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TechNote 201

Working with Microspheres

9025 Technology Dr. • Fishers, IN 46038-2886 800.387.0672 • 317.570.7020 • Fax 317.570.7034 •



I. Introduction

A. Analyte

B. Raw Materials

C. Microsphere Sizes Suggested for Different Test Formats

D. Microsphere Types Suggested for Different Applications

E. Specialized Microspheres for Special Uses

F. Protein or Not

G. Choosing mAb vs. pAb

H. Water (“Are You Using Your Microspheres to Purify Your Water?”)

I. Cleaning Microspheres (Before and After Coating)

J. “Do Nothing” or “NO Washing”

K. “Just Enough Washing”

L. “Washing”

M. Dialysis

N. “Dead-End” or Bed Filtration

O. Cross-Flow Filtration

P. Mixed Ion-Exchange (IX) Resins

Q. Column Methods

R. Ultrasonics

S. Microsphere Characterization

T. Size/Monodispersity

U. Surface Titration

V. Critical Coagulation Concentration

W. Electrokinetics

X. Measuring Bound Protein

II. Coating Microspheres

A. Simple Adsorption

B. Complex Adsorption

C. Reasons for Covalent Attachment (Don’t Get in a Bind Unless You

Need To)

D Carboxylate Covalent Coupling Chemistries

E. Beyond WSC

F. GA Binding

G. Easiest (?) Binding

H. Special Applications

I. Quenching Active Coupling Groups

III. Achieving Optimal Coating for Your Test or Assay

A. Microsphere Capture ELISAs and Tests, Dyed Particle Sandwich

Tests, and Solid Phase Assays

B. Latex Agglutination Tests (LATs) and Immunoassays; Filter

Separation Agglutination Tests and Assays

C. Use of Coadsorbant, Surface Diluent, Filler, or Blocker

IV. Final Formulation of Wet and Dry Reagents

A. Microsphere Agglutination Tests

B. Nephelometric/Turbidimetric Assays

C. Choosing Filter Media and Membranes

D. Membrane or Chromatographic “Strip Tests”

E. More Ideas for Preventing Microspheres from Sticking to the

Membrane, and Other Ideas F. Strip Test References

V. Final Words

A. About Development Strategy B. “Take the Time”

VI. References

I. IntroduCtIon

Many tests and assays use uniform latex particles, or microspheres, as substrates or supports for immunologically based reactions - tests and assays. These range from the original “latex” agglutination tests to more recent assays, such as particle capture ELISAs, turbidimetric immunoassays, dyed particle sandwich tests, and solid phase assays using silica or magnetic microspheres. In all cases, the particles must be prepared for binding and coated with a ligand, usually a protein, before they can be used in the chosen test or assay. One must also consider the microspheres’ interaction with other test elements, like filters, membranes, and magnets.

A. Analyte

The analyte involved will partly determine the type of format that one chooses. For example, molecules with MW < 6000 may be difficult to detect in a sandwich format, since it’s difficult for two antibodies to fit on such a small molecule. Such small analytes require competitive assays. This explains why drug tests are performed via inhibition or competitive binding formats. Large analytes, like proteins, can be measured by either direct or inhibition assays.

Actual clinical samples should be used early in the antibody selection process, to gauge the effect of interferences. “Out of 6-10 good antibodies that have passed all other selection criteria, only one will give these superior results with clinical specimens.”1

Bangs Laboratories, Inc. TechNote 201 Rev. #004, Active: 14/March/2013

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