Indirect enzyme-linked immunosorbent assay for measurement of human immunoglobulins E and G to purified cow's milk proteins: application in diagnosis of cow's milk allergy

Abstract

An indirect double-antibody enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human immunoglobulin E (IgE) and IgG to the cow's milk proteins (CMP) a-casein, a-lactalbumin, and P-lactoglobulin. Human serum albumin was used as the negative-antigen control. Rabbit anti-human IgE or IgG served as the primary antibody, and horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin served as the secondary antibody. Positive control sera were obtained from patients with welldocumented histories of cow's milk allergy, while negative control sera were obtained from cord bloods of healthy full-term infants and from normal adult volunteers without known milk allergy. Test sera were obtained from 41 children (ages, 3 months to 13 years; average age, 2.6 years) with suspected cow's milk allergy and clinical manifestations that included wheezing, rhinitis, atopic dermatitis, urticaria, or gastrointestinal disturbances. The patients were simultaneously evaluated by prick skin testing with scratch test antigen to whole CMP. Although only 13 (32%) of the 41 patients were positive by the prick skin test, 25 (61%) were positive by the IgE ELISA. Of the 25 IgE ELISA-positive patients, 20 were also positive by the IgG ELISA. There was concordance of positive results between skin testing and the IgE ELISA in only 9 patients (22%), and there was concordance of negative results in 12 patients (29%). Discordant results were observed in 20 patients (49%). These results indicate that the ELISA is more sensitive than prick skin testing in the identification of individuals with elevated levels of IgE to CMP. Cow's milk supplementation in infant nutrition is now commonplace and is reflected in increased incidences of cow's milk protein (CMP)-induced adverse reactions which have diverse clinical manifestations (1, 13, 14). The lack of practical, efficient, and definitive diagnostic tests has restricted the identification of the allergy in these patients. Until recently, the techniques for the evaluation of suspected immediate-type hypersensitivity to CMP have been limited to skin testing, the radioallergosorbent test (RAST), and elimination-challenge tests, all of which are either impractical or lacking in both sensitivity and specificity. Enzyme-linked immunoadsorbent assays (ELISAs) are equal to RAST in sensitivity in the detection of allergen-specific immunoglobulin (IgE). ELISAs have several major advantages over RAST, including a long shelf life of reagents, nonradioisotopic probes, and inexpensive instrumentation requirements. These advantages make possible allergenspecific IgE testing in even the most modestly equipped laboratories. We have developed an indirect double-antibody ELISA for the detection of CMP-specific human IgE and IgG. All reagents used in the assay are commercially available, which simplifies their use in routine clinical laboratories. The procedure uses purified CMPs, i.e., ac-casein, oa-lactalbumin, and 3-lactoglobulin, which have been previously described

Anti‐betalactoglobulin IgG antibodies bind to a specific profile of epitopes when patients are allergic to cow's milk proteins

“…The reasons for these discrepancies have never been elucidated. Some studies have found significant associations between IgE and IgG antibodies: ranging from frequently associated detection [ 13], to significant correlation between isotype titres [ 14], and attribution of a predictive value to IgE/IgG ratios for monitoring or predicting the decline of clinical reactivity [ 15]. Other studies report no relationship at all between IgE and IgG antibody levels [ 16].…”

The effects of roasting on the allergenic properties of peanut proteins

“…Protein products modified by the Maillard reaction have been shown to elicit an IgG response, which has been correlated by a number of studies to IgE production. [5][6][7][8] AGEs have been shown to promote monocyte migration 9,10 and cytokine production. 11 Although the effect of the AGEs in association with heightened immuogenicity, aging, and age-enhanced disease states, such as diabetic complications, atherosclerosis, hemodialysis-related amyloidosis, and Alzheimer's disease, have been investigated, few studies have been done to address the role of these products on the allergenic properties of ingested foods.…”

Proteomic Screening Points to the Potential Importance of Ara h 3 Basic Subunit in Allergenicity of Peanut

“…Previous studies [8,21] reported that peanut roasting affects the allergenic properties of peanuts possibly due to the reactions of proteins with sugars to form Maillard reaction products [22]. Further investigations [23][24][25] established a correlation between level of IgE binding and the level of advanced glycation end products (AGE).…”

“…This data implies that AGE adducts may alter the peanut allergens in a way that influences the IgE recognition of the allergens. Maleki et al [8,11,29] has demonstrated the occurrence of structural changes in peanut allergens which contain AGE adducts after roasting or reactions with various sugars. Such allergens have a higher IgE binding than those without AGEs.…”

“…The International Union of Immunological Societies Allergen Nomenclature Subcommittee published 'official list of allergens' as January of 2008 and all 9 peanut allergenic proteins, Ara h 1-9, are recognized in peanuts (http://www.allergen.org). Peanut Ara h 1 was the first allergen described [4] and identified as a major peanut allergen [5] of 63 kDa and has gained a considerable amount of attention in recent years [6][7][8][9]. In addition to Ara h 1, 8 other peanut allergens identified are Ara h 2 [10][11][12] of 17 to 20 kDa that is the most potent peanut allergen [13], Ara h 3 [14,15] consisting of a series of polypeptides ranging from 14 to 45 kDa, Ara h 4 to Ara h 7 [16] of 14 to 36 kDa, Ara h 8 and Ara h 9 [17,18] that are between 7 to 18 kDa.…”