The enzyme-linked immunosorbent assay (ELISA) is a powerful immunological technique for quantifying small amounts of compounds and has been used in research and clinical settings for years. Although there are laboratory exercises developed to introduce the ELISA technique to students, their ability to promote student learning has not been thoroughly assessed. We found that a commercially available ELISA kit increased student performance on pre- and post-tests in three undergraduate college courses, especially in those taught to general-education students. Student confidence levels about ELISA methodology, as well as comfort level in performing the technique, increased significantly in both general- education and biology-major courses.

Immunology is one of the most important fields of study in biology, but it is also one of the most complicated because of the intricate biochemical and cellular mechanisms involved. Immunological probes (antibodies, antigens) provide important tools for scientists studying cell and molecular biology, as well as genetics and microbiology. Students at all educational levels, therefore, need to be aware of the immunological process and the various tools and techniques that immunology provides. A wide array of educational approaches have been developed to teach students about the different aspects of immunology, including wet-lab exercises (Brokaw & Cobb, 2009), semester-long inquiries (Goyette & DeLuca, 2007), dry-lab simulations (Baker & Moore, 1996), interactive “virtual” exercises accessed via computer (Howard Hughes Medical Institute, 2004), and educational videos (DnaTube.com, 2007).

One of the most widespread research tools using immunology is the enzyme-linked immunosorbent assay (ELISA), a technique developed in the early 1970s that allows for quantitative detection of small amounts of almost any molecule with antigenic properties (Engvall & Perlmann, 1971; Engvall, 2010). To illustrate the growing importance of the ELISA technique, a literature search in PubMed (National Library of Medicine) for the years 2006–2010 yielded almost 48,000 research articles that used the term “ELISA.” This continues the sharply upward trend noted by Lequin (2005), showing that the use of this technique is rapidly expanding. ELISAs are used in myriad ways in diverse fields like biotechnology, medicine, agriculture, and environmental science. For example, home testing kits for detecting pregnancy are based on detecting the levels of the hormone human chorionic gonadotropin in women’s urine using an ELISA-like chromatographic immunoassay (Cole et al., 2005). ELISAs are used in developing countries to identify pregnant women infected with HIV (Humphrey et al., 2010) and to detect contamination of soil by dioxin (Trindade et al., 2008).

Instructors generally recognize that incorporation of hands-on activities enriches student learning (Hofstein and Lunetta, 2004). Rather than prepare ELISA exercises from scratch (e.g., Russo et al., 1984; Anderson & McNellis, 1998; Brokaw & Cobb, 2009), many biology instructors are now purchasing commercially prepared ELISA kits offered by science-education companies. These kits are fairly complete, providing most of the required reagents along with instructor directions and student manual, thus providing reliability, lower cost, and instructional ease. Commercially available ELISA kits that utilize real antigens and antibodies are sold by Ward’s Natural Science (Rochester, NY), Modern Biology (Lafayette, IN), and Bio-Rad Laboratories (Hercules, CA), among others. Companies like Carolina Biological Supply Company (Burlington, NC) and Flinn Scientific (Batavia, IL) offer inexpensive classroom kits that simulate ELISAs but don’t actually use antigens or antibodies.

There have also been numerous publications to help instructors generate their own hands-on ELISA experiments for students (Russo et al., 1984; Anderson & McNellis, 1998; Grimes et al., 1998; Gerbig et al., 2000; Chow & Phoon, 2003; Preszler & Marion, 2006). These authors generally focused on the technical and pedagogical aspects of the ELISA technique and presented no substantial assessment of student learning. Haussmann et al. (2007) and Brokaw & Cobb (2009) included evaluations of student learning and success with their classroom ELISA exercises, but they were subjective. Goyette & DeLuca (2007) designed a semester-long lab-research project on immunology that centered on enzyme immunoassays and assessed student learning by monitoring student grades, laboratory reports, and student responses on year-end course evaluations. They reported that students learned more after carrying out student-directed research than when they completed instructor-designed experiments.

Judging from the large number of laboratory kits on the market and the numerous publications, it appears that many biology instructors are bringing the ELISA technique into the classroom. The ability of these lab exercises to aid student learning has not been thoroughly examined, however. Our objective was to assess the effectiveness of a commercial ELISA lab exercise in three different courses: two general-education biology courses, taught at the freshman level to students who were not biology majors, and one sophomore-level course required of biology majors. This assessment was done by giving students a test containing questions about immunology and the ELISA technique, before and after carrying out the lab exercise. Student self-confidence levels and lab success rates were also examined.

Materials & Methods

This study was conducted at Bellarmine University, a small, private liberal-arts institution located in Louisville, Kentucky. Three classes were assessed in this study: Drugs and the Human Body (a general-education course, BIOL 118), Spring 2011, 24 students; Introduction to Forensic Science (a general-education course, BIOL 119), Fall 2010, 37 students; and Cell Biology (a course required of all biology majors, BIOL 231), Fall 2010, 40 students. Each of these courses included one laboratory session per week: 2 hours for the general-education courses, 3 hours for the biology majors’ course.

An ELISA exercise was carried out in each course during the second half of the semester when students were already fairly well acquainted with basic laboratory techniques. The ELISA exercise used was the ELISA Immuno Explorer Kit marketed by Bio-Rad Laboratories (no. 166-2400EDU). This kit comes complete with all the reagents for carrying out the ELISA experiment except for sundry items like marking pens and beakers, and includes a 119-page lab manual with complete instructions for both instructor and student.

Three different scenarios are provided in the manual for the instructor to choose from. The first ELISA scenario provided in this kit has students track the spread of a simulated disease within their own classroom population, an approach described by Grimes et al. (1998). This scenario was used in two of the three classes (Drugs and the Human Body, and Cell Biology). In this approach, each student was given a microcentrifuge tube containing simulated “body fluids” that they successively shared with three other students in the room by pipetting and mixing. One student in the lab was given a tube that was infected with a simulated pathogen for a disease, like anthrax or smallpox. This “carrier” tube actually contained a harmless antigen that reacts with the primary antibody used in the ELISA. After students shared their “body fluids,” they pipetted a portion of their new blends into a 12-well microplate strip. Each student’s sample was pipetted into duplicate wells, to check for pipetting errors. Positive and negative controls were included, also done in duplicate.

The second ELISA scenario in this kit bypasses the epidemiological aspects of the first scenario but provides more information about positive and negative controls. This scenario was used in the Introduction to Forensic Science course, with students using ELISA to identify a simulated bioterrorism threat. The third ELISA scenario is like the second but includes even more details about the immune response and the importance of experimental controls.

Once samples were loaded into the appropriate wells, they were incubated for 5 minutes to allow the antigen to bind. Samples were then washed in buffer, and nonspecific binding sites were blocked with gelatin for 5 minutes. Wells were then washed and incubated with primary antibody for 5 minutes, and washed again before adding secondary antibody (conjugated to horseradish peroxidase). Samples were incubated for 5 minutes and washed again. Reaction substrate was added, which produced a blue color if the secondary antibody–peroxidase conjugate was still present. A blue-colored solution indicates the presence of the antigen bound to the bottom of the well and recognized by the primary antibody, which is subsequently recognized by the secondary antibody conjugate (Figure 1). The results are qualitative as well as quantitative. All steps were conducted at room temperature and can be completed in less than 2 hours, but could be adapted for two shorter periods.

Figure 1.

Example of ELISA results using an epidemiology scenario. All samples were run in duplicate, with “+” indicating the positive control, “−” indicating the negative control, and “In” representing the initial student sample, with subsequent exchanges of simulated bodily fluids indicated. These results show that the infectious agent was contracted from the second exchange and was diluted by the third.

Figure 1.

Example of ELISA results using an epidemiology scenario. All samples were run in duplicate, with “+” indicating the positive control, “−” indicating the negative control, and “In” representing the initial student sample, with subsequent exchanges of simulated bodily fluids indicated. These results show that the infectious agent was contracted from the second exchange and was diluted by the third.

The Bio-Rad ELISA kit manual contains appendices with information about immunology and detailed descriptions of pathogenic diseases. This information was integrated into a 15-minute prelab lecture on immunology and ELISAs delivered to students before they carried out the exercise. Details were provided about the specific immune response only in the prelab of the nonmajor courses, because biology majors study immunology in the microbiology course that they are required to take later. Immunology was not specifically addressed in classroom lectures in either the Cell Biology or the Drugs and the Human Body courses. However, ABO blood typing was briefly covered in the forensics course 9 weeks prior to the laboratory exercise.

Although the kit contains ready-to-use detailed instructions for students, we chose to shorten the introduction and modify the directions. For instance, the Bio-Rad kit has students assay each sample in triplicate (thereby obtaining four results per 12-well microplate strip), but we assayed in duplicate to obtain six results per strip.

To evaluate student learning, a pretest was given to the students 1 week prior to their ELISA laboratory. The pretest consisted of 11 multiple-choice questions (Table 1). To reduce bias on the post-test, pretests were not returned and answers to questions were not disclosed.

Table 1.

List of multiple-choice questions asked on the pre- and post-tests.

No.Question
“ELISA” is an acronym for ____________. 
Which immune cell produces antibodies? 
Which is true about the secondary antibodies used in the ELISA protocol? 
What has to be done after each incubation step of the ELISA protocol? 
A “False Positive” is defined as ___________________. 
What is the purpose of using a “Blocking Agent” during the ELISA protocol? 
Which of the following is the correct series of steps used when performing an ELISA? 
Why were the samples incubated for 5 minutes after the addition of the substrate? 
What does the “negative control” consist of when performing an ELISA? 
10 Based on the table of ELISA results provided, who was the initial carrier of the disease? 
11 Based on the table of ELISA results provided, Student X could have obtained the disease from _____. 
12 How comfortable do you feel with performing an ELISA? 
No.Question
“ELISA” is an acronym for ____________. 
Which immune cell produces antibodies? 
Which is true about the secondary antibodies used in the ELISA protocol? 
What has to be done after each incubation step of the ELISA protocol? 
A “False Positive” is defined as ___________________. 
What is the purpose of using a “Blocking Agent” during the ELISA protocol? 
Which of the following is the correct series of steps used when performing an ELISA? 
Why were the samples incubated for 5 minutes after the addition of the substrate? 
What does the “negative control” consist of when performing an ELISA? 
10 Based on the table of ELISA results provided, who was the initial carrier of the disease? 
11 Based on the table of ELISA results provided, Student X could have obtained the disease from _____. 
12 How comfortable do you feel with performing an ELISA? 

Students were also asked to self-report their confidence level about each of their answers using a 5-point scale, in which 1 was “lowest confidence” and 5 was “highest confidence.” One of the options for each multiple-choice question was “I have no idea,” which was automatically scored as lowest confidence. In addition to the 11 multiple-choice questions, students were asked to rate their level of comfort about performing the ELISA exercise in the lab (question 12). They used the same 5-point scale for this question, with 1 being “lowest comfort” and 5 being “highest comfort.”

To assess learning, the same test was given to students 2–3 weeks after the lab exercise. Students were not graded on the pretest, but the post-tests in the general-education classes were given as separate, graded quizzes. In Cell Biology, the post-test was given during their cumulative final exam but attached as a nongraded supplement. Test scores and confidence levels were statistically compared using paired t-tests and one-way repeated-measures analysis of variance (Jandel Scientific, 1995). Student comfort levels with the lab technique were analyzed using the Wilcoxon signed-rank test. Correlation analysis was performed in SPSS software on self-reported confidence levels versus student performance on test questions.

Results & Discussion

The ELISA experiments were very successful, working 100% of the time in these three courses. A typical result can be seen in Figure 1. Occasionally, a student would observe variation in the color of the duplicated wells. This provided the instructor an opportunity to point out the challenge of consistent pipetting and the importance of replication.

On average, students in these classes came up with correct answers on the ELISA pretest only 42% of the time (Table 2). Students in the Introduction to Forensic Science course had significantly lower pretest scores than those in the Cell Biology course (P < 0.001), whereas the Drugs and the Human Body scores were intermediate and not statistically different from those in the other two courses. Pretest scores in the biology majors’ course were statistically greater than for the combined scores of the two general-education courses (P = 0.003). The most difficult question for all students was question 2 (Table 1). Only 4–8% of the students answered that question correctly on the pretest. There were two questions that nonmajors did relatively poorly on, compared with the majors: 3 and 9. Nonmajors correctly answered these two questions only about 15% of the time, compared with 38% in the majors’ course. Nonmajors also had difficulty with question 6, with 22% and 42% of the general-education students getting the correct answer, compared with 78% correct in the biology majors’ class.

The highest score on the pretest, for all three classes, was for question 10, which asked students to identify the “carrier” of a disease by looking at an example of ELISA results from eight people. Between 84% and 96% of the students answered that question correctly. Students in all three courses also did relatively well on question 11 (62–80% correct), which also involved interpreting epidemiology data, and question 5 (67–84% correct).

Table 2.

Average student responses on the questionnaire before and after the ELISA exercise, from three classes: Introduction to Forensic Science (F; n = 37), Drugs and the Human Body (D; n = 24), and Cell Biology (C; n = 40).

Percent Correct AnswersConfidence Level
PretestPost-testPretestPost-test
QuestionFDCFDCFDCFDC
22 54 60  100 100 65  1.7 3.1 2.6  5.0 4.8 3.7 
 78 88 30  2.3 2.1 2.6  4.0 3.9 3.2 
14 17 38  73 79 48  1.2 1.7 1.8  3.2 3.3 2.7 
22 29 53  98 100 95  1.3 2.0 2.3  4.8 4.8 4.2 
84 67 75  89 88 88  3.9 3.4 3.8  4.7 4.7 4.4 
22 42 78  54 58 83  1.5 2.0 2.7  3.1 3.3 3.4 
38 30  78 88 48  1.2 1.8 2.0  3.5 3.3 2.8 
14 29 33  35 21 33  1.4 2.1 2.1  3.1 3.2 3.1 
14 17 38  78 63 45  1.2 1.3 1.9  3.2 3.2 2.8 
10 84 96 93  100 96 98  3.1 3.2 4.1  4.3 4.7 4.6 
11 62 79 80  92 96 93  2.7 3.1 3.7  4.1 4.5 4.5 
12 – – –  – – –  1.6 2.3 2.2  3.9 4.4 3.5 
Average 32 43 53  80 80 66  1.9 2.3 2.7  3.9 4.0 3.6 
SD ±29.8 ±28.4 ±26.3  ±20.2 ±24.0 ±26.4  ±0.89 ±0.67 ±0.79  ±0.68 ±0.71 ±0.70 
Percent Correct AnswersConfidence Level
PretestPost-testPretestPost-test
QuestionFDCFDCFDCFDC
22 54 60  100 100 65  1.7 3.1 2.6  5.0 4.8 3.7 
 78 88 30  2.3 2.1 2.6  4.0 3.9 3.2 
14 17 38  73 79 48  1.2 1.7 1.8  3.2 3.3 2.7 
22 29 53  98 100 95  1.3 2.0 2.3  4.8 4.8 4.2 
84 67 75  89 88 88  3.9 3.4 3.8  4.7 4.7 4.4 
22 42 78  54 58 83  1.5 2.0 2.7  3.1 3.3 3.4 
38 30  78 88 48  1.2 1.8 2.0  3.5 3.3 2.8 
14 29 33  35 21 33  1.4 2.1 2.1  3.1 3.2 3.1 
14 17 38  78 63 45  1.2 1.3 1.9  3.2 3.2 2.8 
10 84 96 93  100 96 98  3.1 3.2 4.1  4.3 4.7 4.6 
11 62 79 80  92 96 93  2.7 3.1 3.7  4.1 4.5 4.5 
12 – – –  – – –  1.6 2.3 2.2  3.9 4.4 3.5 
Average 32 43 53  80 80 66  1.9 2.3 2.7  3.9 4.0 3.6 
SD ±29.8 ±28.4 ±26.3  ±20.2 ±24.0 ±26.4  ±0.89 ±0.67 ±0.79  ±0.68 ±0.71 ±0.70 

The overall average score on the post-test was 79% higher than that on the pretest (Table 2). The post-test scores for the three courses were not statistically different from each other, although the average scores for the general-education courses were numerically higher than those for the biology majors’ course, 80% versus 66%. The general-education students were graded on their answers to the post-test, whereas the biology majors knew that the post-test was not graded and, thus, might not have taken it as seriously.

The average post-test scores were statistically higher than the pretest scores in all three courses (P < 0.004), with general-education students showing greater improvement (Table 2). Even though the Introduction to Forensic Science course had the lowest pretest score, the post-test scores were as high as, or higher than, those in the other two courses.

Students generally appeared to improve the least (from pretest to post-test) on question 8 (Table 2). In fact, only in the forensics class was there any increase in the score for this question. This might be because students did not adequately understand the concept of secondary antibody conjugation to a detection enzyme. The general-education students also had relatively low success on question 6. Question 2 was the most difficult question on the pretest but was the most improved upon in all classes, even though the score in the biology majors’ course was still relatively low (30%). The correct answer to question 2 on the test was “B-Cells,” with the other multiple-choice options being “Antigen,” “Macrophages,” “T-Cells,” and “I have no idea.” There was greater emphasis on the immune cells during the prelab orientation in the general-education courses. Because biology majors were slated to take a full-semester course in microbiology later, this topic was not emphasized in the prelab for Cell Biology. Question 4 was also much improved in all classes, but in this case students in the biology majors’ course scored high (95%). Therefore, all students appeared to inherently gain an understanding of the importance of washing ELISA wells between each step. Pretest scores on questions 5, 10, and 11 indicate that students were already proficient at interpreting ELISA results, perhaps because it is fairly intuitive. They seemed to have learned the most about ELISA techniques and about antibody production (e.g., question 2).

There were a number of questions on the post-test on which biology majors scored below the general-education students: 1, 2, 3, 7, and 9. As mentioned previously, this could be because the general-education students received a grade for their answers, whereas Cell Biology students answered the questions as a nongraded supplement to their cumulative final exam. This is reflected in questions 3, 7, and 9, on which 20% of Cell Biology students answered “I have no idea,” which they would not have done if they knew that it was graded. On questions 2, 3, 7, and 8, up to half of the students gravitated toward a specific erroneous answer, whereas on the other questions incorrect answers were distributed more evenly.

Students were also asked to rate their confidence level on each of the questions on the test (Table 2). Pretest confidence levels were statistically lower in the Introduction to Forensic Science course than in the other two courses (P < 0.005). This parallels the lower scores seen on the pretest in this course. By the end of the semester, however, the students in the two general-education classes had similar confidence levels, which were about 88% higher than their pretest levels. This improvement was statistically significant (P < 0.001). So, even though two courses carried out the ELISA exercise using the epidemiological scenario, whereas the other used the more immunological approach, the levels of improvement on test scores and confidence were similar. Biology majors’ confidence levels were higher than those of the general-education students on the pretest but were statistically lower on the post-test (P < 0.01). Post-test confidence of students in the biology majors’ course increased by 38% (statistically significant; P < 0.001), even though their ability to correctly answer the post-test questions increased by only 25%.

Overall, there was a positive, linear relationship between student scores and confidence levels on the tests (Figure 2). The correlation coefficient for this relationship was statistically significant (r = 0.88, r2 = 0.77; P < 0.001). Two questions (2 and 8) did not fit this pattern. On the pretest, students performed the poorest on question 2, with less than 10% of the students answering correctly, while rating their self-confidence fairly high (above 2.0). On the post-test, student confidence was also relatively high (above 3.0) on question 8, even though only ≤35% of the students answered it correctly. The Cell Biology students also continued to rate their self-confidence about question 2 high (3.2) on the post-test, even though 70% of the students answered incorrectly.

Figure 2.

Correlation between student confidence level and student ability to answer test questions correctly. Average pre-test and post-test results for the 11 questions from all three courses are included (n = 66). The linear regression line for this data is statistically significant (r = 0.88, r2 = 0.77; P < 0.001).

Figure 2.

Correlation between student confidence level and student ability to answer test questions correctly. Average pre-test and post-test results for the 11 questions from all three courses are included (n = 66). The linear regression line for this data is statistically significant (r = 0.88, r2 = 0.77; P < 0.001).

Average student comfort level about executing the ELISA in the laboratory (question 12) increased 67% after carrying out this exercise (Table 2). This increase in comfort level was statistically significant for all three classes (P < 0.001). Before carrying out the exercise, the students in Introduction to Forensic Science had significantly lower comfort levels (P < 0.05) than in the other two courses, but after the exercise it was the Cell Biology students who had the lowest average comfort level (P < 0.05).

This exercise seemed to have a greater impact on general-education students (nonmajors) taking introductory freshman courses than on biology students taking Cell Biology, a sophomore-level course in their major. This can be seen in the higher relative rise in the post-test scores, confidence levels, and lab comfort levels. By the end of the semester, biology majors scored lower than the general-education students for all three of these parameters. It could be that the high confidence levels the biology majors expressed at the beginning of the exercise caused them to focus more on the myriad other challenging topics that are covered in the Cell Biology course. Student interest in this exercise in the Cell Biology lab appeared to be high, but perhaps the activity needs to be modified to provide more of a challenge, such as measuring the color change using a spectrophotometer or microplate reader. The kit manual contains extension exercises, including the use of a standard curve to quantify antigen concentrations using semilog graph paper. Also, using the introductory material of the third scenario of the kit would have delivered more detail about immunology and the ELISA technique. Perhaps the wider blend of students in the general-education courses contributed to their success. The Cell Biology class was almost entirely composed of sophomores who had already taken several other courses together, whereas the general-education courses typically have a mixture of college level and majors. There is contradictory evidence in the literature about student success in major versus nonmajor courses. Sundberg & Dini (1993) reported that in freshman lecture courses, nonmajors started the semester with lower scores on pretests than biology majors, but they found that students performed similarly by the time they took the post-test. Knight & Smith (2010), however, compared separate lecture courses in genetics taught to biology majors versus nonmajors and reported significantly higher motivation, interest, study time, and learning gains among the biology majors. Perhaps more research comparing student learning of majors versus nonmajors needs to be pursued. The use of a single test given to students before and after a learning experience, as done here, is a valuable way of accomplishing this. Assessment of student self-confidence about individual test questions, as well as about performing the laboratory procedure, is recommended.

This ELISA laboratory exercise appeared to be successful, especially in general-education courses. We believe that the lower-than-expected performance by biology majors on the post-test is due to (1) their not being graded on it, (2) less emphasis on immunological principles during the prelab orientation, and (3) not including supplementary exercises. Students seemed to learn a great deal about basic immunology, ELISA techniques, and interpretation of results after carrying out this exercise. Confidence levels also increased. We have used this kit in these same courses for several years and have consistently seen 100% success in the laboratory. An advantage of this kit is that it uses research-grade ELISA plates and students can dispense reagents using adjustable pipettors or the disposable micropipettes supplied with the kit. This allows students to have a state-of-the-art ELISA experience. The three alternate laboratory scenarios and supplementary exercises included in this kit provide the flexibility to adjust the exercise to the specific needs of the course. This exercise offers the kind of inquiry-based approach that is recommended for laboratory courses (Hofstein & Lunetta, 2004) and is adaptable to many types of courses and students.

Acknowledgments

The authors wish to thank Mary Huff and Crista Riggs for allowing us to assess the students in their Cell Biology labs.

References

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