title: Use of o-phthalaldehyde assay to determine protein contents of Alhydrogel-based vaccines.
abstract: Aluminum based adjuvants (alum), including aluminum hydroxide (Alhydrogel) and aluminum phosphate are the most commonly used adjuvant in the US. In order to ensure quality of vaccines, regulatory authorities require evaluation of antigen content in final vaccine products. Currently, there are no generic methods available for the determination of protein content in alum-based vaccines. Aluminum hydroxide gels exist as particles in solution, which interfere with direct quantitation of protein content in formulations using assays such as Lowry, BCA or Bradford protein assay. The present study adapts a simple fluorescent assay to directly (without the need for antigen extraction) determine antigen content on Alhydrogel with accuracy and sensitivity using the o-phthalaldehyde (OPA) reagent. Malaria vaccine candidates AMA1-C1/Alhydrogel, AMA1-C2/Alhydrogel, MSP1(42)-3D7/Alhydrogel, MSP1(42)-C1/Alhydrogel or BSAM-2/Alhydrogel were used as model formulations. The results of the present study show that the OPA assay is highly accurate (87-100%), reproducible, and simple with a linear detection range of 25-400 microg/mL for Alhydrogel vaccines (except for MSP1(42)-C1, which has a linear detection range of 31.25-500 microg/mL). This assay has proven to be highly useful in our laboratory and been used in routine vaccine quality control processes.
authors: Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Disease, National Institutes of Health, 5640 Fishers Lane, Rockville, MD 20852, USA. dzhu@niaid.nih.gov
authors: Daming
authors: D
authors: Zhu
authors: Allan
authors: A
authors: Saul
authors: Shuhui
authors: S
authors: Huang
authors: Laura B
authors: LB
authors: Martin
authors: Louis H
authors: LH
authors: Miller
authors: Kelly M
authors: KM
authors: Rausch
mesh_terms: D000276:Adjuvants, Immunologic / Q000032:analysis; D000536:Aluminum Hydroxide / Q000032:analysis*; D000953:Antigens, Protozoan / Q000032:analysis*; D005453:Fluorescence; D017780:Malaria Vaccines / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D015800:Protozoan Proteins / Q000032:analysis*; D011786:Quality Control; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D009764:o-Phthalaldehyde / Q000737:chemistry*
chemical_list: D000276:Adjuvants, Immunologic; D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C090077:apical membrane antigen I, Plasmodium; D000536:Aluminum Hydroxide; D009764:o-Phthalaldehyde