How do I run an Immunoassay?

Typical Immunoassay Protocol

Preparation

In this example, 1 blank (which is simply the sample diluent), 7 standards, 3 controls, and 37 unknown samples are being tested in duplicate in 1 microtiter plate MoAb-PoAb sandwich immunoassay.

Standards

As each kit typically comes with only 1 vial of a high-concentrate standard which is lyophilized (freeze dried into a powder), it must be reconstituted, and then further diluted with sample diluent. If the lyophilized standard was at 5000pg, when reconstituted with 5mL, it would have a concentration of 5000pg/5mL or 1000pg/mL. To generate a standard curve, it should be serially diluted 1:2 (500uL standard with 500uL sample diluent) to create standards at 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, and 15.13pg/mL.

Controls

The controls may or may not come with a kit, but are often lyophilized and must also be reconstituted, typically with 2mL sample diluent. Controls are usually not diluted further.

Unknown Samples

Any samples thought to have a value higher than the reconstituted standard (at 1000pg/mL) should be diluted with the sample diluent so that it falls between the high standard (at 1000pg/mL) and the low standard (at 15.13 pg/mL). If they do not need to be diluted, samples often can be run neat. However, if the samples have some sort of interfering substance, such as rheumatoid factors, or complement, they may need to be treated prior to running in the assay. If you do not know about your samples, simply run them in the assay once and see where they fall (neat and diluted samples can be run at the same time). If the values are too high, just dilute, and/or treat the samples and run the assay again. Each investigator must determine their own protocols for sample dilutions and pre-assay treatments (see Consultation for assistance from ICT).

Immunoassay Procedure

• Step 1: Add 50 uL per well of assay diluent into every well of the plate.
• Step 2: Add 200 uL per well of your blanks, standards, controls, or samples onto the plate. Each item should be tested in duplicate (in 2 wells).
• Step 3: Cover with plate sealer and incubate 2 hours at room temperature.
• Step 4: Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4 cycles. Blot on paper towels.
• Step 5: Add 200uL per well of PoAb-HRP conjugate solution into every well of the plate.
• Step 6: Cover with plate sealer and incubate 2 hours at room temperature.
• Step 7: Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4 cycles. Blot on paper towels.
• Step 8: Add 200uL per well of TMB substrate solution into every well of the plate.
• Step 9: Incubate 20 minutes at room temperature.
• Step 10: Add 50 uL per well 2N HCl stop solution into every well of the plate.
• Step 11: Read plate at 450nm while subtracting a reference wavelength of 540nm.
• Step 12: Calculate data based on OD values of the unknown samples compared to the known values of the standard curve.

Typical Plate Map

Coating Plates

The first step in making a reliable ELISA is proper coating of the antibody or antigen onto the plate. ICT has specifically formulated two plate coating buffers for coating antibodies and antigens onto polystyrene microtiter ELISA plates. Both coating buffers stabilize coated proteins by maintaining their tertiary three-dimensional structure, allowing for greater binding reactivity with the detection molecule, thereby enhancing the specific signal. 


ICT has developed two coating buffers based on the type of ELISA being developed. For antibody-sandwich ELISAs, use ICT's 5x Universal Antibody Plate Coating Buffer (CB1). CB1 is a unique buffer to coat antibodies onto polystyrene microtiter ELISA plates. It can also be used for antigen-down ELISAs as well. However, for most antigen-down ELISAs, we recommend ICT's 5x Antigen Coating Buffer (CB2). This buffer is targeted for antigen-down ELISAs.

Simply dilute the coat buffer 1:5 (for example, add 100mL 5x coat buffer to 400mL diH20 for a final volume of 500mL), add your protein antigen or antibody, let the solution stir for 15 minutes, and pipette onto the plate. As both coat buffers are concentrated 5x, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm the buffer until all crystals are dissolved. Do not let it boil.

5x Universal Antibody Coating Buffer CB1:

The pH and ionic strength of this buffer have been specifically formulated to create an optimal coating environment which maximizes the adsorption efficiency of most antibodies and some protein antigens onto polystyrene plate surfaces.

This buffer also stabilizes coated antibodies by maintaining their tertiary three-dimensional structure during the adsorption process. By stabilizing the adsorbed antibody, antigenic regions are preserved, allowing for greater binding reactivity with the detection molecule, thereby enhancing the specific signal.


By generating a higher specific signal, a lower concentration of coating antibody may be needed when this buffer is used, thereby saving valuable reagents. There is a finite quantity of antibody that may be coated effectively onto microtiter plates. Use of antibody concentrations in excess of the optimal range will result in surface saturation and clumping on the plate surface. This produces lower specific signals, and higher nonspecific signals.

5x Antigen Coating Buffer CB2:

This buffer is well suited to coat all antigens onto polystyrene plates. CB2 enhances the coating adsorption of antigens onto polystyrene plates by using an alkaline pH binding environment so that this buffer can be used for all antigen-down ELISAs. During the adsorption process, CB2 stabilizes and hydrates coated antigens without compromising their three-dimensional structure. This allows for greater accessibility to detector antibody that are present in sample solutions, thereby enhancing the specific signal. By generating a higher specific signal, a lower concentration of coat antigen may be needed when this buffer is used, thereby saving valuable reagents.

ICT’s proprietary antibody coating buffers contain an antimicrobial agent to retard bacterial growth during the plate coating process, allowing the procedure to be performed at room temperature. Depending on the activity of the coated antibody or antigen, plates may be stored at 2°-8°C (or even at room temperature for hardy proteins) for several months, or even years. 


Both of ICT's coating buffers are supplied as a 5x concentrate to conserve valuable storage space. Simply add 4 parts deionized water to 1 part buffer, mix, and it’s ready to use. No pH adjustment is required. After dilution, the 1x buffer will suppress bacterial growth for up to 1 month at room temperature and 6 months at 2°-8°C. The concentrated 5x buffer will suppress bacterial growth up to 18 months at 2°-8°C. During refrigeration, some salts in the buffer may precipitate. Simply warm the bottle in a water bath (do not boil) and mix. The crystals should go into solution. Which plates should I use?
 Be sure to use a 96-well plate specific for ELISA applications, as 96-well tissue culture plates will cause background problems. For antibody sandwich ELISAs, try Costar EIA/RIA Stripwell plates, and secondarily Immulon II HB plates. For antigen-down ELISAs, we have found that Immulon II plates tend to work best.

How much coating buffer do I need?

Plates can be coated at many different volumes depending on the protein you are coating with. For example, if you coat with 100 uL/well (in a 96-well plate), you will need 9.6 mL of 1x buffer per plate. The 25 mL trial size is enough for 125 mL of 1x buffer = 12-13 plates. You can coat 51 plates with 100mL of 5x size, and 520 plates with the 1L bottles. If you are coating 1000s of plates, we offer each coat buffer in 10L carboys = 5,200 plates.
 If you coat with 200 uL/well, you will need 19.2 mL of 1x coat buffer per 96-well microtiter plate. Therefore, 6 plates can be coated with the 25 mL trial size; 25 plates can be coated with 100 mL of 5x coat buffer; 125 plates can be coated with 500 mL 5x, and 250 plates can be coated with the 1L size. If you are coating 1000s of plates, we offer each coat buffer in 10L carboys = 2,600 plates.

How do I coat plates?

1. Dilute the 5x coat buffer by adding 1 part buffer to 4 parts deionized water (100 mL coat buffer to 400 mL diH2O, yielding a total volume of 500 mL) and mix for 15 minutes.

2. Dilute your antigen or antibody into the coating buffer; coating concentration varies significantly from less than 0.1 ug/mL to over 10 ug/mL.
3. Let the solution stir (10 - 15 minutes) and pipette onto the plate (coating volume generally ranges between 50-300 uL per well).

4. Once added to the plate, incubate the coating solution from 3 - 24 hours at room temperature (RT) protected from light. Minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a covered, humidified storage box.
5. After incubation, dump or aspirate the coating solution out of the wells. 

6. Wash the plate 2 - 4 times with ICT’s ELISA wash buffer.

7. Aspirate and pipette one of ICT’s blocking buffers onto the plate at a higher volume than the coating solution (300-400 uL per well). 

8. Once added to the plate, incubate the blocking buffer from 3 - 24 hours at RT protected from light. Minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a covered, humidified storage box. 

9. Aspirate the block buffer. 

10. The assay can be run at this point, or the plate can be dried and packaged for later use.

11. Dry the plate by letting it sit on the bench top from 2 - 24 hours (but protected from light – loosely cover with aluminum foil), or dry in a drying oven from 2 - 24 hours at RT or warmer.

12. When dry, seal the plate in an air-tight foil pouch with a desiccant and store at RT or 2°-8°C protected from light.

Help! I can't get my assay to work!


Just give us a call at 1-800-829-3194. To help you develop your assay, ICT has provided some background information on ELISA technology online and offers consultation services in assay development.