Section11:Initial Concept and Method Development of a Sandwich Immunoassay

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Initial Development Experiment

Goal: Develop a basic working method by determining the antibody which should be the primary/capture antibody and which antibody should be the secondary or detection antibody. Determine the optimum antibody concentrations for both the primary and secondary antibody.

Experiment: Coat the ELISA plate with several dilutions of both antibodies that will be used as part of the sandwich assay. Add the standard to be measured at a high, low and zero concentration. Use both of the antibodies at several concentrations as a secondary antibody. The results of this experiment will determine which antibody is best for both the capture antibody and the secondary antibody. The dilution needed for both antibodies will also be determined.

Reagents: Listed below is the plate type and buffers that will work for the majority of immunoassays. Use these buffers as a starting point.

1. Two antibodies that recognize different epitopes on the analyte.
2. A monoclonal for the capture and polyclonal for the detection antibody tend to yield the best sandwich assay.
3. Greiner immunoassay plate.
4. Coating buffer: 50mM sodium carbonate pH 9.6.
5. Blocking buffer: 1% BSA, TBS, 0.1% Tween-20.
6. Antibody diluent buffer: 1% BSA, PBS or TBS, 0.1 % Tween-20.
7. Wash buffer: PBS or TBS, 0.1% Tween-20.
8. TMB and HRP are used for enzyme/substrate readout.
9. Acid stop buffer.

Protocol:

1. Titrating both the primary capture antibody and the secondary detection antibody are made across a plate using a high, low and zero level of the analyte.
2. Determine the desired working range of the analyte. This will give you the high and low concentrations.
3. Dilute both antibodies in coating buffer at 0.5, 1 and 2 mg/ml and add 100 µl to each well of the 96-well microtiter plate.
4. Incubate the plate containing the primary capture antibody overnight at 4°C then use the next day.
5. Stability of the primary capture antibody bound to the plate can be determined in later experiments.
6. Remove the primary capture antibody solution from the microtiter plates by aspirating or dumping the plate.
7. Add 200 µl of blocking buffer to each well of the 96-well microtiter plate.
8. Incubate the plate for one hour at RT.
9. Remove the blocking buffer from the plate by aspirating or dumping the plate.
10. Dilute the standard in dilution buffer to give a high and low concentration.
11. Zero concentration will give you the NSB.
12. Add 100 µl of the standard to each well in the microtiter plate and incubate for 2.5 hours at RT.
13. Wash the plates 3 times with wash buffer.
14. Dilute the secondary antibody serially at 1:200, 1:1000, 1:5000 and 1:25,000 in diluent.
15. Add 100 µl of detection antibody to each well of the microtiter plate and incubate for 1 1/2 hours at RT.
16. Wash the plates 3 times with wash buffer.
17. Dilute streptavidin-HRP according to manufacture instructions in antibody diluent and add 100 µl to each well in the microtiter plate and incubate for 1 hr at RT.
18. For HRP readout add either OPD or TMB as substrate to allow color development and incubate for 10-20 minutes at RT.
19. Add acid stop reagent to stop the enzyme reaction.
20. Read at 405 nm for TMB/HRP.

Plate Layout for Initial Experiment
Plate 1

Primary capture antibody A
Secondary Antibody	5 µg/ml			2 µg/ml			1 µg/ml			0.5 mg/ml
1:200	H	L	0	H	L	0	H	L	0	H	L	0
	H	L	0	H	L	0	H	L	0	H	L	0
1:1000	H	L	0	H	L	0	H	L	0	H	L	0
	H	L	0	H	L	0	H	L	0	H	L	0
1:5000	H	L	0	H	L	0	H	L	0	H	L	0
	H	L	0	H	L	0	H	L	0	H	L	0
1:25000	H	L	0	H	L	0	H	L	0	H	L	0
	H	L	0	H	L	0	H	L	0	H	L	0

Plate 2

Primary capture antibody B
Secondary Antibody	5 µg/ml			2 µg/ml			1 µg/ml			0.5 mg/ml
1:200	H	L	0	H	L	0	H	L	0	H	L	0
	H	L	0	H	L	0	H	L	0	H	L	0
1:1000	H	L	0	H	L	0	H	L	0	H	L	0
	H	L	0	H	L	0	H	L	0	H	L	0
1:5000	H	L	0	H	L	0	H	L	0	H	L	0
	H	L	0	H	L	0	H	L	0	H	L	0
1:25000	H	L	0	H	L	0	H	L	0	H	L	0
	H	L	0	H	L	0	H	L	0	H	L	0

Results: H=High and L=Low: High ligand concentration in combination with the low ligand concentration will give an indication of the dynamic range.

L=Low: Low ligand concentration will give indication of sensitivity.
0=Zero: Zero ligand will give the non-specific binding (NSB) and indicate if there are background issues.

Determine the Absorbance units (A.U.) that yield the maximum signal to noise ratio or the greatest difference between the high and low analyte concentrations with the lowest variability. These are the conditions that will be selected for the antibody to be used as the primary capture antibody and the dilution of the antibodies to be used in the next experiment.

• If the background signal is unacceptably high (greater than 0.2 A.U.) then run additional experiments varying the plate type, blocking buffers, blocking buffers with a diluent agent like species specific IgG, antibody diluent buffers, wash buffers, and the reporter type.
• If the above general conditions have an acceptable NSB then determine if the dynamic range and sensitivity are in the appropriate range. To improve the sensitivity of the assay, the buffers, timing of incubations and matrix conditions can be varied in the next experiment.
• Antibodies are the reagents that play a major role in the sensitivity and dynamic range of an immunoassay. This is due to the actual antibody affinity for the analyte. If after attempting to develop the assay the sensitivity is still not in the desired range, different antibody pairs will need to be evaluated.

Example: An ELISA was set up to measure the amounts of an LP protein where there is only one polyclonal antibody available. The polyclonal antibody was used as both the primary capture antibody and the secondary detection antibody. Biotinylated antibody was used as the detection antibody.

Reagents:

1. Pab- 0172B 140 µg/ml affinity pure antibody.
2. LP276 230 µg/ml.
3. LP276BT 400 µg/ml biotinylated affinity pure antibody.

Experiment: Follow the above protocol and plate layout

1. Coated the affinity purified antibody at 3 levels: 2, 1 and 0.5 µg/ml
2. Diluted the biotinylated antibody at 3 levels: 1:1000, 1:5000, and 1;25,000
3. Diluted the standard LP276 protein in buffer to 50 ng/ml and 1 ng/ml