Comparative accuracy of 6 common allergy panels

The article is sponsored by nextmune.

Background

Allergen-specific IgE serology has been available since the 1990s. Most commercially available serological tests now use ELISA plates coated with crude allergen extracts and one or more monoclonal antibodies or soluble IgE receptors to capture IgE.

One of the challenges in evaluating the accuracy of serology testing is knowing the real IgE sensitization profile of each patient.The availability of canine specific monoclonal IgE now allows for the creation of artificial samples with a known, predefined,IgE sensitization profile, thus facilitating the evaluation of serological test performance.

Objectives

In this study, we aimed to compare the accuracy of six allergen specific IgE serological assays available in the US, using individual and mixed canine IgE monoclonal antibodies specific for different allergens.

Methods

Three dog allergen-specific monoclonal antibodies were created either by caninization of human allergen-specific monoclonal antibodies (two antibodies: Bio-Rad Abd Serotec, Neuried, Germany) or by substituting the dog IgG backbone with the dog epsilon chain in an allergen-specific IgG monoclonal antibody (one antibody; Protogenix, Schiltigheim, France). These two antibodies were specific for Phl p 5 from Timothy grass (Phleum pratense), Der f 2 from house dust mite (Dermatophagoides farinae) and a subfamily of non-specific lipid-transfer proteins (nsLTPs) from various plants. The single antibody recognizes Pla a 3 from both American Sycamore (Platanus acerifolia) and Oriental plane tree (Platanus orientalis). It also recognizes Mal d 3 from apple (Malus domesticus) and Zea m 14 from corn (Zea mays).

Artificial“sera” were generatedin Nextmune’s Spainlab by adding3% of bovine serumalbumin (Omnilab, Germany)tophosphate-buffered salineandcaninered blood cells for a more natural color.

The “sera” were spiked with the three monoclonal allergen-specific mAbs, alone or in combination, to create samples with the following characteristics and initial values on the PAX(table 1A,B).We also created two negative controls:one without anyI gE monoclonal antibodies and one with a high concentration of Der f 2-specific IgG-B monoclonal antibody to verify the specificity of the IgE capture reagents used by the serological tests.

The “sera” were then sent to our Phoenix, Arizona laboratory aliquoted, blinded and coded.The PAX test was repeated on the coded samples received in September& November. The coded sample aliquots were also sent out via veterinary dermatologists for further serological allergen panels.

The following additional serological tests were performed:

September

• Allergen Regional Panel(IDEXX)
• Allercept Companion Animal (HESKA)
• Liquid Gold(VARL)

December

• NelcoVetAllergyPanel(NELCO)
• Extended Universal Panel(PetPreferred Dx)
• Liquid Gold(VARL; second test)

The analysis was limited to the evaluation of environmental panels and that of reactivities (components, if available, or extracts otherwise) corresponding to the monoclonal allergen-specific IgE present in the samples. For the PAX, we thus reviewed the results for the Phl p 5, Der f 2, and Pla a 3 components; for the two other extract based tests, we used the results for Timothy grass, Sycamore tree and Dermatophagoides farinae. Each test’s positivity was determined according to the manufacturer’s recommendation:

• PAX >= 28ng/mL
• Allergen Regional Panel (IDEXX) >= 80 EAU
• Allercept (HESKA) >= 11 HERBU
• Liquid Gola VARL >= Class 1
• Nelco-Vet: at least LP (low positive)
• Pet Preferred Dx: Intensity >= 5

In this study, we labeled the result as “CORRECT” if the serological test was positive for the component (s) or extract(s) corresponding to the added allergen-specific IgE monoclonal antibodies, Any other scenario (e.g., an added IgE monoclonal testing negative or one not included testing positive) was categorized as “INCORRECT.” Finally, since the same amount of monoclonal allergen-specific IgE was added to four of the 12 samples (all those with “low” amounts), we could then calculate the coefficients of variation (reproducibility) for each of the three serological tests.

Results

The expected and obtained reactivities for the different mixes are described below for each lab.

Nextmune Results

In blinded tests conducted in September and November 2025, the PAX test correctly identified all expected reactivities, identifying both the correct mAb composition of the artificial sera, and which vial contained the higher IgE level when the tubes contained only a single IgE mAb.

The coefficient of variations were excellent, ranging from 15% to 35%, indicating that the PAX test has high reproducibility.

IDEXX Results

The IDEXX test reported results matching the original compositions in only one-third of samples. Among the correct identifications were the two vials containing only low and high concentrations of Timothy grass (Phl p 5) IgE, with the test correctly identifying which sample had the higher concentration. The Sycamore(Pla a 3) results were consistently negative, even with the high concentration sample, indicating that their Sycamore extract contained low levels of Pla a 3. The D. farinae extract yielded only one positive, even though samples 1, 7, and 10 had the same low amounts of Der f 2 IgE. Importantly, even though sample 2 contained a higher concentration of Der f 2 mAb than samples 1, 7, 8, and 10, the result was negative for that sample. One can infer that the D. farinae extract used in the IDEXX test contains low amounts of Der f 2. The coefficient of variations ranged from 42% to 135%, indicating low to average test reproducibility.

HESKA Results

HESKA’s Allercept test correctly identified the specific IgE composition in 42% of the samples. The results for sample 10, which contained a mix of all three mAbs, had to be labeled “CORRECT” per our definition, but this was due to the Allercept test yielding positives for all 82 out of 83 possible results - including Timothy, Sycamore and D. farinae. One could argue that these results should be marked “INCORRECT” as most were false positives.

HESKA Results Continued

An unexpected result was that 4/12 (33%) samples were positive for nearly all allergens in the Allercept test, even though the sample contained only one, two or three IgE mAbs. This very high number of false positives was not attributable to a specific mAb, as sample 8

contained a similar amount of nsLTP IgE as samples 5, 9, and 10, but it only had 12/83 positives (14%) instead of 74-82 of 83 (89-99%) for the other three. The coeffcient of variations ranged from 12% to 60%, indicating that the Allercept test has a mediocre reproducibility level.

VARL Results

The VARL test was repeated on both sets of artificial sera, as the first results (October 2025) all came back negative, which suggested sample degradation. The second test also came back fully negative, suggesting that the extracts of Timothy gass, Sycamore tree, and D. farinae mite contained low amounts of Phl p 5, Pla a 3, and Der f 2 respectively.

Despite these consistently negative results, we had to mark the results for the negative controls (samples 8 and 7 in the new coding) as correctly identified, even though the test had yielded false negative results for all other samples. The coeffcient of variation could not be assessed due to all results being negative.

NELCO Results

The NELCO test correctly identified 1 of 12 samples, but this was likely due to false-positive results in 10 of 12 tests (83%) for the mite mix extract. Curiously, sample 5 showed low reactivity (LP, low positive) to the mite mix even though it contained four times more Der f 2 IgE than sample 6, which had a high positive (HP) result. Sample 8 only contained saline and albumin, yet it returned a positive result to sycamore extract. Similarly, sample 7, which only contained saline, albumin and Der f IgG1, was negative for sycamore, but positive for timothy grass and mite mix. This discrepancy, along with the positivity of negative controls, casts doubt on this test’s accuracy.

NELCO Results Continued

The reproducibility of the test could not be assessed as numerical values were not reported.

Pet Preferred Diagnostics

This test correctly identified the mAb mixes in 5 of 12 samples (42%), two of which were the negative controls. The results were positive for the two samples containing only Der f 2 IgE, but the values were the same despite a fourfold increase in Der f 2 IgE concentrations.

Remarkably, the D. farinae results were negative for samples 9, 11, and 12, which contained Der f 2 IgE identical to that of sample 6; sample 1 was positive for D. farinae even though it had no Der f 2 IgE. Samples 1, 3 and 11 were positive for sycamore, although sample 3 did not contain sycamore (Pla a 3) IgE. On the other hand, the test did a good job correctly identifying positivity for Timothy grass.

The coefficient of variation was good for Timothy grass (15%) and poor for the other 2 mAbs (98% to 119%).

Conclusion

The use of allergen-specific dog IgE monoclonal IgE in this study represents a “first-in-kind” study with samples having known reactivities. The results of the blinded analysis confirmed that the PAX was the only test able to identify all sample reactivities correctly.

The other tests either yielded completely negative results (VARL, twice)or positive results in nearly allergens tested (HESKA,4/12samples;33%).The other tests identified only some samples correctly and yielded inconsistent results for some allergen extracts, even though samples had the same concentrations of the three mAbs.

The most likely reason for the negative results for some of the allergen extracts is that the crude extracts used in these tests contained low concentrations of relevant allergens, which were insufficient for detection by the specific IgE monoclonals used,evenwhenaddedathighconcentrationsinthesamples.Alowconcentrationofimportant allergenic molecules is aknown limitation of extract-based tests(2).

Because four samples contained similar concentrations of each monoclonal IgE, the intra-assay reproducibility of the tests could be calculated. The PAX was found to be the most reproducible of the five measurable tests, with the lowest coefficient of variation (CV%) for the 3 monoclonals.

References

1. Olivry, T.; Mas-Fontao, A.; Aumayr, M.; Ivanovova, N.P.; Mitterer, G.; and Harwanegg, C. Validation of a Multiplex Molecular Macroarray for the Determination of Allergen-Specific IgE Sensitizations in Dogs. Vet. Sci. 2024, 11, 482. https://doi.org /10.3390/vetsci11100482
2. Welters, M.; Mas-Fontao, A.; Auxilia, S.; and Olivry, T. Composition heterogeneity and low-molecular-weight allergen content of Dermatophagoides farinae house dust mite allergen extracts used in veterinary medicine. Vet. Sci. 2025, 12: 824. https://doi.org /10.3390/vetsci11100482