Laboratory Experiment pubs.acs.org/jchemeduc
A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules Jennifer L. Powers,* Karen Duda Rippe, Kelly Imarhia, Aileen Swift, Melanie Scholten, and Naina Islam Department of Chemistry & Biochemistry, Kennesaw State University, Kennesaw, Georgia 30144, United States S Supporting Information *
ABSTRACT: ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A competitive, direct ELISA used to detect and quantify levels of digoxin, a cardiac glycoside, is described. Unique features of this lab include collecting data in quadruplicate followed by statistical analysis of replicates using a Q-test. Use of a microplate reader for measuring absorbances makes data collection extremely quick. Students plot their average absorbance versus log concentration digoxin and fit data to a third- or fourth-order polynomial. They also examine the maximum and minimum absorbance for the assay, determine the region of linearity, and then fit the linear region to a straight-line equation that can be used to determine the concentration of an unknown. The experiment can be completed in a 3-h period and is suitable for upper-level biochemistry, chemistry, and biology students. Although students find understanding a competitive ELISA more challenging than some other experiments, they enjoy learning about this commonly used laboratory technique. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Bioanalytical Chemistry, Drugs/Pharmaceuticals, Enzymes, Laboratory Equipment/Apparatus, UV−Vis Spectroscopy
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above. There have been several journal articles focused on teaching that summarize ELISA principles9,14 or provide a procedure for detection of antibody15,16 or other protein.17,18 Of those emphasizing small molecule detection, they rely on a kit19−22 and most do not use a microplate reader.19−21 Of the two articles that use a microplate without a kit, one is qualitative23 and the other detects a DNP-derivative of the amino acid alanine.24
n enzyme-linked immunosorbent assay, ELISA, is a technique used to quantitatively detect the quantity of antigen or antibody in a sample. In an immunological context, the antigen is the molecule that results in antibody production; in other applications, the antigen is simply any molecule one wishes to detect and quantify. This assay is particularly useful in clinical chemistry and is widely used in the diagnosis of several medical conditions including pregnancy, HIV,1 hyperthyroidism,2 and hepatitis C.3 However, ELISAs are useful for a variety of other applications such as detection of contaminated or genetically modified foods,4,5 screening for drugs of abuse in body fluids or hair samples,6−8 quantifying levels of drugs whose concentrations need to be monitored for therapeutic reasons,9 and screening for or quantifying environmental contaminants in soil or wastewater.10−12 Despite the widespread use of ELISA for many applications, published chemistry and biochemistry lab manuals do not normally include this procedure. This could be due to the time involved for an accurate quantitative study or lack of a microplate reader, the standard equipment used for analysis, which has only recently become standard equipment in biochemistry laboratories. The only published biochemistry lab manual procedure for ELISA we have found involves determination of titer and quantification of levels of an enzyme.13 Neither of these applications relates to the use of antibodies as an analytical tool to quantify levels of a small molecule, which is common in many contexts mentioned © 2012 American Chemical Society and Division of Chemical Education, Inc.
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GOALS AND SIGNIFICANCE This ELISA experiment was developed for quantitative detection of small molecules in a short period of time. Students prepare serial dilutions, collect data points in quadruplicate, analyze data for outliers using a Q-test, collect data over a wide range of concentrations, perform a nonlinear curve fit, determine the region of linearity and concentration range in which the assay is useful, fit the linear region to a straight line, and use it to determine concentration on an imaginary unknown with a stated absorbance. Thus, the experiment presents ELISA in a manner that captures its use and limitations as a quantitative tool. Although typically used in biochemistry laboratories, it should also be useful for forensic, pharmaceutical, clinical chemistry, or bioanalytical chemistry laboratories as well as various upper-level biology laboratories. Published: September 27, 2012 1587
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Table 1. Sample Platea Layout for an ELISA Ab Bb Cb Db E F G H
1
2
3
4
5
6
7
8
9
10
11
12
10000 10000 10000 10000 ec e e e
5000 5000 5000 5000 e e e e
1000 1000 1000 1000 e e e e
500 500 500 500 e e e e
100 100 100 100
50 50 50 50
10 10 10 10
5 5 5 5
1 1 1 1
0.5 0.5 0.5 0.5
0.1 0.1 0.1 0.1
0 0 0 0
a
The MaxiSorp plate used is a 96-well plate. bSamples are done in quadruplicate (rows A−D). Exact concentrations may vary in any manner desired, but should span the range 0.10−10,000 ng/mL. Zero digoxin is optional, but will show the maximum absorbance when no competition is occurring. c Wells labeled “e” are extras that the students can prepare to use in case of possible pipetting mistakes during the additions of digoxin and HRPantidigoxin or for an unknown solution if desired.
between the therapeutic dose and toxic dose and its concentration must be monitored in patients to maintain safe levels. The therapeutic plasma concentration range of digoxin is 0.8−2.0 ng/mL.9 However, this procedure could be adapted for other small molecules such as ones of environmental or forensic interest as long as an antibody and a protein conjugate of the small molecule are available.
In addition to teaching students about using antibodies as an analytical tool, this quantitative procedure exposes students to modern technology (a microplate reader) and use of a 96-well plate for samples. Plate readers have become commonplace in biochemistry laboratories and industry because absorbance measurements of many samples can be taken in a few minutes.25 This technology allows students to quickly collect data from four replicates for each data point.
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EQUIPMENT AND MATERIAL A microplate spectrometer is required for reading absorbances in a multiwell format. BioTek PowerWave XS is used here, but many are available. Flat-bottomed 96-well plates are available from various suppliers. For additions of the same solution to each well, it is convenient, but not required, to deliver with a multichannel pipet. Suggested reagent suppliers are listed in Supporting Information.
OVERVIEW OF THE PROCEDURE All ELISA procedures employ the tight binding interactions between an antigen and its antibody. Either one or two antibodies may be used depending on the type of ELISA, but one must be conjugated to an enzyme. At the end of the procedure, a substrate for the enzyme is added and a colored product is formed. Absorbance at a visible wavelength is then measured. In all ELISA procedures, some component of the assay will be adsorbed to the microplate. For persons unfamiliar with ELISA terminology or needing more detailed descriptions of various types of ELISA and typical uses of each, there is detailed information in the Supporting Information, including a link to a Web animation that visualizes the various steps of a typical protocol. The procedure described here detects the compound digoxin by the use of a competitive ELISA. The antibody is enzyme linked, so it is similar to a direct ELISA in this manner, but because small molecules do not bind well to microplates, a competition component was added to the assay. In this procedure, digoxin is covalently attached to the protein bovine serum albumin (digoxin-BSA), which is adsorbed to the plate at a fixed concentration. Then, both enzyme-linked antidigoxin (the antibody) and separate solutions of increasing concentrations of digoxin are added. Thus, the digoxin being added to the well and the digoxin already bound to the plate compete for the available antibody binding sites. When no digoxin solution is added with the antibody, the maximum signal is obtained upon addition of substrate to the wells. For other wells, competition occurs and decreasing absorbance signals result.
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STUDENT PROTOCOL A detailed procedure can be found in the Supporting Information. Students made digoxin dilutions to give several data points that span the range from 10,000 to 0.10 ng/mL so that they may identify the concentration range over which the assay is usable. Plates previously incubated overnight with digoxin-BSA were washed by decanting and filling with PBST (phosphate buffered saline containing 0.05% Tween-20), then the wash was repeated three times. SuperBlock blocking buffer (Thermo Scientific) was added to each well and incubated at room temperature for 10 min. After washing as before, varying concentrations of digoxin were added to the wells, in quadruplicates (Table 1) using a micropipet, and immediately the HRP-antidigoxin was added using a multichannel pipet. The plate was incubated with shaking for 1 h. After washing again as before, freshly prepared substrate, ortho-phenylamine diamine (OPD), was added to each well and allowed to incubate for 10−30 min at room temperature. After stopping the reaction with H2SO4, absorbances were read using a microplate reader at 490 nm within 15 min of the addition of H2SO4 and data exported for further analysis. The students use the entire 2 h and 45 min period. The plates must be prepared a day ahead with the digoxin-BSA conjugate.
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THE SMALL MOLECULE The pharmaceutical agent digoxin was chosen as this was likely to appeal to the interests of the chemistry and biochemistry majors, many of whom are premed or on a pharmaceutical or forensic chemistry track. Digoxin, obtained from the leaves of the plant Digitalis lanata and used to treat heart failure and a particular type of irregular heartbeat,9 is interesting due to its small therapeutic window. This means there is little difference
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HAZARDS Normal laboratory safety protocols should be observed and care should be taken when dispensing the H2SO4. OPD is a possible carcinogen and can cause damage to various organs if inhaled, absorbed, or swallowed; use of OPD tablets minimizes this risk. The 10× peroxide buffer should also be handled with 1588
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Figure 1. Sample student and instructor data. Data points are the average of four replicates. Error bars represent the standard error of the mean. (A) A typical student result from spring 2009. The plate was coated with 0.5 μg/mL digoxin-BSA (Meridian Biosciences). All other conditions were as listed in the text. (B) A typical instructor result from summer 2010 using plates coated with 1.0 μg/mL digoxin-BSA (Fitzgerald Industries). Volumes of 50 μL were used for digoxin, antibody, OPD, and H2SO4. Incubation with OPD was approximately 5 min.
majority of the student pairs, the linear region spanned 2 to 3 orders of magnitude, but occasionally a pair had a lower or higher range of linearity. More details of student results are available in Supporting Information.
care during preparation of the OPD solution as it can cause damage to the respiratory tract and eyes.
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DATA ANALYSIS AND TYPICAL STUDENT RESULTS This experiment has been used with minor variations in volumes and reagent suppliers for over three semesters. It was used in labs for biochemistry majors as well as nonbiochemistry majors with no differences in their ability to perform and understand the experiment. Students examined their replicates prior to graphing. If they suspected any outliers, they used the Q-test and the 90% confidence value to omit any outliers (see the Supporting Information). Typically, students only found 1− 3 data points (out of 44) to be discarded. For nonbiochemistry majors (chemistry and biology students), data were examined from spring 2009 and spring 2010 labs. In both semesters, four out of six pairs of students achieved a R2 of 0.99 for their curve fit to a polynomial. For the others, the R2 ranged from 0.91 to 0.96. For the biochemistry majors, data from two sections of lab from fall 2009 were examined. Of the 13 pairs of students, 10 pairs had a R2 of 0.99 for their curve fit to a polynomial. Usually the polynomial fit was to a third- or fourth-order equation (Figures 1A and 1B). However, in spring 2010, using a new supplier of digoxin-BSA resulted in a curve with no true minimum. Thus, these students fit their data to a second-order polynomial. A lowered concentration of digoxin-BSA or antibody (or lower activity of antibody) would have allowed the minimum to be seen. As mentioned earlier, we wanted students to see that this type of assay has a maximum and minimum above and below a region of linearity. This is only possible if a wide concentration of digoxin is used. Students were asked to determine the region of linearity and replot that data, fitting it to a straight-line equation. They then used that equation to calculate the concentration of an “unknown” that gave an absorbance of a certain value in the assay. (If desired, an actual unknown could be provided for them and they would measure the absorbance.) There was again no difference in data for biochemistry majors and nonmajors. Students were able to pick a linear region and generate a straight line with an R2 of 0.96−0.99. For the
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VARIABILITY AND TYPICAL MISTAKES Student-to-student variability and error comes from differences in pipetting, timing, or making their digoxin serial dilutions. Occasionally, a student will not read directions clearly and fail to wash the plate prior to addition of OPD resulting in no differences in absorbance across the plate. The maximum and minimum absorbances obtained with the assay can depend on several factors, and these will not necessarily remain the same even if the exact concentrations and volumes are used for all solutions from semester to semester. Some factors include different activity of antibody solutions over time or from different batches, when the substrate was prepared, and the temperature of the room. Whenever changing suppliers or using new lots of reagents, it is recommended that the procedure be tested to assess the maximum and minimum. A slight increase or decrease in concentration of digoxin-BSA, digoxin, or antibody may be needed to achieve a curve where the maximum and minimum are observed. Alternately, the range of digoxin used could be adjusted. Data from two different semesters show typical variability. Figure 1A shows a typical student result from spring semester 2009; the maximum absorbance ranged from approximately 1.0 to 1.7 for all of the students. Figure 1B shows a typical instructor result (summer 2010) with a different digoxin-BSA supplier at a higher concentration and a shorter substrate incubation time. Regardless of the maxima and minima observed, over 70% of students had a curve with an R2 of 0.99 and a linear region of two or 3 orders of magnitude. When the linear region was used to determine the concentration of an “unknown” with an absorbance of 0.639, there was variability. Much of the variation can be attributed to students stopping the reaction at different times (15−30 min) after addition of substrate. Additional student results showing variations in the region of linearity and using the linear plot to 1589
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determine the concentration of digoxin in a sample with a stated absorbance are given in the Supporting Information.
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STUDENT COMMENTS AND ASSESSMENT Anecdotal evidence showed that students were excited about learning a technique that can be used as a diagnostic tool, but found it more challenging than some of the labs they have done during the semester. Results from the lab final exam showed that students learned the principles involved. (See the Supporting Information for questions used.) On the spring 2009 final exam, 10 out of 13 students correctly identified two changes that would result in an increased signal for the assay when presented with a list of possible changes to the procedure. The other students identified only one correct item out of the list. Similar results (seven out of 11 students answering correctly) were seen in spring 2010. An additional question (see Supporting Information) about ELISA was given in spring 2009 with 100% of students answering correctly. ASSOCIATED CONTENT
S Supporting Information *
Student handout; instructor information for suppliers of equipment, reagents, and solution preparation; sample answers to questions from the report instructions; assessment questions; detailed descriptions of various types of ELISA and typical uses of each. This material is available via the Internet at http://pubs.acs.org.
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REFERENCES
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ADAPTATION IDEAS This experiment could be easily modified in a variety of ways depending on the emphasis of the instructor. This procedure uses a large concentration range to allow students to observe the linear and nonlinear regions for the assay. Because of this, students are asked to use a logarithmic x axis when graphing. By using this large concentration range in a competition assay, students can also be introduced to the concept of the halfmaximal inhibitory concentration (IC50), if desired, which is a common value reported from competition-type binding experiments. However, more replicates over a smaller concentration range could be done in the same amount of time with the focus only on the linear region of the assay. Also, an actual unknown sample (rather than an imaginary unknown) could be provided to the students so that they measure the absorbance of the unknown and then determine its concentration. Additionally, in an advanced lab, this procedure could serve as a basic guide to development of an ELISA for other small molecules.
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Laboratory Experiment
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank the Department of Chemistry and Biochemistry for the purchase of equipment and supplies, the students of various sections of CHEM 3500L and 3501L at KSU for sharing their data. We also thank Jonathan McMurry for trying this procedure in his lab sections. 1590
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