Establishment of a Highly Sensitive Sandwich Enzyme-Linked Immunosorbent Assay Specific for Ovomucoid from Hen’s Egg White

Abstract

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hen's egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hen's egg proteins.

The establishment of a highly sensitive ELISA for detecting bovine serum albumin (BSA) based on a specific pair of monoclonal antibodies (mAb) and its application in vaccine quality control

“…The quantitative range of BsA concentration is from 0.5 to 40 µg/L, which is comparable to the currently used polyclonal antibody (pAb) eLIsA. Intra-assay and inter-assay coefficient variations are both lower than 10% at the three concentrations used (10,20 and 40 µg/L). Thus, the mAb sandwich eLIsA developed herein may provide a stable, precise and highly sensitive method for quantifying BsA, which is very useful in the quality control of some vaccines.…”

“…Coupling of HRP to BSA mAbs was performed according to a routine protocol employed in our laboratory. 20 Each of the four mAbs was labeled with HRP and then utilized as either the capture or the detection antibody to determine the optimum combination. Briefly, 100 µL of coating BSA mAb (20 mg/L) was added to each well of an ELISA plate and incubated overnight at 4°C.…”

Application and bioactive properties of proteins and peptides derived from hen eggs: opportunities and challenges

“…ovalbumin, ovomucoid, ovotransferrin and lysozyme, are strong and well‐known allergens in egg white. However, compared with the other three proteins, ovomucoid is considered a dominant allergen because of its relatively higher stability to treatments by heat or digestive enzymes . Ovomucoid is a heat‐resistant glycoprotein (22–29% carbohydrate) with 186 AA residues and is an inhibitor of trypsin, catalase and chymotrypsin .…”

Characteristics of anaphylaxis‐inducing IgG immune complexes triggering murine passive systemic anaphylaxis

“…7 and No. 9 against ovomucoid (OVM) were provided by Li et al (16); mAbs against human serum albumin (HSA) and recombinant mannoprotein 1 of penicillium (MP1P) were provided by Ma et al (17) and Wang et al (18); mAbs against recombinant human haemoglobin f (rHb zeta), human haemoglobin f (Hb zeta), human haemoglobin A2 (HbA2) and advanced oxidation protein products (AOPP) were prepared in our laboratory (19)(20)(21)(22). The specificity and isotype of mAbs are listed in Table S1.…”