Multiplexed Microsphere‐Based Flow Cytometric Immunoassays

Kathryn L. Kellar1, Aida J. Mahmutovic1, Kakali Bandyopadhyay1

1 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.1
DOI:  10.1002/0471142956.cy1301s35
Online Posting Date:  February, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Multiplexed microsphere‐based immunoassays can be developed to simultaneously measure multiple analytes in a biologic system by flow cytometric resolution of spectrally distinct microspheres coupled with capture molecules and reporter fluorochromes bound to detection antibodies. A multiplexed sandwich immunoassay is based on an ELISA format that is transferred directly to microspheres to quantitate multiple antigens. These assays require smaller sample volumes, are less expensive, and are as reproducible, reliable, and sensitive as ELISAs. However, potential cross‐reactivities between multiplexed antibodies, antigens, and specimens need to be systematically eliminated during the validation process. Sandwich and competitive immunoassays, which require only one antigen‐specific antibody, can be combined in the same multiplexed array. Antibody‐capture immunoassays are used to detect multiple antibodies from a specimen for diagnostic or surveillance purposes. The protocols for these three multiplexed immunologic assays are accompanied by methods for coupling analytes to microspheres and biotinylation of antibodies with a water‐soluble derivative.

Keywords: microsphere‐based immunoassays; multiplexed immunoassays; biotinylation; signal‐to‐noise ratios; sandwich immunoassays; competitive immunoassays; antibody capture immunoassays

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Covalently Coupling Analytes to Microspheres
  • Support Protocol 1: Assessment of Microsphere Coupling Efficiency
  • Basic Protocol 2: Biotinylation of Antibodies
  • Basic Protocol 3: Multiplexed Antibody‐Capture Assays
  • Support Protocol 2: Titration of Detection Antibodies
  • Basic Protocol 4: Multiplexed Sandwich Immunoassays
  • Support Protocol 3: Titration of Detection Antibodies and SA Conjugate
  • Support Protocol 4: Cross‐Reactivity Testing for Validation of Multiplexed Sandwich Immunoassays
  • Support Protocol 5: Spiking and Recovery Experiments for Validation of Multiplexed Sandwich Immunoassays
  • Basic Protocol 5: Multiplexed Competitive Immunoassays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Covalently Coupling Analytes to Microspheres

  Materials
  • Capture analytes: purified antigens or affinity‐purified antibodies to be coupled
  • MES buffer (see recipe)
  • Microsphere populations with carboxyl functional groups (Luminex)
  • Activation buffer (see recipe)
  • Sulfo‐NHS (N‐hydroxy‐sulfosuccinimide; Pierce Chemical; store desiccated at 4°C)
  • EDC [1‐ethyl‐3‐(3‐dimethyaminopropyl)‐carbodiimide hydrochloride; Pierce Chemical or Sigma; store desiccated at −20°C]
  • PBN (see recipe)
  • Dialysis thimbles (Slide‐A‐Lyzer Mini Dialysis Units) or dialysis cassettes (Slide‐A‐Lyzer Dialysis Cassettes; both from Pierce Chemical)
  • Bath sonicator
  • 1.5‐ml microcentrifuge tubes (no. 1415‐2500; USA Scientific)
  • Microcentrifuge with horizontal rotor
  • Hematology rocker
  • Additional reagents and equipment for cell counting ( appendix 3A)
  • NOTE: This protocol was developed for Luminex xMAP microspheres, but can be modified for use with microspheres from other sources, e.g., Bangs Quantum Plex, Duke Scientific Cyto‐Plex, or Spherotech PAK.

Support Protocol 1: Assessment of Microsphere Coupling Efficiency

  Materials
  • Analyte‐coupled microsphere populations (see protocol 1)
  • PBN (see recipe)
  • 10 µg/ml biotinylated (see protocol 3) or directly labeled IgG antibody against the coupled analyte for each microsphere population
  • 75 µg/ml streptavidin‐conjugated fluorochrome (SA conjugate) as reporter (for use with biotinylated antibody)
  • 1% formaldehyde (optional; see recipe)
  • Microtiter plates
  • Microtiter plate shaker (e.g., Titer Plate Shaker, Lab‐line Instruments)
  • Flow cytometer equipped with lasers specific for the fluorochromes embedded in the microspheres and for the detection antibodies or reporter molecules, and with a microtiter plate handler

Basic Protocol 2: Biotinylation of Antibodies

  Materials
  • Affinity‐purified IgG antibody preparations
  • PBS, pH 7.4 (see recipe)
  • Biotin sodium sulfosuccinimide ester (sulfo‐NHS‐LC‐LC‐biotin; Molecular Probes; mol. wt. 669.74; store desiccated at −20°C)
  • Microspheres coupled with capture antibodies specific to the biotinylated products to be tested (also see protocol 1)
  • PBN (see recipe)
  • Streptavidin R‐phycoerythrin (SA‐PE; Molecular Probes)
  • 1% formaldehyde (see recipe)
  • PBN/2.5% BSA (see recipe)
  • 2% sodium azide in deionized H 2O
  • Dialysis thimbles (Slide‐A‐Lyzer Mini Dialysis Units) or dialysis cassettes (Slide‐A‐Lyzer Dialysis Cassettes; both from Pierce Chemical)
  • Microcentrifuge tubes
  • Additional reagents and equipment for flow cytometry (see protocol 2)

Basic Protocol 3: Multiplexed Antibody‐Capture Assays

  Materials
  • Normal sera from the different animal species that are the source of the multiple detection antibodies
  • Antigen‐coupled microspheres for each antibody to be captured (see protocol 1)
  • Samples for analysis
  • Positive and negative control samples
  • PBN (see recipe)
  • Detection antibody (titrated as in protocol 5): biotinylated or fluorochrome‐conjugated polyclonal antibody specific to the source of the sample antibodies; i.e., anti–human IgG, IgA, or IgM for human samples
  • Streptavidin‐conjugated fluorochrome (SA conjugate) as reporter (for use with biotinylated antibody)
  • Vacuum grease
  • PBN/2.5% BSA (see recipe)
  • Wash buffer (see recipe)
  • Multiscreen microtiter filter plates (MABVN 1.2 µm; Millipore)
  • Pressure‐sensitive film (Falcon)
  • Multiscreen vacuum manifold (Millipore) with vacuum source
  • Additional reagents and equipment for titration of detection antibodies (see protocol 5) and flow cytometry (see protocol 2)

Support Protocol 2: Titration of Detection Antibodies

  Materials
  • Normal sera from the different animal species that are the source of the capture and detection antibodies
  • Serum, plasma, or other samples from eight normal donors
  • PBN (see recipe)
  • PBN/2.5% BSA (see recipe)
  • Antibody‐coupled microspheres for each antigen (see protocol 1)
  • 20 µg/ml stocks of recombinant or purified antigens in PBN (see recipe) as standards (store in aliquots at −20°C)
  • Affinity‐purified biotinylated or directly conjugated detection antibodies specific for each antigen to be multiplexed
  • Vacuum grease
  • Wash buffer (see recipe)
  • TiterTubes (Bio‐Rad)
  • Multiscreen microtiter filter plates (MABVN 1.2 µm; Millipore)
  • Pressure‐sensitive film (Falcon)
  • Multiscreen vacuum manifold (Millipore) with vacuum source
  • Additional reagents and equipment for antibody and SA conjugate titration (see protocol 7) and flow cytometry (see protocol 2)
NOTE: Prior to running samples, the detection antibodies should be titrated by preparing standard curves and blanks for testing against various dilutions of the detection antibodies. The titrations can be performed with each analyte separately as in steps to of this protocol, which is recommended, or with multiplexed analytes (see protocol 7).

Basic Protocol 4: Multiplexed Sandwich Immunoassays

  Materials
  • Antigen‐coupled microspheres (see protocol 1)
  • Antigen‐containing samples, including ones with known antigen concentration to serve as positive controls
  • Additional reagents and equipment for multiplexed sandwich immunoassay (see protocol 6), preparing filter plate (see protocol 4, steps to ), and titration of detection antibodies (see protocol 7)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Biagini, R.E., Sammons, D.L., Smith, J.P., MacKenzie, B.A., Striley, C.A.F., Semenova, V., Steward‐Clark, E., Stamey, K., Freeman, A.E., Quinn, C.P., and Snawder, J.E. 2004. Comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme‐linked immunosorbent assay for measurement of human immunoglobulin G antibodies to anthrax toxins. Clin. Diag. Lab. Immunol. 11:50‐55.
   Camilla, C., Mély, L., Magnan, A., Casano, B., Prato, S., Debono, S., Montero, F., Defoort, J‐P., Martin, M., and Fert, V. 2001. Flow cytometric microsphere‐based immunoassay: Analysis of secreted cytokines in whole‐blood samples from asthmatics. Clin. Diagn. Lab. Immunol. 8:776‐784.
   Carson, R.T. and Vignali, D.A.A. 1999. Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay. J. Immunol. Methods 227:41‐52.
   Chen, R., Lowe, L., Wilson, J.D., Crowther, E., Tzeggai, K., Bishop, J.E., and Varro, R. 1999. Simultaneous quantification of six human cytokines in a single sample using microparticle‐based flow cytometric technology. Clin. Chem. 45:1693‐1694.
   Faucher, S., Martel, A., Sherring, A., Ding, T., Malloch, L., Kim, J.E., Bergeron, M., Sandstrom, P., and Mandy, F.F. 2004. Protein bead array for the detection of HIV‐1 antibodies from fresh plasma and dried‐blood‐spot specimens. Clin. Chem. 50:1250‐1253.
   Joubert, O., Keller, D., Pinck, A., Monteil, H., and Prévost, G. 2005. Sensitive and specific detection of staphylococcal epidermolysins A and B in broth cultures by flow cytometry‐assisted multiplex immunoassay. J. Clin. Microbiol. 43:1076‐1080.
   Kellar, K.L., Kalwar, R.R., Dubois, K.A., Crouse, D., Chafin, W.D., and Kane, B‐E. 2001. Multiplexed fluorescent bead‐based immunoassays for quantitation of human cytokines in serum and culture supernatants. Cytometry 45:27‐36.
   Lal, G., Balmer, P., Stanford, E., Martin, S., Warrington, R., and Borrow, R. 2005. Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes. J. Immunol. Methods 296:135‐147.
   Martins, T.B. 2002. Development of internal controls for the Luminex instrument as part of a multiplex seven‐analyte viral respiratory antibody profile. Clin. Diagn. Lab. Immunol. 9:41‐45.
   Martins, T.B., Pasi, B.M., Pickering, J.W., Jaskowski, T.D., Litwin, C.M., and Hill, H.R. 2002. Determination of cytokine responses using a multiplexed fluorescent microsphere immunoassay. Am. J. Clin. Pathol. 118:346‐353.
   Oliver, K.G., Kettman, J.R., and Fulton, R.J. 1998. Multiplexed analysis of human cytokines by use of the FlowMetrix system. Clin. Chem. 44:2057‐2060.
   Pickering, J.W., Martins, T.B., Schroder, M.C., and Hill, H.R. 2002. Comparison of a multiplex flow cytometric assay with enzyme‐linked immunosorbent assay for quantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type B. Clin. Diagn. Lab. Immunol. 9:872‐876.
   Wong, S.J., Demarest, V.L., Boyle, R.H., Wang, T., Ledizet, M., Kar, K., Kramer, L.D., Fikrig, E., and Koski, R.A. 2004. Detection of human anti‐flavivirus antibodies with a West Nile virus recombinant antigen microsphere immunoassay. J. Clin. Microbiol. 42:65‐72.
Key References
   Kellar, K.L. and Douglass, J.P. 2003. Multiplexed microsphere‐based flow cytometric immunoassays for human cytokines. J. Immunol. Methods 279:277‐285.
  A methods paper that outlines procedures for multiplexed microsphere‐based sandwich immunoassays.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library