reproducible results for quantitation require technical skill, precise timing, and/or automated procedures; for this reason the qualitative ELISA as outlined here is probably more appropriate for freshman-level and high school courses. However, if students are familiar with Beer’s law, they can easily appreciate how the qualitative ELISA shown here can be extrapolated to quantitative detection. The ELISA experiment has several advantages: (i) it utilizes state-of-the art diagnostic procedures used routinely in clinical diagnoses; (ii) it reinforces concepts learned in accompanying lectures such as the specificity of enzymes, enzyme assays to detect minute levels of compounds, and the chemistry of peroxidase; and (iii) it introduces new concepts and procedures such as the specificity of antibodies and the micro scale of assays often used in clinical situations. Acknowledgment The development and testing of this laboratory experiment was funded in part by a Curriculum Development Grant from Indiana University South Bend. Literature Cited 1. Van Weeman, B. K.; Schuurs, A. FEBS Lett. 1971, 15, 232–236. 2. Engvall, E.; Perlmann, P. Immunochemistry 1971, 8, 871–874. 3. Rubenstein, K. E.; Schneider, R. S.; Ullman, E. F. Biochem. Biophys. Res. Commun. 1972, 47, 846–851. 4. Monroe, D. Anal. Chem. 1984, 56, 920A–931A. 5. Antibodies: A Laboratory Manual; Harlow, E.; Lane, D., Eds.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988. 6. Calbreath, D. F. Clinical Chemistry: A Fundamental Textbook; Saunders: Philadelphia, 1992; pp 153–177. 7. Anderson, S. C.; Cockayne, S. Clinical Chemistry: Concepts and Applications; Saunders: Philadelphia, 1993; pp 92–106. 8. Antibody Techniques; Malik, V. S.; Lillihoj, E. P., Eds.; Academic: San Diego, 1994. 9. Crowther, J. R. ELISA: Theory and Practice; Humana: Totow, NJ, 1995.
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