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Figure 1: Enlarged view. FIG. 1. Two-dimensional representation of cells bound to a microtiter well, showing three different types of epitopes (antigenic determinants, , , ) expressed on each cell. (Samuel Fan, Bradley University, Peoria, IL)

Figure 2: Enlarged view. FIG. 2. The microtiter well from Figure 1, with blocking agent () added in excess. (Samuel Fan, Bradley University, Peoria, IL)

Figure 3: Enlarged view. FIG. 3. The microtiter well from Figure 2, after washing. Previously free binding sites on the well have now bound blocking agent, and are no longer available to bind proteins from this point on. (Samuel Fan, Bradley University, Peoria, IL)

Figure 4: Enlarged view. FIG. 4. The microtiter well from Figure 3, with antibodies ( ) added. The antibodies (primary antibodies) are reactive with only one of the three epitopes ( ) on the plate-bound cells. (Samuel Fan, Bradley University, Peoria, IL)

Figure 5: Enlarged view. FIG. 5. The microtiter plate from Figure 4, after washing. Only the primary antibodies that have reacted with the cell-bound epitope remain. Antibody-antigen binding being dependent on equilibrium, a small fraction of specific epitopes would also be unbound, but there are not enough epitopes shown here to show that effect. (Samuel Fan, Bradley University, Peoria, IL)

Figure 6: Enlarged view. FIG. 6. The microtiter plate from Figure 5, with secondary antibodies () added. The secondary antibody specifically reacts with the primary antibody, and is conjugated to an enzyme (peroxidase in this case,), and is added in excess of the first. Therefore, nearly all the available binding sites on the plate-bound primary antibodies should be bound to second antibody. (Samuel Fan, Bradley University, Peoria, IL)

Figure 7: Enlarged view. FIG. 7. The microtiter plate from Figure 6, after washing. Excess secondary antibodies have been washed away. The amount of enzyme indirectly bound to the plate is directly related to the amount of primary antibodies bound. (Samuel Fan, Bradley University, Peoria, IL)

Figure 8: Enlarged view. FIG. 8. The microtiter plate from Figure 7, with substrate () for the enzyme just added, before products are formed. (Samuel Fan, Bradley University, Peoria, IL)

Figure 9: Enlarged view. FIG. 9. The microtiter plate from Figure 8, after incubation. Products () have been formed. The colored product can be quantified by spectrophotometry, either in a microtiter plate reader or by loading the product into a micro-volume cuvette (or a 4 mL cuvette) with appropriate dilution to fill the cuvette. The rate of product formation is dependent on the amount of enzyme bound, which is dependent on the amount of secondary antibodies bound, which is in turn dependent on the amount of primary antibodies bound to their cognate epitope. (Samuel Fan, Bradley University, Peoria, IL)

Figure 10: Enlarged View. FIG. 10. A sample result from an ELISA using ABTS as substrate for peroxidase. In the center of the plate are 4 sets of 4 wells each (rows C – F, columns 5 - 8), showing a gradation of green product. The intensity of the green color indicates the level of expression of the epitope detected.  (Samuel Fan, Bradley University, Peoria, IL)

Figure 11: Enlarged View. FIG. 11. Two-dimensional representation of "capture antibodies" () bound to a microtiter well. These antibodies specifically react with a single epitope on the antigen to be detected. (Samuel Fan, Bradley University, Peoria, IL)

Figure 12: Enlarged View. FIG. 12. The microtiter well from Figure 11, with blocking agent ( ) added in excess. (Samuel Fan, Bradley University, Peoria, IL).

Figure 13: Enlarged View. FIG. 13. The microtiter well from Figure 12, after washing. Previously free binding sites on the well have now bound blocking agent, and are no longer available to bind proteins from this point on. (Samuel Fan, Bradley University, Peoria, IL)

Figure 14: Enlarged View. FIG. 14. The microtiter well from Figure 13, with a limiting amount of a sample added. The sample contains 3 types of molecules, including the antigen to be detected (), and two that are not reactive with the plate-bound antibody ( , ). The detected antigen carries three different types of epitopes (antigenic determinants, , , ), but the plate-bound antibodies (capture antibodies) are reactive with only one of the three epitopes ( ). (Samuel Fan, Bradley University, Peoria, IL)

Figure 15: Enlarged View. FIG. 15. The microtiter plate from Figure14, after washing. Only the antigens that have reacted with the cell-bound antibodies remain. (Samuel Fan, Bradley University, Peoria, IL)

Figure 16: Enlarged View. FIG. 16. The microtiter plate from Figure 15, with secondary antibodies () added. The secondary antibody specifically reacts with the primary antibody, and is conjugated to an enzyme (peroxidase in this case), and is added in excess of the first. Therefore, nearly all the available binding sites on the plate-bound primary antibodies should be bound to second antibody. (Samuel Fan, Bradley University, Peoria, IL)

Figure 17: Enlarged View. FIG. 17. The microtiter plate from Figure 6, after washing. Excess secondary antibodies have been washed away. The amount of enzyme indirectly bound to the plate is directly related to the amount of antigen bound. (Samuel Fan, Bradley University, Peoria, IL)

Figure 18: Enlarged View. FIG. 18. The microtiter plate from Figure 17, with substrate (, ) for the enzyme just added, before products are formed. (Samuel Fan, Bradley University, Peoria, IL)

Figure 19: Enlarged View. FIG. 19. The microtiter plate from Figure 18, after incubation. Products ( ) have been formed. The colored product can be quantified by spectrophotometry, either in a microtiter plate reader or by loading the product into a micro-volume cuvette (or a 4 mL cuvette) with appropriate dilution to fill the cuvette. The rate of product formation is dependent on the amount of enzyme bound, which is dependent on the amount of secondary antibodies bound, which is in turn dependent on the amount of antigen bound to their capture antibody. (Samuel Fan, Bradley University, Peoria, IL)

Figure 20: Enlarged View. FIG. 20. The microtiter plate from Figure 19, after the reaction has been stopped. The blue product has been converted by the stop solution to yellow (). (Samuel Fan, Bradley University, Peoria, IL)

Figure 21: Interactions between antigen and antibodies during the steps of an ELISA Assay. FIG. 21. The Enzyme-Linked Immunosorbent Assay (ELISA) is a technique that can be used for a variety of purposes, including the detection of specific antigens in serum through the use of antibodies. This type of ELISA, known as an "indirect ELISA" is an immunological technique used in medical diagnoses to detect the presence of pathogens. Serum is plated into wells where antigens will attach (step A). A primary antibody that can recognize the antigen is then added. If the antigen is present, the primary antibody will bind to the antigen (step B). A wash step (step C) is then performed to wash away unbound primary antibody. Antibodies will remain in the well where the antigen for detection is present. Antibodies in the wells where the antigen is not present will be washed away. A secondary antibody conjugated to an enzyme (i.e. horseradish peroxidase, symbolized by a red dot in this figure) that can recognize the primary antibody is then added (step D). A subsequent wash step is performed to wash away unbound secondary antibody. A second wash step (step E) is performed to wash away unbound secondary antibody. Secondary antibodies in the wells where primary antibodies are present will remain. Secondary antibodies in the wells where primary antibodies are not present will be washed away. A substrate for the enzyme is then added (step F). The enzyme-substrate reaction serves to show whether the secondary antibody has bound to the primary antibody which has bound to the antigen. One substrate for horseradish peroxidase is 3,3',5,5' – tetramethylbenzidine (TMB) – a colorless solution. In the presence of HRPO, TMB will turn blue. The presence of a color change indicates presence of the antigen. The colorimetric change can be measured using a spectrophotometer and a standard curve to determine the concentration of antigen present in the serum sample. (Erica English, Alexandra Jantorno, and Brian M. Forster, Saint Joseph's University, PA)

Figure 22: ELISA result using Bio-Rad's ELISA Immuno Explorer Kit. FIG. 22. This kit uses as its primary antibody a rabbit anti-chicken antibody.  This antibody recognizes chicken gamma globulin. The secondary antibody is goat anti-rabbit antibody conjugated to HRPO.  Wells 1 and 2 are positive control serum. The blue color in the wells shows that chicken gamma globulin was detected. Wells 3 and 4 are negative control serum. The lack of blue color in the wells shows that chicken gamma globulin was not detected. Wells 5 through 8 are "unknown" serum samples to determine whether chicken gamma globulin can be detected. The image shows that chicken gamma globulin was not detected in sample 1, but was detected in sample 2. (Erica English, Alexandra Jantorno, and Brian M. Forster, Saint Joseph's University, PA)

This kit uses as its primary antibody a rabbit anti-chicken antibody. This antibody recognizes chicken gamma globulin.  The secondary antibody is goat anti-rabbit antibody conjugated to HRPO. Wells 1 and 2 are positive control serum.  The blue color in the wells shows that chicken gamma globulin was detected.  Wells 3 and 4 are negative control serum. The lack of blue color in the wells shows that chicken gamma globulin was not detected.  Wells 5 through 8 are "unknown" serum samples to determine whether chicken gamma globulin can be detected.  The image shows that chicken gamma globulin was not detected in sample 1, but was detected in sample 2. Normal 0 false false false EN-US X-NONE X-NONE

Figure 23: ELISA result using Bio-Rad's ELISA Immuno Explorer Kit (Labeled view). FIG. 23. (Labeled view). This kit uses as its primary antibody a rabbit anti-chicken antibody. This antibody recognizes chicken gamma globulin. The secondary antibody is goat anti-rabbit antibody conjugated to HRPO.  Wells 1 and 2 are positive control serum. The blue color in the wells shows that chicken gamma globulin was detected. Wells 3 and 4 are negative control serum.  The lack of blue color in the wells shows that chicken gamma globulin was not detected. Wells 5 through 8 are "unknown" serum samples to determine whether chicken gamma globulin can be detected. The image shows that chicken gamma globulin was not detected in sample 1, but was detected in sample 2. Normal 0 false false false EN-US X-NONE X-NONE