Tips For Performing Immuno-PCR
August 8th, 2017
What is Immuno-PCR?
Immuno-PCR is an extremely powerful technique that combines the specificity of an enzyme linked immunosorbent assay (ELISA) with the signal amplification of the polymerase chain reaction (PCR). Antibodies specific to the protein target are immobilized onto a microplate, the analyte is captured, and then a second specific antibody which has been coupled to DNA is added. The DNA is amplified and detected by real-time PCR (RT-PCR).
Why is Immuno-PCR Such a Good Technique?
While RT-PCR provides exponential signal amplification, it cannot be used directly for antigen detection. An ELISA is adaptable to any protein, however its sensitivity is not adequate for the detection of analytes of low abundance. Immuno-PCR therefore offers a number of key advantages:
- Extremely low limit of detection
- Suitable for small sample volumes
- Compatible with complex samples such as serum
- Amenable to multiplexing
- Fewer incubation steps than an ELISA, providing higher assay reproducibility
- Rapid time to results
What are the Main Steps of an Immuno-PCR Assay?
An immuno-PCR assay is typically carried out as follows:
- Immobilization of antibodies specific for the protein target to the surface of a vessel
- Washing to remove unbound capture antibody
- Addition of sample
- Washing to remove unbound sample
- Addition of a second specific antibody, coupled to a DNA molecule
- Washing to remove unbound detection antibody
- DNA amplification and detection.
What are the Key Considerations When Setting Up an Immuno-PCR Assay?
When developing an immuno-PCR assay it is suggested that, if possible, the parameters of an existing immunoassay are adapted and optimized. The aim of the optimization process is to maximize the specific readout while keeping the background signal to a minimum.
The success of any immunoassay is dependent on the quality of the reagents that are employed. Immuno-PCR requires the use of highly specific antibodies for capture and detection of the antigen. If either antibody detects multiple epitopes, the background signal of the assay will be increased considerably. Efficient conjugation of the detection antibody to the marker DNA is also essential. Attachment of the antibody to the DNA should be simple to perform, and result in maximal recovery of reagents.
Which Factors Should be Taken Into Account When Developing an Immuno-PCR Assay?
- Plate coating. The capture antibody is bound to the microplate by nonspecific adsorption. Both the choice of coating buffer and the concentration of the capture antibody require optimization. Typically a carbonate or bicarbonate buffer at pH>9 is used for plate coating, and it is usual to use an overnight incubation at 4°C for the coating step. The antibody concentration will vary from one antibody to another, and will be dependent on the affinity of the antibody for its target
- Wash buffer. Once the capture antibody has been successfully coated on to the microplate, the plate should be washed to remove any unbound antibody. Typical wash buffers include Phosphate Buffered Saline (PBS) or Tris Buffered Saline (TBS), often with the addition of a low concentration of a detergent such as Tween 20. Efficient washing is essential to minimize nonspecific binding, and the number and duration of the washes should be optimized
- Blocking solution. After coating the microplate with the capture antibody, the coated wells should be blocked to prevent nonspecific binding and maximize the signal to background ratio. Typical blocking buffers include weak solutions of proteins such as Bovine Serum Albumin (BSA) or fish skin gelatin, diluted in wash buffer. The choice of blocking solution and the duration of the blocking step are variables that should be optimized during assay development
- Sample incubation. Because immuno-PCR is highly sensitive, the sample can often be diluted; this significantly reduces any background effects caused by the nature of the sample material. The sensitivity of immuno-PCR also means that short incubation times are often sufficient for antibody:antigen binding, however the duration of the sample incubation step should always be evaluated during assay development
- Inclusion of relevant controls. As for any well-designed experiment, the inclusion of relevant controls is critical for sensible interpretation of results. A positive control would typically consist of spiked samples containing known concentrations of the antigen, while a negative control would contain the sample material without any antigen. For quantitation of samples containing an unknown concentration of antigen it is necessary to include a standard curve. For accurate results, controls and standards should be diluted in the same matrix as the test samples. Ideally all samples, whether controls or unknowns, should be analyzed in duplicate
- Detection antibody. Conjugation of the detection antibody to DNA is a notoriously difficult and time consuming process, which can result in significant loss of reagents. Our Thunder-Link® PLUS Oligo Conjugation System enables quick and easy covalent attachment of antibodies to oligonucleotides, and the resulting conjugates are ideally suited to immuno-PCR.
Why use Thunder-Link® PLUS for Antibody–Oligo Conjugation?
Our Thunder-Link® PLUS Oligo Conjugation System enables simple and rapid covalent attachment of antibodies to oligonucleotides. The kit overcomes the time consuming and lengthy protocols which are associated with standard conjugation methods, and provides numerous advantages:
- Quick and easy to use – Save time, no specialist knowledge required
- High levels of antibody and oligo recovery
- Covalent bond – Highly stable conjugates produced
- Use your own oligo and antibody, at your desired ratio
- Freeze dried – Ships at ambient temperature, long shelf life
- Stringently QC tested – Consistent high quality, excellent batch-to-batch reproducibility
- Unidirectional chemistry, no risk of crosslinking
- Linking chemistry works at both the 5’ and the 3’ end, providing the ability to combine with other modifications
- Optional postconjugation cleanup step for removal of unbound oligo
- Positive control antibody and oligo included to confirm conjugation success.
What Does the Thunder-Link® PLUS Conjugation Procedure Involve?
The Thunder-Link® PLUS antibody–oligo conjugation procedure involves just a few simple steps. The oligo is added to the oligo activation reagent vial, and the antibody to the antibody activation reagent vial, then both vials are incubated for 30 minutes at room temperature. During this time two desalt columns are washed. The activated oligo mix is passed through one of the columns, and the activated antibody mix is passed through the other column. The activated antibody and oligo are next incubated together for one hour at room temperature, and the conjugate is then ready to use.
Antibody–oligonucleotide conjugates have huge potential to be the platform tool in multiplexed protein diagnostic assays, and Thunder-Link® PLUS provides an ideal solution for the quick and easy generation of these extremely powerful reagents. For any questions regarding our products please contact our technical support team.