2D4 vs R5: Choosing the Right Gluten ELISA Technology

2D4 vs R5: Choosing the Right Gluten ELISA Technology

If you test food for gluten, you need to see both sides of the story – native gluten and the deamidated forms that show up after processing. R5 antibody does well on native gluten but loses affinity when deamidation is present. 2D4 was designed to keep an R5-like profile on wheat, barley, and rye, and it also sees deamidated gluten [1]. That’s the practical difference.

Thermal/chemical steps and functional ingredients can deamidate gluten (e.g., wheat protein isolate used as an emulsifier or texturiser in sauces, fillings and even some pasta applications). Deamidation alters epitopes so that R5’s affinity is markedly reduced, risking under-reporting in processed matrices [1]. In contrast, 2D4 binds native and deamidated prolamins across wheat/rye/barley.

Below is what an accredited external lab found when they compared our InviLisa® Gluten ELISA (2D4) against kits based on the R5 and G12 antibodies across several reference materials [2]. The study normalised units and ran two independent analysts.

Key takeaways at a glance

• Blank matrix: InviLisa® reported <2.5 mg/kg; the R5 kit reported <5 mg/kg. The G12 kit gave measurable signals (~4.6–5.1 mg/kg), suggesting background on a gluten-free breadcrumb blank.
• Proficiency (cake mix, assigned 41.6 mg/kg): InviLisa® means were 42.2 and 50.6 mg/kg (two prep/dilution entries). R5 means were 35.1 and 45.6 mg/kg. G12 trends were higher with broader spread.
• Internal QC (incurred biscuit crumb): InviLisa® mean 20.4 mg/kg; R5 23.9 mg/kg; G12 37.0 mg/kg with higher variation.
• PWG-gliadin calibrant: PWD-gliadin is a reference material that has been produced under the guidance of the Prolamin Working Group (PWG). InviLisa® targeted 25 mg/kg and read 23.6–25.1 mg/kg. R5 targeted 40 mg/kg (separate setup) and read ~32.7–33.9 mg/kg. The G12 kit targeted 80 mg/kg but read roughly ~105–123 mg/kg depending on analyst and calculation method. In short: InviLisa® tracked closest to its target; G12 over-estimated; R5 trended low vs its target.

And when a separate FAPAS dataset looked only at precision on the same cake mix: 2D4 CV ≈ 11.5% vs R5 CV ≈ 25.5% (means 46.4 vs 40.3 mg/kg; SD 5.4 vs 10.3). Same sample,  better precision (or ‘tighter results’) with 2D4.

What this means in day-to-day testing

• Precision you can see. Across analysts, InviLisa® gluten results showed lower %CVs than R5 on the proficiency and calibrant materials. That’s useful when you need repeatability as results near 20 ppm (parts per million or mg gluten/kg).
• Trueness to calibrant. On PWG-gliadin, InviLisa® readings sat at the top of the target range; R5 skewed low vs its own target; G12 skewed high. If you consider bias, this matters.
• Blanks stay blank. InviLisa® and R5 held at “below limit” on the gluten-free breadcrumb blank. Whereas, low positives were observed on G12 . That can waste time on needless investigations.
  
  

Why 2D4 helps with processed foods

R5’s affinity is observed to reduce by about 125-fold with deamidated gluten detection. So if your matrix or ingredient includes wheat protein isolate or other deamidated forms, an R5-only assay may under-report. 2D4 recognises a consensus motif (XPXQPFX) and maintains detection on native and deamidated gluten [1]. That’s the design goal, and it’s backed by peer-reviewed data.

Where does deamidated gluten show up? In common places: emulsifiers, gelling aids, fortifiers, meat binders, baked goods, sauces, soups, even wine fining – often labelled as wheat protein isolate. These are not isolated cases; they’re common place ingredients in certain foods.

What R5 is (still) great for – and where scope matters

R5 remains the Codex Type 1/AOAC OM method for gluten measurement in defined matrices and is widely recognised in standards and audits. Final Action (2016) narrowed the matrix claim to rice- and corn-based foods – appropriate for many dry gluten-free categories, but not a blanket endorsement for heavily processed or deamidated systems [3].

Practical take-away: keep using R5 where it is mandated or clearly within scope. For high-processing/likely deamidation scenarios, add a method that can see those forms – this is where 2D4 helps.

Practical notes on the InviLisa® Gluten ELISA

Beyond antibody specificity, the 2D4 ELISA simplifies routine throughput:

• Faster extraction: approx. 30 min for 10 samples vs. ~120 min for a typical R5 workflow compared side-by-side.
• Odourless reducing extractant (no fume hood) and tannin-binder option for polyphenol-rich matrices (e.g., tomato-based sauces).

• Reported LOD/LOQ window and shorter incubations support higher sample coverage per shift.

• Reportable range: 2.5–50 mg gluten/kg under the standard protocol; extendable by dilution when needed (e.g. 5–100 mg/kg).

Bottom line

If your products can contain deamidated gluten – and many processed foods can – R5 alone leaves a blind spot. 2D4 closes it. In the independent study, the InviLisa® Gluten ELISA showed tighter precision, closer alignment to the calibrant, and clean blanks – without inflating results. It sees what R5 can miss and holds steady on the everyday samples you test.

Notes: The results above relate to the materials and methods used in the study. They’re shown here to help you compare kit behaviour in typical food testing scenarios.

Why we keep saying this:  The science is clear – R5 loses affinity on deamidated epitopes, while 2D4 covers both states and targets a consensus motif. That’s the core reason InviLisa® reads the whole picture. 

REFERENCES

1. 1. Benoit L, Ryan K, Treloar T, Tranquet O, Masiri J, Ryan A, Ryan M, Samadpour M. Dual detection of native and deamidated gluten residues using the novel monoclonal antibody, 2D4. Journal of Cereal Science. 2022 Nov;108:103585. doi:10.1016/j.jcs.2022.103585.
   2. Invitek Diagnostics. InviLisa® ELISA Kits Whitepaper. October 2025.
   3. Lacorn M, Dubois T, Weiss T, Zimmermann L, Schinabeck T-M, Loos-Theisen S, Scherf K. Determination of Gliadin as a Measure of Gluten in Food by R5 Sandwich ELISA RIDASCREEN® Gliadin Matrix Extension: Collaborative Study 2012.01. Journal of AOAC INTERNATIONAL. 2022 Mar–Apr;105(2):442–455. doi:10.1093/jaoacint/qsab148.