Proficiency monitoring of monoclonal antibody cocktail–based enzyme-linked immunosorbent assay for detection of allergen-specific immunoglobulin E in dogs

Abstract

Abstract. The purpose of our study was to document the continued comparative proficiency of different laboratories that perform a monoclonal antibody-based enzyme-linked immunosorbent assay (macELISA) for detection of allergenspecific immunoglobulin (Ig)E in dogs. Replicate samples of 18 different sera pools were independently evaluated in a single blinded fashion by each of 16 different operators functioning in 10 different laboratories. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.0% (range: 2.7-16.1%), while the average intralaboratory interassay variance was 7.5% (range: 3.9-10.9%). The overall interassay interlaboratory variance was consistent among laboratories and averaged 11.4% (range: 8.5-12.5%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Considering the positive or negative results, interassay interlaboratory concordance of results exceeded 90%. Correlation of optical density values between and among all laboratories was strong (r > 0.9, P < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory by differing operators but also among laboratories using the same monoclonal-based ELISA.

Proficiency Monitoring of Allergen-Specific IgE macELISA – 2020

“…The inaugural proficiency evaluations, initiated in 2009 [1] and repeated in 2010 [2], in six different laboratories documents that inter-laboratory standardization and quality control measures in the veterinary arena were on the immediate forefront and that uniformity, consistency, and reproducibility of results between laboratories is achievable. The reproducibility of results among different laboratories documented in the subsequent proficiency evaluations completed in 2013 [3], 2016 [4], 2018 [5], and 2019 [6] confirm that consistent and reproducible results using this assay have been evident for…”

“…Stallergenes Greer Laboratories (Lenoir, NC) served as the prototype for evaluation of macELISA; the 2020 evaluations included results reported by three separate and independent operators. Because the performance characteristics of macELISA in Stallergenes Greer's VRL have been well documented for use over an extended period [1][2][3][4][5][6] all results observed in the other participating laboratories were compared directly with the results observed in Stallergenes Greer's reference laboratory.…”

“…The buffers used throughout have been previously described [1][2][3][4][5][6][7] and included: a) well coating buffer: 0.05 M sodium carbonate bicarbonate buffer, pH 9.6; b) wash buffer: phosphate buffered saline (PBS), pH 7.4, containing 0.05% Tween 20, and 0.05% sodium azide; c) reagent diluent buffer: PBS, pH 7.4, containing 1% fish gelatin, 0.05% Tween 20 and 0.05% sodium azide. The buffer used for dilution of serum samples was identical to the reagent diluent buffer, but it has been supplemented (2.5 mG/mL) with a cross-reactive carbohydrate inhibitor derived from the carbohydrate components present in bromelain (BROM-CCD) [7].…”

“…The allergen panel was a 24 allergen composite derived from the array of allergens that are included in the specific panels routinely evaluated in the various laboratories; the composite allergen panel consisted of 4 grasses, 6 weeds, 6 trees, 5 mites, and 3 fungi. The protocol for coating and storage of wells has been previously described [1][2][3][4][5][6][7].…”

A selective and sensitive nanosensor for fluorescent detection of specific IgEs to purified allergens in human serum

“…The ELISA tends to be laborious and requires highly trained personnel and numerous operation steps, thereby failing to allow the timely design of treatments to prevent allergic patients from life-threatening events. 24,25 Recently, nanomaterials have increasingly been used for food allergy detection. [27][28][29][30] Although the detection limit for the application of photonic crystal enhanced uorescence toward detection of sIgE is very low (0.048 ng mL À1 ), 30 the detection procedures are cumbersome, and substantial, expensive equipment is required, which limit its clinical application.…”