The "ACID" solution is sufficiently concentrated to damage whatever it touches (eyes, skin, clothing). Wear goggles and handle with care. Rinse immediately should it get on your skin or clothing. It is phosphoric acid, so it is nontoxic when diluted. (Phosphoric acid is a major ingredient in soft drinks.)
The "SUB" (substrate/peroxide) solution contains organic chemicals and peroxide that are skin irritants and suspected carcinogens. Wear goggles, handle with care, and rinse any spills immediately.
Proteins spontaneously bind strongly and nonspecifically to chemically clean plastic (or glass) surfaces. Most ELISA assays depend upon the ability of protein antigen, or antibody, to bind in this manner to the bottoms of plastic wells in 96-well plates. This is called coating the well.
After the coat, or first layer of protein, is bound, additional layers of antibody or antigen are bound. These, however, must bind specifically only to the protein in the first layer. Therefore it is crucial to block the spontaneous nonspecific binding of these additional layers to the plastic well. One method of blocking nonspecific binding utilizes a protein such as bovine serum albumin to coat any plastic left exposed after the initial coating. A less expensive blocking reagent is a non-ionic detergent with the trade name of Tween-20. It coats bare plastic and prevents protein from binding, but does not remove previously bound protein -- and it does not inhibit antibody:antigen reactions nor the enzymatic reactions employed.
One method for ELISA is to coat the well directly with unknown IgG, which is reported with an enzyme-conjugated anti-IgG antibody (or conjugated second antibody, "direct" or "indirect" ELISA respectively). However, the binding of IgG to the plastic well is greatly dependent upon purity. For example, it is inhibited over 80% when mixed with 20 molecules of albumin per molecule of IgG. Since we wish to quantitate IgG in samples of varying purity, direct coating of the well is unsatisfactory.
We shall use a competition ELISA. All wells will be coated with the same amount of rabbit IgG. The unknown samples or known standards ("competitors" for short) will be mixed in advance with a constant amount of horseradish peroxidase-conjugated goat anti-rabbit IgG ("conjugate" for short). This mixture will be applied to the IgG-coated well. In the absence of competitor, a maximum amount of conjugate will bind to the well (giving maximum absorbance). As the concentration of IgG in the competitor increases, more and more of the conjugate is neutralized in solution, leaving less and less to bind to the well. So a low absorbance means a high concentration of IgG in the competitor. The method in outline:
Half-maximum (I50). The final absorbance is most sensitive to the concentration of the competitor at the half-maximum inhibition by competitor, I50. Often the maximum inhibition that can be obtained with high concentrations of competitor is less than 100%. Therefore it is important to determine it, since half-max is half-way between (i) the maximum absorbance (no competitor), and (ii) the minimum absorbance at maximum competitor.
Standard vs. unknown. Suppose we determine that pure IgG ("standard") produces I50 at 0.65 mg IgG/ml. Now, if we find the dilution of an unknown that also gives I50, at that dilution, there must be 0.65 mg IgG/ml. Then, the concentration of IgG in the unknown is simply the dilution (e.g. 9,500 fold) times 0.65 mg IgG/ml.
Standard Curves. Standard curves are used to determine the concentration of IgG that gives half-maximal inhibition. Results from a series of dilutions of known IgG standard can be plotted, and the concentration giving half-maximal inhibition (I50) determined by interpolation. I50 will occur at approximately 0.5 µg IgG/ml (concentration before mixing with conjugate). This will vary within a several-fold range from plate to plate, day to day, person to person. Therefore a standard curve must be included on each plate!
Unknowns. The goal with each unknown is to use a series of dilutions giving a curve that crosses I50.
Blanks on 96-well Reader. The program we'll use for reading your plates on the 96-well reader will average wells A1, A2, and A3, and the average will be subtracted from the absorbances of the remaining 93 wells.
You will do a series of experiments. Each experiment is done in one 96-well plate. For each plate, you must first list the questions you want the plate to answer. Then you plan the plate in detail, and execute it. After you have the results, you may find that you need to do the plate again, either because your technique was not good enough, or because the design of the plate wasn't good enough. Once your experiment has answered the original question(s), you can complete the design details for a new plate that addresses additional questions. Your goal is to do enough plates to determine the concentrations of IgG in your unknown samples. The number of plates will vary from person to person depending on design and technique.
The first step is to plan your first plate. The ultimate question for the first plate is What concentration of IgG gives half-maximal inhibition? In order to answer this question, you'll have to answer some key questions about the method itself by the use of controls. Your first plate should have only controls plus a standard curve, all in quadruplicate. After you have succeeded in getting satisfactory results you can begin doing plates with unknowns. (What does "satisfactory" mean here?)
Controls. Think about what controls you need and what information each control gives you. In this type of experiment, a "control" is a test that establishes how much certain component(s) of the reaction contribute to the absorbance, in the absence of other component(s). Therefore each control involves omitting some component(s) of the reaction. Decide which of the possible controls are essential and which are optional.
Assay parameters. In the detailed procedure below, most of the assay parameters have already been worked out for you. But you still need to make decisions such as:
Check the design of each of your plates with an instructor before executing it. Every plate must include enough of a standard curve to confirm the I50.
Premixing conjugate with competitors.
Binding conjugate to the coat.