Indirect ELISA protocol

This assay kit uses the Indirect-ELISA principle. This variation of the ELISA assay is similar to the Western Blot, where a secondary antibody binds to a primary antibody. The indirect technique can offer splendid flexibility. With this type of ELISA, several primary antibodies can be used with one labelled secondary one. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of the target protein. You can calculate the concentration of the protein of interest in the samples by comparing the OD of the samples to the standard curve.

Materials

ELISA microtiter plate
Antigen/Analyte
Wash buffer
Detection antibody
Standard
Standard/Sample diluent
Antigen/Analyte diluent
Streptavidin-HRP diluent
Substrate
Stop solution
Plate sealers
Microplate reader
Pipettes and pipette tips
Microplate washer
Micro-oscillator
Deionized water graduated cylinder
Polypropylene test tubes
Incubator

Sample collection and storage

1. Serum
Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

2.Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately, or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

3.Urine
Use a sterile container to collect urine samples. Remove particulates by centrifugation for 15 minutes at 1000×g at 2-8°C. Collect the supernatant to carry out the assay

4. Other biological fluids
Centrifuge samples for 20 minutes at 1000×g. Collect the supernatant and assay immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles. Avoid haemolytic and hyperlipidaemia samples for serum and plasma. Dilution: Dilute samples at the appropriate multiple (recommend carrying out a pre-test to determine the dilution factor).

Reagent preparation

Bring all reagents to room temperature before use. If crystals have formed in the concentrate bring the reagent to room temperature and mix gently until the crystals have completely dissolved. It is recommended to test in duplicates.

Standard: Add Standard/Sample Diluent into freeze-dried standard, sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Prepare EP tubes containing Standard/Sample Diluent and carry out a serial dilution. Any remaining standard solution can be aliquoted and stored at -20°C to -70°C.

Washing

Aspirate each well and wash, repeating the process 2 times for a total of 3 washes. Wash by filling each well with Wash Buffer using a squirt bottle, manifold dispenser, or auto washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining wash buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

Assay procedure

1. Coat the plate with the antigen, cover and incubate overnight at 4 C.

2. Wash three times with wash buffer.

3. Add the blocking buffer and incubate for 1 hr at 37 C.

4. Wash four times with wash buffer.

5. Add the samples and standards to the chosen wells.

6. Incubate for 90 mins at 37 C/overnight at 4 C.

7. Wash three times with wash buffer.

8. Add the detection antibody in wash buffer to the chosen wells.

9. Incubate for 1 hr at 37 C.

10. Wash three times with wash buffer.

11. Add the substrate.

12. Incubate at room temperature until satisfactory colour change can be seen.

13. Add the stop solution.

14. Read the absorbance values.