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 which are skin irritants and suspected carcinogens. Wear goggles, handle with care, and rinse any spills immediately.

The goals of this exercise are to determine the concentrations of rabbit IgG in several unknown samples, such as your starting normal rabbit serum, your dialyzed salt precipitate, your DEAE pool, and if there is time, your sephadex pools. To accomplish this, a standard curve will be determined using IgG of known concentration. Since you already know the total protein concentration in each sample based on A₂₈₀, after you determine [IgG] you can calculate the purity at each stage. This will enable you to see whether each step succeeded in increasing the purity significantly.

One method for ELISA is to coat the well directly with unknown IgG, which is reported with an enzyme-conjugated anti-IgG antibody. 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 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 (maximum signal). 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 signal means a high concentration of IgG in the competitor. The method in outline:

1. In a separate container (not the final test well), mix variable concentrations of rabbit IgG "competitor" with a constant amount of "conjugate".
2. Coat the test wells with a constant amount of rabbit IgG, rinse.
3. Add conjugate-competitor mixture to coated well, incubate, rinse.
4. Add constant amount of substrate to well, allow color to develop.
5. Add acid to well to stop reaction.
6. Read absorbances of wells.

• COAT (rabbit IgG in PBS azide).
• PBST (phosphate buffered saline/tween; diluent for competitors). Tween-20 is a nonionic detergent which coats bare plastic and prevents protein from binding. It does not remove previously bound protein, and does not inhibit antibody:antigen binding or the enzymatic reactions employed.
• STD (standard IgG competitor, µg/ml will be announced).
• CONJ (horseradish peroxidase conjugate of goat anti-rabbit IgG or F(ab')₂).
• SUB (substrate for peroxidase, tetramethyl benzidine + peroxide in dimethylformamide).
• ACID (1 M phosphoric).
• Samples from each stage of your work with rabbit immunoglobulin ("unknowns").

Endpoint. I recommend 35% inhibition as an endpoint ("I₃₅"). In other words, when the unknown gives 35% inhibition, it is at the same concentration as a known standard that gives 35% inhibition. (Often inhibition reaches a plateau at around 70%, hence a 50% inhibition endpoint may be problematic.)

Standard Curves. Results from a series of dilutions of known IgG standard can be plotted, and the concentration giving 35% inhibition determined by interpolation. I₃₅ will occur at approximately 0.5 µg/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 35% inhibition. To minimize confusion, in plans and graphs, express all IgG concentations and competitor dilutions as they are BEFORE MIXING WITH CONJUGATE.

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 you have answered the question(s), you can complete the design details for a new plate that addresses new questions. Your goal is to do enough plates to determine the concentrations of IgG in your 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 inhibits the signal by 35%? In order to answer this question, you'll have to answer some more basic 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 which establishes how much certain component(s) of the reaction contribute to the signal, 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:

• Exactly which controls actually to do.
• How many replicates for each combination: singlicate, duplicate, triplicate, quadruplicate, quintuplicate?
• For each unknown, you'll do a serial dilution. What is the highest concentration of each unknown you need to test? In other words, what dilution do you use at the start of the serial dilution?
• How many dilution steps do you need in each serial dilution?
• What size dilution step in the serial dilutions: 2-fold, 3-fold, 5-fold, or 10-fold steps?
• What kinds of containers to do your dilutions in: test tubes or wells?
• How to arrange all the tests in each plate (the "plate layout").

Check the design of each of your plates with an instructor before executing it. Every plate must include enough of a standard curve to verify the I₃₅.

1. Wear goggles at all times (acid droplets might inadvertantly come sailing through the air from someone else ...).

   Premixing conjugate with competitors.

2. Make your serial dilutions of the competitors.
3. Mix each competitor with an equal volume of conjugate. Conjugate is expensive. For each mixture, don't make more than twice the volume that you plan to consume in the wells.
4. Premixtures of competitors with conjugate should be allowed to react at room temperature for at least 20 minutes. Longer is OK (even hours), but be aware that inhibition of signal will be slightly greater with longer times. (What is happening during this time?)

   Coating wells.

5. Put only the wells to be coated with IgG in the well holder. Push the wells down firmly (so they won't flip out during rinses).
6. Add 50 µl "COAT" to each well. Tap the plate sideways against the bench to make sure the entire bottom of each well is covered.
7. Incubate 5 minutes. (The time here is not critical because at the concentration used, by 5 minutes, the well bottom is saturated.)
8. Rinse. Flip out the well contents into sink. Fill the wells with tap water, and flip out. Repeat 4 times for a total of 5 rinses. (It is OK if the coated well bottoms dry, but for reproducibility, it is best if all wells are uniformly dried or not. If you need to hold coated wells for later use on the same day, keep them filled with tap water and flip them out just before use. If you are going to store the coated wells between classes, store them dry.)

Binding conjugate to the coat.

9. Put 50 µl of premixed competitor + conjugate per well according to your plan. Change pipet tips for each series. Within each series, if you start with the highest concentration of competitor and move towards lower concentrations of competitor, you can use one tip for the whole series.
10. Incubate at room temperature for 15 minutes. (Timing should be accurate here since conjugate binding is not saturated under these conditions.)
11. Rinse 5 times with tap water.

    Enzymatic reaction.

12. Add 50 µl "SUB" (TMB/P substrate) per well.
13. Incubate 15 minutes, mixing several times during the incubation by tapping the plate sideways against the bench. Time here should be controlled carefully.
14. Add 50 µl "ACID" to each well. (Squirt into well from above to avoid contaminating the tip with color, and to allow you to use one tip for all wells.)
15. Read absorbances at 450 nm.
16. When you are sure you have your data, flip well contents into sink, rinse several times, and push the wells out of the well holder into the wastebasket. Save the well holder!

Data analysis.

17. Plot your data on semilog graph paper: concentration of known (µg IgG/ml) or fold-dilution of unknown on the abcissa (bottom axis), and A₄₅₀ on the ordinate (side axis).
18. Interpolate to find the concentration or fold-dilution giving I₃₅.
19. Calculate the IgG concentrations in your unknowns.
20. You now have total protein (from A280) and IgG (from ELISA). For each unknown, calculate the percentage of total protein which is IgG.

    Lab notebook.

21. There is no formal report for your ELISA work. Your lab notebook will be handed in and graded for the semester's work. For ELISA, you should have a clear summary list of questions and answers, with brief comments and interpretation. Each answer should cite a particular plate number in the series of plates you did.
22. You and your lab partner will be analyzing the same samples. Include a table comparing results, and briefly discuss any signficant discrepancies.
23. Before handing in your lab notebook, add a table of contents with page numbers so, for example, we can find your ELISA summary easily, then find the section for each cited plate easily. Review the Policies guide handed out at the beginning of the semester (and on the web) for other lab notebook requirements.