Expert Coffee Chat Webinar: Bio-Plex Multiplex Immunoassays in Vaccine Research and Development

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Our customers asked our Bio-Rad application scientists about how multiplex assays are being used on the front lines to understand patient immune response during infection. A lively discussion was held about sample preparation, system maintenance, targets, panels, data analysis, coronavirus research, and more. Watch the video of this session and read the questions and answers below.

Air Date: June 3, 2020

 

We had so many great questions asked by the audience. Watch the whole video, or quickly jump to a specific question:

  • 00:10  Introduction
  • 3:27  What are multiplex Immunoassays? And why are they helpful for vaccine research?
  • 7:19  Can multiplex immunoassays be used to look at patients exposed to coronavirus?
  • 10:28  What sort of adjuvants is being used for COVID-19 vaccines? Are new ones being developed? Can multiplex immunoassays be used to help test which adjuvant is best?
  • 13:42  Regarding antibody specificity and the ability to bind to a specific target or its fragment, what are the differences between an ELISA and a multiplex assay?
  • 16:24  What ways are vaccines evaluated to ensure they are safe and nontoxic?
  • 20:22  Will we need to optimize the assay to run patients’ samples infected with COVID-19? What are your recommendations?
  • 23:24  I use these assays in preclinical assays can I use them in clinical trials too?
  • 25:00  What types of positive and negative controls are essential to obtain when developing an in-house MIA (microsphere-based immunoassay)?
  • 27:09  Are multiplex immunoassays used in QC'ing of manufactured products? What is the benefit of ELISAs?
  • 29:47  Can someone use the plate more than once, for example, divide the plate twice?
  • 33:00  What type of samples do you recommend to run with Bio-Plex assays? Do you think that serum would be a good choice knowing that most of the assays start with nasal swabs?
  • 34:25  One of the challenges in MIA for serology purpose is finding that sample high enough for all the Ab of interest. What is the highest multiplex assay for antibodies screening have you ever developed? How did you look for a reference standard curve sample? How did you test the cross-reactivity?
  • 39:48  Do various antibody affinities (attached to beads) affect the overall outcome?
  • 41:06  Can you describe the assay to test for various antibody isotypes after COVID disease. Antigen (spike protein) coated to bead, add patient sample, then add beads coated with antibody to detect various isotypes? Is this a kit?
  • 43:25  How do I compare sample data collected across different days? What controls can I use?
  • 47:21  Is throughput an issue? What about data management?
  • 49:22  Could you elaborate a little on the choice of clonality of Ab for selection for capture and detection? What are the trade-offs of using a polyclonal ab for capture?
  • 52:53  Has anyone used these assays in their vaccine development?
  • 55:54  Closing

Bio-Plex Questions Answered by Our Experts

Audience questions and answers from our Bio-Rad application scientist and Bio-Plex specialists grouped by common topics.

Bio-Plex Multiplex Immunoassays

 What are multiplex immunoassays? How are they helpful for vaccine research?

 Regarding antibody specificity and ability to bind to a specific target or its fragment, what are the differences between an ELISA and a multiplex assay?

 What types of positive and negative controls are essential to obtain when developing an in-house MIA (microsphere-based immunoassay)?

 Are multiplex immunoassays used for QC in manufacturing? What are the benefits of Bio-Plex assays vs. ELISAs?

 Can a Bio-Plex assay plate be used more than once? For example, subdivide the plate in half and use it twice?

 One of the challenges in MIA for serology purposes is finding the signal high enough for all the antibodies of interest. What is the highest number of multiplex assays for antibody screening you have developed? How did you look for a reference standard curve sample? How did you test the cross-reactivity?

 Do various antibody affinities (attached to beads) affect the overall outcome?

 Could you elaborate a little on the clonality of antibody selection for capture and detection? What are the trade-offs of using a polyclonal anitbody for capture?

Bio-Plex Immunoassay System

 How do I compare sample data collected across different days? What controls can I use?

 Is throughput an issue? What about data management?

Sample Preparation for Bio-Plex Assays

 What type of samples do you recommend to run with Bio-Plex assays? Do you think that serum would be a good choice given that most of the COVID-19 assays start with nasal swabs?

 What are your recommendations for sample/virus inactivation that will not diminish cytokine levels?

Our recommendations for inactivation of virus for use on the Bio-Plex system are citations that document using a 2% paraformaldehyde solution to fix the sandwich assay to insure that any infectious agents left behind will be inactivated.

For customers concerned about possible infectious agents left behind in each well they can add the 1-4% paraformaldehyde solution after the assay is complete, then read it. NOTE: 20 ml per liter paraformaldehyde is 2%.

References on fixing sandwich assay with paraformaldehyde solution:

  • Beadlyte Stop Solution, Catalog # 43-001. One vial containing 2.5ml of solution: 0.2% (v/v) formaldehyde in PBS, pH 7.4.
  • Oliver KG et al. Multiplexed Analysis of Human Cytokines by Use of the FlowMetrix System. Clinical Chemistry 1998 44(9) 2057–2060. DOI: /10.1093/clinchem/44.9.2057
    After the incubations, samples were diluted to 150 uL with PBS containing 20 mL/L formaldehyde and analyzed on a standard Becton Dickinson FACScan equipped with a Luminex FlowMetrix System.
  • Faucner S et al. Protein Bead Array for the Detection of HIV-1 Antibodies from Fresh Plasma and Dried-Blood-Spot Specimens. Clinical Chemistry 2004 50(7) 1250–1253. DOI: 10.1373/clinchem.2004.032995
    The beads were resuspended in 20 mL/L paraformaldehyde in phosphate-buffered saline and analyzed directly from the filter-bottomed, 96-well plate with a Luminex-100 System equipped with the Luminex Data Collector software ver. 1.7.

Bio-Plex Clinical Applications

 I use these assays in preclinical assays; can I use them in clinical trials too?

COVID-19 Research

 Can multiplex immunoassays be used to look at patients exposed to coronavirus?

 Will we need to optimize the assay to run patient samples infected with COVID-19? What are your recommendations?

 Can you describe the assay to test for various antibody isotypes after COVID disease? Would it be antigen (spike protein) coated onto beads, add patient sample, then add beads coated with antibody to detect various isotypes? Is this available as a kit?

Vaccine Development

 What sort of adjuvants are used for COVID-19 vaccines? Are new ones being developed? Can multiplex immunoassays be used to help test which adjuvant is best?

 What ways are vaccines evaluated to ensure they are safe and nontoxic?

 Has anyone used these assays in their vaccine development?

Video time index (minutes:seconds) 52:53

Several recent publications have reported using our off-the-shelf Bio-Plex assays, for example, to quantitate cytokines and other targets in immune response profiling. Researches can also use the Bio-Plex platform to create custom assays by coupling their own antibodies and antigens to Luminex beads, for example, to create a new serology assay.

Both off-the-shelf and custom coupled beads have been used in vaccine development throughout the whole development process, from pre-clinical studies through clinical trials and after-market studies. Bio-Plex assays can be used in vaccine research to determine whether or not you're eliciting the intended immune response, either by directly quantitating neutralizing antibodies, as for example with the Gardasil HPV vaccine, or by using cytokine levels to monitor host immune response indirectly. We offer species-spefiic assays for the rat and mouse models often used in pre-clinical trials, and our human cytokine assays can be used in the clinical phases.

The following publications highlight some of the possibilities for Bio-Plex applications in vaccine studies:

  • Takahashi Yet al. Immunological studies of cerebrospinal fluid from patients with CNS symptoms after human papillomavirus vaccination. J Neuroimmunol. 2016;298:71–78. PMID: 27609278
  • Smith JF et al. Evolution of type-specific immunoassays to evaluate the functional immune response to GARDASIL, a vaccine for Human Papillomavirus types 16, 18, 6, and 11. Hum Vaccin. 2008;4(2):134–142. DOI: 10.4161/hv.4.2.5261
  • Nygard M et al. Evaluation of the long-term anti-human papillomavirus 6 (HPV6), 11, 16, and 18 immune responses generated by the quadrivalent HPV vaccine. Clin Vaccine Immunol. 2015;22(8):943–948 PMCID: PMC4519713

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Application Notes