Abstract
The quantification of antigens adsorbed to aluminum-based adjuvants (alum) typically involves a method that first extracts antigen from the alum followed by the quantification of the antigen available in the extract. Extraction procedures often result in less than 100 % desorption of the antigen from the alum adjuvant and may alter the conformation of the antigen, reducing the accuracy of the subsequent method used for quantification. There is no generic method available for directly assessing the protein content when formulated on alum. Here we offer a method that can directly quantify protein adsorbed to Alhydrogel® using a simple fluorescence assay that is highly accurate and reproducible for Alhydrogel® formulations containing 25–400 μg/mL of antigen.
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References
Zhu D, Saul A, Huang S, Martin LB, Miller LH, Rausch KM (2009) Use of o-phthalaldehyde assay to determine protein contents of Alhydrogel-based vaccines. Vaccine 27(43):6054–6059
Shore PA, Burkhalter A, Cohn VH (1959) A method for the fluorometric assay of histamine in tissues. J Pharmacol Exp Ther 127:182–186
Cohn VH Jr, Shore PA (1961) A microfluorometric method for the determination of agmatine. Anal Biochem 2(3):237–241
Benson JR, Hare PE (1975) O-phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc Natl Acad Sci 72(2):619–622
Avarez-Coque MCG, Hernández MJM, Camañas RMV, Fernández CM (1989) Studies on the formation and stability of isoindoles derived from amino acids, o-phthalaldehyde and N-acetyl-cysteine. Anal Biochem 180:172–176
Kang SL, Dennis GD (1978) Fluorometric amino-acid analysis with o-phthaldialdehyde (OPA). Int J Biochem 9(7):457–467
Molnár-Perl I (2001) Derivatization and chromatographic behavior of the o-phthaldialdehyde amino acid derivatives obtained with various SH-group-containing additives. J Chromatogr A 913(1–2):283–302
Church FC, Porter DH, Catignani GL, Swaisgood HE (1985) An o-phthalaldehyde spectrophotometric assay for proteinases. Anal Biochem 146(2):343–348
Hapuarachchi S, Janaway GA, Aspinwall CA (2006) Capillary electrophoresis with a UV light-emitting diode source for chemical monitoring of native and derivatized fluorescent compounds. Electrophoresis 27(20):4052–4059
Go K, Horikawa Y, Garcia R, Villarreal FJ (2008) Fluorescent method for detection of cleaved collagens using O-phthaldialdehyde (OPA). J Biochem Biophys Methods 70(6):878–882
Frank MP, Powers RW (2007) Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids. J Chromatogr B 852(1–2):646–649
Roth M (1971) Fluorescence reaction for amino acids. Anal Chem 7:880–8822
Wong OS, Sternson LA, Schowen RL (1985) Reaction of o-phthalaldehyde with alanine and thiols: kinetics and mechanism. J Am Chem Soc 107(22):6421–6422
Acknowledgement
This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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Rausch, K.M., Zhu, D. (2017). Determination of Protein Content in Alhydrogel®-Based Vaccines by O-Phthalaldehyde Assay. In: Fox, C. (eds) Vaccine Adjuvants. Methods in Molecular Biology, vol 1494. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6445-1_19
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DOI: https://doi.org/10.1007/978-1-4939-6445-1_19
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