A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA)as a Quantitative Technique for Small MoleculesJennifer 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 usedtechnique with applications in disease diagnosis, detection of contaminated foods,and screening for drugs of abuse or environmental contaminants. However, publishedprotocols with a focus on quantitative detection of small molecules designed forteaching laboratories are limited. A competitive, direct ELISA used to detect andquantify levels of digoxin, a cardiac glycoside, is described. Unique features of this labinclude collecting data in quadruplicate followed by statistical analysis of replicatesusing a Q-test. Use of a microplate reader for measuring absorbances makes datacollection extremely quick. Students plot their average absorbance versus logconcentration digoxin and fit data to a third- or fourth-order polynomial. They alsoexamine the maximum and minimum absorbance for the assay, determine the region oflinearity, and then fit the linear region to a straight-line equation that can be used todetermine the concentration of an unknown. The experiment can be completed in a 3-hperiod and is suitable for upper-level biochemistry, chemistry, and biology students. Although students find understanding acompetitive ELISA more challenging than some other experiments, they enjoy learning about this commonly used laboratorytechnique.

KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives,Bioanalytical Chemistry, Drugs/Pharmaceuticals, Enzymes, Laboratory Equipment/Apparatus, UV−Vis Spectroscopy

An enzyme-linked immunosorbent assay, ELISA, is atechnique 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 onewishes to detect and quantify. This assay is particularly useful inclinical chemistry and is widely used in the diagnosis of severalmedical conditions including pregnancy, HIV,1 hyperthyroid-ism,2 and hepatitis C.3 However, ELISAs are useful for a varietyof other applications such as detection of contaminated orgenetically modified foods,4,5 screening for drugs of abuse inbody fluids or hair samples,6−8 quantifying levels of drugswhose concentrations need to be monitored for therapeuticreasons,9 and screening for or quantifying environmentalcontaminants in soil or wastewater.10−12

Despite the widespread use of ELISA for many applications,published chemistry and biochemistry lab manuals do notnormally include this procedure. This could be due to the timeinvolved for an accurate quantitative study or lack of amicroplate reader, the standard equipment used for analysis,which has only recently become standard equipment inbiochemistry laboratories. The only published biochemistrylab manual procedure for ELISA we have found involvesdetermination of titer and quantification of levels of anenzyme.13 Neither of these applications relates to the use ofantibodies as an analytical tool to quantify levels of a smallmolecule, which is common in many contexts mentioned

above. There have been several journal articles focused onteaching that summarize ELISA principles9,14 or provide aprocedure for detection of antibody15,16 or other protein.17,18

Of those emphasizing small molecule detection, they rely on akit19−22 and most do not use a microplate reader.19−21 Of thetwo articles that use a microplate without a kit, one isqualitative23 and the other detects a DNP-derivative of theamino acid alanine.24

■ GOALS AND SIGNIFICANCE

This ELISA experiment was developed for quantitativedetection of small molecules in a short period of time. Studentsprepare serial dilutions, collect data points in quadruplicate,analyze data for outliers using a Q-test, collect data over a widerange of concentrations, perform a nonlinear curve fit,determine the region of linearity and concentration range inwhich the assay is useful, fit the linear region to a straight line,and use it to determine concentration on an imaginaryunknown with a stated absorbance. Thus, the experimentpresents ELISA in a manner that captures its use andlimitations as a quantitative tool. Although typically used inbiochemistry laboratories, it should also be useful for forensic,pharmaceutical, clinical chemistry, or bioanalytical chemistrylaboratories as well as various upper-level biology laboratories.

Published: September 27, 2012

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In addition to teaching students about using antibodies as ananalytical tool, this quantitative procedure exposes students tomodern technology (a microplate reader) and use of a 96-wellplate for samples. Plate readers have become commonplace inbiochemistry laboratories and industry because absorbancemeasurements of many samples can be taken in a fewminutes.25 This technology allows students to quickly collectdata from four replicates for each data point.

■ OVERVIEW OF THE PROCEDUREAll ELISA procedures employ the tight binding interactionsbetween an antigen and its antibody. Either one or twoantibodies may be used depending on the type of ELISA, butone must be conjugated to an enzyme. At the end of theprocedure, a substrate for the enzyme is added and a coloredproduct is formed. Absorbance at a visible wavelength is thenmeasured. In all ELISA procedures, some component of theassay will be adsorbed to the microplate. For persons unfamiliarwith ELISA terminology or needing more detailed descriptionsof various types of ELISA and typical uses of each, there isdetailed information in the Supporting Information, including alink to a Web animation that visualizes the various steps of atypical protocol.The procedure described here detects the compound digoxin

by the use of a competitive ELISA. The antibody is enzymelinked, so it is similar to a direct ELISA in this manner, butbecause small molecules do not bind well to microplates, acompetition component was added to the assay. In thisprocedure, digoxin is covalently attached to the protein bovineserum albumin (digoxin-BSA), which is adsorbed to the plate ata fixed concentration. Then, both enzyme-linked antidigoxin(the antibody) and separate solutions of increasing concen-trations of digoxin are added. Thus, the digoxin being added tothe well and the digoxin already bound to the plate compete forthe available antibody binding sites. When no digoxin solutionis added with the antibody, the maximum signal is obtainedupon addition of substrate to the wells. For other wells,competition occurs and decreasing absorbance signals result.

■ THE SMALL MOLECULEThe pharmaceutical agent digoxin was chosen as this was likelyto appeal to the interests of the chemistry and biochemistrymajors, many of whom are premed or on a pharmaceutical orforensic chemistry track. Digoxin, obtained from the leaves ofthe plant Digitalis lanata and used to treat heart failure and aparticular type of irregular heartbeat,9 is interesting due to itssmall therapeutic window. This means there is little difference

between the therapeutic dose and toxic dose and itsconcentration must be monitored in patients to maintain safelevels. The therapeutic plasma concentration range of digoxin is0.8−2.0 ng/mL.9 However, this procedure could be adapted forother small molecules such as ones of environmental or forensicinterest as long as an antibody and a protein conjugate of thesmall molecule are available.

■ EQUIPMENT AND MATERIALA microplate spectrometer is required for reading absorbancesin a multiwell format. BioTek PowerWave XS is used here, butmany are available. Flat-bottomed 96-well plates are availablefrom various suppliers. For additions of the same solution toeach well, it is convenient, but not required, to deliver with amultichannel pipet. Suggested reagent suppliers are listed inSupporting Information.

■ STUDENT PROTOCOLA detailed procedure can be found in the SupportingInformation. Students made digoxin dilutions to give severaldata points that span the range from 10,000 to 0.10 ng/mL sothat they may identify the concentration range over which theassay is usable. Plates previously incubated overnight withdigoxin-BSA were washed by decanting and filling with PBST(phosphate buffered saline containing 0.05% Tween-20), thenthe wash was repeated three times. SuperBlock blocking buffer(Thermo Scientific) was added to each well and incubated atroom temperature for 10 min. After washing as before, varyingconcentrations of digoxin were added to the wells, inquadruplicates (Table 1) using a micropipet, and immediatelythe HRP-antidigoxin was added using a multichannel pipet.The plate was incubated with shaking for 1 h. After washingagain as before, freshly prepared substrate, ortho-phenylaminediamine (OPD), was added to each well and allowed toincubate for 10−30 min at room temperature. After stoppingthe reaction with H2SO4, absorbances were read using amicroplate reader at 490 nm within 15 min of the addition ofH2SO4 and data exported for further analysis. The students usethe entire 2 h and 45 min period. The plates must be prepareda day ahead with the digoxin-BSA conjugate.

■ HAZARDSNormal laboratory safety protocols should be observed andcare should be taken when dispensing the H2SO4. OPD is apossible carcinogen and can cause damage to various organs ifinhaled, absorbed, or swallowed; use of OPD tablets minimizesthis risk. The 10× peroxide buffer should also be handled with

Table 1. Sample Platea Layout for an ELISA

1 2 3 4 5 6 7 8 9 10 11 12

Ab 10000 5000 1000 500 100 50 10 5 1 0.5 0.1 0Bb 10000 5000 1000 500 100 50 10 5 1 0.5 0.1 0Cb 10000 5000 1000 500 100 50 10 5 1 0.5 0.1 0Db 10000 5000 1000 500 100 50 10 5 1 0.5 0.1 0E ec e e eF e e e eG e e e eH e e e e

aThe 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.cWells labeled “e” are extras that the students can prepare to use in case of possible pipetting mistakes during the additions of digoxin and HRP-antidigoxin or for an unknown solution if desired.

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care during preparation of the OPD solution as it can causedamage to the respiratory tract and eyes.

■ DATA ANALYSIS AND TYPICAL STUDENTRESULTS

This experiment has been used with minor variations involumes and reagent suppliers for over three semesters. It wasused in labs for biochemistry majors as well as nonbiochemistrymajors with no differences in their ability to perform andunderstand the experiment. Students examined their replicatesprior to graphing. If they suspected any outliers, they used theQ-test and the 90% confidence value to omit any outliers (seethe Supporting Information). Typically, students only found 1−3 data points (out of 44) to be discarded. For nonbiochemistrymajors (chemistry and biology students), data were examinedfrom spring 2009 and spring 2010 labs. In both semesters, fourout of six pairs of students achieved a R2 of 0.99 for their curvefit to a polynomial. For the others, the R2 ranged from 0.91 to0.96. For the biochemistry majors, data from two sections of labfrom fall 2009 were examined. Of the 13 pairs of students, 10pairs had a R2 of 0.99 for their curve fit to a polynomial. Usuallythe polynomial fit was to a third- or fourth-order equation(Figures 1A and 1B). However, in spring 2010, using a newsupplier of digoxin-BSA resulted in a curve with no trueminimum. Thus, these students fit their data to a second-orderpolynomial. A lowered concentration of digoxin-BSA orantibody (or lower activity of antibody) would have allowedthe 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 aregion of linearity. This is only possible if a wide concentrationof digoxin is used. Students were asked to determine the regionof linearity and replot that data, fitting it to a straight-lineequation. They then used that equation to calculate theconcentration of an “unknown” that gave an absorbance of acertain value in the assay. (If desired, an actual unknown couldbe provided for them and they would measure the absorbance.)There was again no difference in data for biochemistry majorsand nonmajors. Students were able to pick a linear region andgenerate a straight line with an R2 of 0.96−0.99. For the

majority of the student pairs, the linear region spanned 2 to 3orders of magnitude, but occasionally a pair had a lower orhigher range of linearity. More details of student results areavailable in Supporting Information.

■ VARIABILITY AND TYPICAL MISTAKES

Student-to-student variability and error comes from differencesin pipetting, timing, or making their digoxin serial dilutions.Occasionally, a student will not read directions clearly and failto wash the plate prior to addition of OPD resulting in nodifferences in absorbance across the plate.The maximum and minimum absorbances obtained with the

assay can depend on several factors, and these will notnecessarily remain the same even if the exact concentrationsand volumes are used for all solutions from semester tosemester. Some factors include different activity of antibodysolutions over time or from different batches, when thesubstrate was prepared, and the temperature of the room.Whenever changing suppliers or using new lots of reagents, it isrecommended that the procedure be tested to assess themaximum and minimum. A slight increase or decrease inconcentration of digoxin-BSA, digoxin, or antibody may beneeded to achieve a curve where the maximum and minimumare observed. Alternately, the range of digoxin used could beadjusted. Data from two different semesters show typicalvariability. Figure 1A shows a typical student result from springsemester 2009; the maximum absorbance ranged fromapproximately 1.0 to 1.7 for all of the students. Figure 1Bshows a typical instructor result (summer 2010) with a differentdigoxin-BSA supplier at a higher concentration and a shortersubstrate incubation time. Regardless of the maxima andminima 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 theconcentration of an “unknown” with an absorbance of 0.639,there was variability. Much of the variation can be attributed tostudents stopping the reaction at different times (15−30 min)after addition of substrate. Additional student results showingvariations in the region of linearity and using the linear plot to

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 aslisted in the text. (B) A typical instructor result from summer 2010 using plates coated with 1.0 μg/mL digoxin-BSA (Fitzgerald Industries). Volumesof 50 μL were used for digoxin, antibody, OPD, and H2SO4. Incubation with OPD was approximately 5 min.

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determine the concentration of digoxin in a sample with astated absorbance are given in the Supporting Information.

■ ADAPTATION IDEASThis experiment could be easily modified in a variety of waysdepending on the emphasis of the instructor. This procedureuses a large concentration range to allow students to observethe linear and nonlinear regions for the assay. Because of this,students are asked to use a logarithmic x axis when graphing. Byusing this large concentration range in a competition assay,students can also be introduced to the concept of the half-maximal inhibitory concentration (IC50), if desired, which is acommon value reported from competition-type bindingexperiments. However, more replicates over a smallerconcentration range could be done in the same amount oftime 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 theabsorbance of the unknown and then determine itsconcentration. Additionally, in an advanced lab, this procedurecould serve as a basic guide to development of an ELISA forother small molecules.

■ STUDENT COMMENTS AND ASSESSMENTAnecdotal evidence showed that students were excited aboutlearning a technique that can be used as a diagnostic tool, butfound it more challenging than some of the labs they have doneduring the semester. Results from the lab final exam showedthat students learned the principles involved. (See theSupporting Information for questions used.) On the spring2009 final exam, 10 out of 13 students correctly identified twochanges that would result in an increased signal for the assaywhen presented with a list of possible changes to the procedure.The other students identified only one correct item out of thelist. Similar results (seven out of 11 students answeringcorrectly) were seen in spring 2010. An additional question(see Supporting Information) about ELISA was given in spring2009 with 100% of students answering correctly.

■ ASSOCIATED CONTENT*S Supporting Information

Student handout; instructor information for suppliers ofequipment, reagents, and solution preparation; sample answersto questions from the report instructions; assessmentquestions; detailed descriptions of various types of ELISAand typical uses of each. This material is available via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*E-mail: jpowers@kennesaw.edu.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe would like to thank the Department of Chemistry andBiochemistry for the purchase of equipment and supplies, thestudents of various sections of CHEM 3500L and 3501L atKSU for sharing their data. We also thank Jonathan McMurryfor trying this procedure in his lab sections.

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