BSA Interference in Immunoassays in Individuals with Egg Allergy

Yıl 2023, Cilt: 5 Sayı: 2, 187 - 91, 15.05.2023

Yücehan Yılmaz , Faruk Dişli , Fatih Kaplan , Sedat Yıldız

https://doi.org/10.37990/medr.1134367

Öz

Aim: The aim of current study was to determine interference by bovine serum albümine (BSA) as blocking agent in enzyme-linked immunosorbent assay (ELISA) carried out in individuals with egg allergy.
Material and Methods: 14 people diagnosed with egg allergy and 7 people without allergy were included. The sample were studied with an indirect ELISA method for egg-white IgG antibody developed in our laboratory. Effect BSA on interference was studied by manipulating antigene coating (none vs. egg white extract), blocking (1% BSA vs. Tween 80), and sample diluent (PBS vs. PBS + 0.5% BSA).
Results: In wells that were blocked with 1% BSA without being coated with antigen, positive samples cross-reacted with BSA to give an optical density (OD) of 0.99 ± 0.16, while negative samples gave an OD of 0.08±0.01 (p<0.05). However, when the same samples were diluted with 0.5% BSA, the OD of positive samples decreased (from 0.99±0.16 to 0.08±0.01), and the statistical difference with negative samples disappeared. It was observed that tween, which was used as a blocking and diluting agent, did not cross-react with the samples. Positive samples gave an OD of 0.66±0.07 in antigen (egg white extract) coated and tween-blocked wells, and 1.01±0.11 OD in BSA blocking (p<0.05). When the samples were diluted with 0.5% BSA, positive samples gave 0.18±0.01 OD on the antigen coated plate, while negative samples gave 0.12±0.04 OD (p<0.05).
Conclusion: Ovalbumin, which is found in high levels in eggs, has a similar molecular structure to BSA, and some antibodies produced against ovalbumin in people with egg allergy may also cross-react against BSA. Therefore, it was concluded that the use of BSA in both dilution and blocking solution should be avoided if the samples of individuals with egg sensitivity are to be analyzed by ELISA method. It has been observed that Tween can be easily applied as an alternative blocking agent in allergy ELISA tests.

Anahtar Kelimeler

Egg, allergy, BSA, IgG

Kaynakça

• Peters RL, Krawiec M, Koplin JJ, Santos AF. Update on food allergy. Pediatr Allergy Immunol. 2021;32:647-57.
• Werfel T. Food allergy in adulthood. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz. 2016;59:737-44.
• Scheurer S, Toda M, Vieths S. What makes an allergen?. Clin Exp Allergy. 2015;45:1150-61.
• Lei DK, Grammer LC. An overview of allergens. Allergy Asthma Proc. 2019;40:362-65.
• Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides. 2015;72;4-15.
• Hornbeck PV. Enzyme‐linked immunosorbent assays. Curr Protoc Immunol. 2015;110:2-1.
• Lin AV. (2015). Direct ELISA. In ELISA. Methods Mol Biol. 2015;61-7.
• Kohl TO, Ascoli CA. Indirect immunometric ELISA. Cold Spring Harb Protoc. 2017;5:pdb-prot093708.
• Steinitz M. Quantitation of the blocking effect of tween 20 and bovine serum albumin in ELISA microwells. Anal Biochem. 2000;282:232-8.
• Xiao Y, Isaacs SN. Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine serum albumin (BSA)—not all BSAs are alike. J Immunol Methods. 2012;384:148-51.
• Konstantinou GN. Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol. 2017;1592:79-94.
• Yu W, Freeland DMH, Nadeau KC. Food allergy: immune mechanisms, diagnosis and immunotherapy. Nat Rev Immunol. 2016;16:751-65.
• Urisu A, Kondo Y, Tsuge I. Hen's egg allergy. Chem Immunol Allergy. 2015;101:124-30.
• Hermanson GT. Chapter 19 - Vaccines and Immunogen Conjugates. Bioconjugate Techniques. Third edition. Academic Press, Boston, 2013;839-65.
• Tomičić S, Norrman G, Fälth‐Magnusson K, Jenmalm MC, Devenney I, Böttcher MF. High levels of IgG4 antibodies to foods during infancy are associated with tolerance to corresponding foods later in life. Pediatr Allergy Immunol. 2019;20:35-41.
• Zhang J, Shen Y, Li J, Li H, Si P. Component-resolved diagnostic study of egg allergy in northern Chinese children. Biomed Res Int. 2020;1-9.
• Zhang JW, Mine, Y. Characterization of IgE and IgG epitopes on ovomucoid using egg-white-allergic patients' sera. Biochem Biophys Res Commun. 1998;253:124-7.
• McKendry RT, Kwok M, Hemmings O, James LK, Santos AF. Allergen‐specific IgG show distinct patterns in persistent and transient food allergy. Pediatr Allergy Immunol. 2021;32:1508-18.
• Vance GHS, Grimshaw KEC, Briggs R, Lewis SA, Mullee MA, Thornton CA, Warner JO. Serum ovalbumin‐specific immunoglobulin G responses during pregnancy reflect maternal intake of dietary egg and relate to the development of allergy in early infancy. Clin Exp Allergy. 2004;34:1855-61.
• Suprun M, Getts R, Grishina G, Tsuang A, Suárez‐Fariñas M, Sampson HA. Ovomucoid epitope‐specific repertoire of IgE, IgG4, IgG1, IgA1, and IgD antibodies in egg‐allergic children. Allergy. 2020;75:2633-2643.