ELISA vs. Immuno-PCR vs. SIMOA: Comparison of Protein Detection Tools

Introduction

ELISA, or enzyme-linked immunosorbent assay, is the “gold standard” method for protein detection.1 In this blog, three popular ELISA platforms are reviewed: standard ELISA, immuno-PCR, and single molecule array (SIMOA). These ELISAs will be compared regarding their design principles, sample volumes, instruments, and sensitivities.

How ELISA works

The basic design principle of ELISA depends on the stability and specificity of antibody-antigen interactions. Briefly, a capture antibody that is immobilized to a solid substrate, such as a bead or microplate, binds to a protein-of-interest in a variety of sample types. Such samples may include plasma, serum, cell and tissue lysates, conditioned media, or urine.

When ELISA was first introduced in 1971, an enzyme-labeled antigen was captured by an antibody on cellulose particles.2 The antibody-antigen conjugate was then isolated through repeated centrifugation and washing. Since then, different ELISA formats have been developed, including the sandwich ELISA where a target protein is “sandwiched” between a capture antibody and a labeled detection antibody (Figure 1). This ELISA format is highly specific since two antibodies are required to bind to a protein for detection. Moreover, protein concentrations (i.e., quantitative data) can be determined with a standard curve.

Figure 1. Sandwich ELISAs use two antibodies to target a protein, resulting in high specificity.

The ELISA platforms discussed here – standard ELISA, immuno-PCR, and single molecule array (SIMOA) – are sandwich ELISAs that generate quantitative data. However, they differ in terms of their substrates, detection methods, instruments, sample volumes, and sensitivities.

Standard ELISA

Standard ELISA (sELISA) immobilizes capture antibodies onto a clear plastic 96-well microplate (Figure 2, Table 1). The detection antibody is conjugated to horseradish peroxidase (HRP), which is an enzyme that reduces its substrate, 3,3’,5,5’-tetramethylbenzidine (TMB), to its blue and oxidized form. The change in color is proportional to the amount of HRP and, as a corollary, the amount of target protein in the sample. The HRP-TMB reaction is stopped with the addition of sulfuric acid, which causes the color to change from blue to yellow. The absorbance is measured at 450 nm using a microplate reader.

Figure 2. How standard ELISA works. Standard ELISAs use an HRP-conjugated detection antibody and a colorimetric substrate to detect captured proteins.
Standard Immuno-PCR SIMOA
Substrate 96-well microplate 96-well PCR plate Beads
Detection method Colorimetric PCR Fluorescent
Instrument Plate reader Real-time PCR instrument Dedicated instrument
Sample volume* 100 µL 10-25 µL 125 µL
Sensitivity High Higher Highest
Cost $ $$ $$$

Table 1. A comparison of standard ELISA, immuno-PCR, and single molecule array (SIMOA). * = final volume per replicate after sample dilution.

The sensitivity of sELISAs generally ranges from 1 pg/ml to 100 pg/ml. However, some sELISAs have lowest limits of quantitation (LLOQs) that are as low as sub-pg/ml or as high as 10 ng/ml. As with all ELISAs, the sensitivity is dependent on the sensitivities of the antibodies that are employed.

With a standard 96-well plate, the final sample volume is 100 µL per well. Less volume (50 µL) is possible with “half area” plates where the diameter of the wells is half as wide, but assay sensitivity may also be reduced. The original sample volume used for any ELISA will depend on the optimal sample dilution, which will vary across proteins, sample types, and experiments.

Results are obtained in ~5 hours once the sample has been added to the plate. However, faster turnaround times are possible when the samples and antibodies are added to the plate at the same time. This reduces the number of incubation steps and washing steps. For example, SpeedELISA, which has a 3-hour processing time, incubates the capture and detection antibodies on the plate simultaneously before the sample is added. Other approaches include adding the samples and both antibodies in a single step or adding the sample and detection antibody together. However, decreasing the processing time will decrease the sensitivity (Figure 3).

Figure 3. A comparison of multi-step sELISA and SpeedELISA in terms of sensitivity and detection range. Standard curve data targeting human (A) CD26 and (B) soluble IL-6 receptor (IL-6 sR). The same standard and antibody pairs were used for both assays. Human CD26 and IL-6 sR were analyzed with sELISA and SpeedELISA kits from RayBiotech Life, Inc. (Peachtree Corners, GA; USA) (CD26: sELISA kit cat no. ELH-CD26, SpeedELISA kit cat no. ELHS-CD26) (IL-6 sR: sELISA kit cat no. ELH-IL6sR, SpeedELISA kit cat no. ELHS-IL6sR). OD = optical density.

A primary advantage of sELISA is that there are low-cost kits available that can target thousands of different proteins from numerous species. In addition, sELISAs require little training, produce easy-to-interpret data, and use an affordable microplate reader that is compatible with other assays. These benefits have led to the prevalent use of sELISA in research.

Immuno-PCR (IQELISATM)

Immuno-PCR, also known as immuno-quantitative ELISA (IQELISA), adheres capture antibodies onto a plastic 96-well PCR plate while the detection antibody is conjugated to a unique DNA barcode (Figure 4, Table 1). Using PCR, the double-stranded (ds) DNA barcode is amplified and stained in the presence of barcode-specific primers and a fluorescent dye that binds to dsDNA, respectively. The fluorescence is proportional to the amount of dsDNA and, as such, the amount of target protein in the sample. The fluorescence is measured using a real-time PCR instrument.

Figure 4. How immuno-PCR works. Immuno-PCR uses a DNA barcoded detection antibody and a dsDNA fluorescent dye to detect captured proteins.

An advantage of IQELISA is its small volume requirements, with as little as 10 µL final volume per replicate. For this reason, IQELISA is a particularly attractive option when the sample volume is limited or when samples are hard to obtain. However, IQELISA is more technically challenging than sELISA because it requires more precision pipetting. Furthermore, samples should be run in at least triplicate since the small volume increases the chance of outliers.

DNA is exponentially increased through amplification cycles with PCR. Accordingly, the LLOQ of IQELISA increases 23-fold, on average, compared to sELISA using the same antigen and antibody pair. The increase in sensitivity is antibody-dependent, ranging from no change to 105-fold (Figure 5). 

From sample incubation through data collection, IQELISA takes approximately 5.5 hours. This turnaround time is similar to sELISA.

Figure 5. A comparison of sensitivity for 28 proteins with sELISA and immuno-PCR. The same standard and antibody pairs for each target were used for both sELISA and immuno-PCR ELISA. This figure and Figure 8 used the same sELISA kits and IQELISA kits from RayBiotech Life, Inc.

Single Molecule Array (SIMOATM)

SIMOA is an ultrasensitive ELISA that uses antibody-coated beads and a fluorescently conjugated detection antibody (Figure 6, Table 1). After the beads, sample, and detection antibody have been mixed together, the solution is applied to a cartridge with over 235,000 microwells. Each microwell can hold only one bead. Oil is then added to the cartridge, which pushes the sample across the surface of the microwell array and forms a liquid-tight seal of the wells.

The fluorescence is measured using a fully or semi-automated system equipped with a fluorescent microscope; the instrument is manufactured by the developer of SIMOA (Quanterix; Billerica, MA; USA). Since each well can contain only one bead, the number of microwells with fluorescence is proportional to the amount of target protein in the sample. This enables single molecule detection.

Figure 6. How the single molecule array works. SIMOA uses a fluorescently-conjugated detection antibody and microwells to detect captured proteins.

A key advantage of SIMOA is that it can detect proteins in the femtomolar range. That is, the sensitivity typically ranges from 10 fg/ml to 1 pg/ml. Compared to sELISA and IQELISA, the average fold increase in sensitivity with SIMOA is 465 and 63, respectively.

Once the reagents are loaded into the Quanterix instrument, it takes approximately 3 hours to obtain data. Therefore, the procedural time for SIMOA is shorter than both sELISA and IQELISA.

While SIMOA achieves some of the highest sensitivity of the three platforms reviewed here, this technology also has several drawbacks. First, the number of proteins analyzed with this technology is currently low (~165). Second, SIMOA kits cost more than sELISA and IQELISA kits. Third, the detection instrument is expensive and can only be used for SIMOA, making it a dedicated instrument that requires specialized training to run.

Figure 7. A comparison of sELISA and SIMOA in terms of sensitivity and detection range. Standard curve data generated by spiking purified human (A) IL-6 or (B) TNA alpha into buffer. The same standard and antibody pairs for each target from RayBiotech Life, Inc. were used for both sELISA and SIMOA (IL-6, sELISA cat no. ELH-IL6; TNF alpha, sELISA cat no. ELH-TNF). Y-axis = normalized signals in which the signal of the blank was subtracted. Signal = OD at 450 nm for sELISA or the Average Enzyme per Bead (AEB) for SIMOA. Blank = buffer with no target protein.

Multiplex ELISA

It is worth mentioning that both IQELISA and SIMOA can analyze as many as 3 and 6 targets at one time, respectively. In addition to needing less sample, multiplexing is often more cost effective than single-plex assays for analyzing the same number of proteins. Importantly, multiplexing decreases IQELISA sensitivity while the sensitivity remains uncompromised with SIMOA using the SR-XTM Biomarker Detection System.

Other quantitative multiplexed ELISA options are available, including Quantibody® and RayPlexTM antibody arrays. Quantibody can analyze up to 40 proteins in quadruplicate within 6 hours using 100 µL of diluted sample. Data are acquired using a compatible laser scanner. RayPlex can measure as many as 25 proteins with 25 µL of diluted sample per replicate within 4 hours using flow cytometry. Both antibody arrays have sensitivities similar to sELISA.

Learn how to identify the right antibody array for multiplexed protein detection in our blog, “Multiplex Protein Detection with Antibody Arrays,” or in our video, “Identifying the Right Antibody Array for Your Research.”

Summary

Standard ELISA, immuno-PCR (IQELISA), and SIMOA have unique advantages and disadvantages when compared to each other:

  • sELISA is a simple colorimetric assay with easy-to-interpret data, thus requiring little training. Data are obtained in 5 hours with a microplate reader capable of measuring absorbance at 450 nm. Thousands of different proteins can be targeted with kits that are less expensive than IQELISA and SIMOA kits. However, sELISA has the lowest sensitivity compared to IQELISA and SIMOA.
  • Immuno-PCR uses a real-time PCR instrument with data collected within 6 hours. It has the lowest volume requirements of the three ELISA platforms covered here, but requires more technical expertise for pipetting and data analysis than sELISA. On average, its sensitivity is 23-fold higher than sELISA. Multiplexed detection of 3 proteins is possible, but with decreased assay sensitivity.
  • SIMOA has the highest sensitivity, with an average increase in sensitivity of 465-fold and 63-fold than sELISA and IQELISA, respectively. Data are obtained in 3 hours with an expensive dedicated instrument that requires specialized training. It also requires more sample volume than sELISA and IQELISA. As many as 6 proteins can be analyzed at one time without compromising sensitivity.

Figure 8 demonstrates that, for many proteins, the general improvement in the LLOQ follows this trend: SIMOA > IQELISA > sELISA. However, there are exceptions. For example, IQELISA has the highest sensitivity for human IL-1β at 0.0064 pg/ml, whereas the LLOQ for sELISA and SIMOA is 0.3 and 0.04 pg/ml, respectively.

Figure 8. A comparison of fold increases in sensitivity between sELISA, immuno-PCR, and SIMOA. The same standard and antibody pairs for each target from RayBiotech Life, Inc. were used for all platforms. The targets included 28 human proteins, 3 mouse proteins, and 1 rat protein. (A) 17 proteins with a 0 – 100-fold increase in sensitivity; not including proteins in (B) or (C). (B) Nine proteins with a 0 – 1,000-fold increase in sensitivity; not including proteins in (A) or (C). (C) Two proteins with a 0 – 10,000-fold increase in sensitivity; not including proteins in (A) or (B). Y-axis = fold increase in sensitivity of the first ELISA platform compared to the second ELISA platform described below the X-axis. Horizontal line = median.

Conclusions

One of the most common protein detection tools is ELISA. Here, we compared three popular sandwich ELISA platforms: standard ELISA, immuno-PCR, and single molecule array (SIMOA). Their substrates, detection methods, instruments, sample volumes, sensitivities, and costs are important considerations when selecting the appropriate ELISA for a study. Standard ELISA is the most popular method because low-cost kits are available that can target thousands of different proteins from numerous species. Moreover, it requires little training to run, its data is easy to interpret, and it uses a non-dedicated microplate reader that is compatible with other assays. A key takeaway is that there is a trade-off between ELISA sensitivity and cost: higher sensitivity results in higher cost. The cost considers the price of a kit, training, labor, and required instrumentation. Thus, sELISA is the most affordable option whereas SIMOA is the most sensitive.

For laboratories that do not have the necessary expertise or equipment to analyze their samples with the ELISA platforms discussed here, full testing services are available at RayBiotech Life, Inc. The services provide a full report, including the raw data, standard curve, and quantified protein concentrations.

Do you need help identifying the right ELISA for your study? Please contact us at [email protected].

References
  1. Aydin S. A Short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides (2015)
  2. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry (1971).

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