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Published on: April 19, 2017
Flow cytometry: an alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant
Mildred Ugozzoli1, Donatello Laera, Sandra Nuti
1Novartis Vaccines and Diagnostics, Cambridge, MA 02139, USA. mildred.ugozzoli@novartis.com
Insights
This study introduces a novel flow cytometry (FC) method for direct vaccine antigen quantification on aluminum hydroxide (AH) adjuvants. This technique simplifies vaccine characterization by avoiding antigen desorption steps.
Area of Science:
- Immunology
- Analytical Chemistry
- Biotechnology
Background:
- Flow cytometry (FC) is a prevalent tool in biological research but has seen limited application in vaccine characterization.
- Characterizing vaccine antigens adsorbed onto adjuvants like aluminum hydroxide (AH) typically involves complex and potentially denaturing desorption processes.
Purpose of the Study:
- To develop and validate a direct flow cytometry (FC) method for quantifying Neisseria meningitidis vaccine antigens adsorbed onto aluminum hydroxide (AH) suspensions.
- To establish a sensitive and specific assay that bypasses the need for antigen desorption from the adjuvant.
Main Methods:
- Development of an antibody-based flow cytometry (FC) assay for direct analysis of antigens adsorbed on AH particles.
- Utilizing FC's particle examination capability to analyze the entire AH suspension without prior antigen desorption.
- Quantification and distribution analysis of vaccine antigens on AH particles.
Main Results:
- The developed FC method demonstrated specificity and sensitivity for quantifying two Neisseria meningitidis vaccine antigens.
- High correlation coefficients (r(2)) were achieved across a wide range of antigen doses (0-150 μg/dose).
- The assay successfully determined antigen concentration, identity, and distribution on AH particles.
Conclusions:
- This novel FC-based assay provides a direct, efficient, and sensitive method for vaccine antigen characterization on AH adjuvants.
- The method circumvents the limitations of traditional assays, such as antigen denaturation and inefficient desorption.
- This technique offers a significant advancement for vaccine quality control and development.
Abstract:
Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r(2)) were routinely achieved for a broad range of antigen doses from 0 to 150 μg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen-adjuvant desorption methods.
