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Conjugation of Proteins to Zepto Ultra Express Fluorescent Microspheres


Zepto™ Ultra Express Microspheres are metal-coated fluorescent polystyrene beads designed for fast surface functionalization with molecules containing primary amines, tiol or dithiol groups. The procedure is simply a mix-and-use process.

The following protocol provides general guidelines for conjugation of antibodies to Zepto™ Ultra Express Microspheres, and their use in downstream assays. Multiple antibodies of different specificities, or other molecules of interest, may be conjugated to microspheres emitting different fluorescent colors, allowing for multiplexed detection capabilities in a single assay.

Conjugation Protocol

Materials:

Washing buffer (PBST): 0.1X PBS containing 0.05% TWEEN 20 
Storage buffer: 1% bovine serum albumin (BSA) in PBST (Sigma, A3059)
Protein to be conjugated (e.g. IgG): 5 µg/mL in 0.5X PBS
Fluorescent Microspheres:  Zepto™ Ultra Express Microspheres

  1. Vortex the product bottle before use to ensure homogeneous suspension of the microspheres.
  2. Immediately aliquot 1 million microspheres (10 µL of stock solution as provided).
  3. Add 10 µL of IgG .*
  4. Mix well and incubate for 2 hours at room temperature with constant rotation.
  5. Add 100 µL of washing buffer and mix well.
  6. Gently pellet at 100 g for 5 min and discard supernatant. **
  7. Add 100 µL of storage buffer and mix well.
  8. Incubate for 1 hour at room temperature with constant rotation.
  9. Conjugated microspheres are now ready for your use in your assay. Alternatively, store at 4°C until use.
*The optimal amount of IgG or other protein may vary. Titration of the optimal amount of protein is recommended.

** The centrifugation speed for pelleting microspheres is a range between 100 to 500 g. A lower/more gentle centrifugation speed with longer time is recommended to form a good microsphere pellet, especially after conjugation to a protein. Prolonged centrifugation at high speed may cause irreversible aggregation and loss of performance.

Detection Assay

The following procedure is based on a sandwich assay for single analyte detection using flow cytometry. Capture antibodies are conjugated to the microsphere surface as described above. A reporter is typically a fluorophore-labeled antibody that recognizes and binds to a different domain or epitope on the analyte than that of the capture antibody. In the presence of analyte, a sandwich is formed when the microspheres coated with the capture antibody and the reporter antibody bind the analyte.

The protocol below is a general guideline and users may need to optimize assay parameters for their particular application to obtain optimal performance.

  1. Aliquot 0.5 µL of conjugated spheres as prepared above (equals about 5,000 microspheres)
  2. Add 5 µL of reporter antibody (50 µg/mL in TBS, 0.1% BSA)
  3. Add 100 µL of analyte standard or sample solution and mix well
  4. Incubate for 1 hour at room temperature with constant rotation
  5. Add 500 µL of washing buffer and mix well
  6. Gently pellet at 100 g for 5 min
  7. Discard supernatant, add 500 µL of washing buffer and mix well
  8. Gently pellet at 100 g for 5 min
  9. Resuspend microspheres in washing buffer and analyze by flow cytometry

Microsphere based immunoassay example results
Figure 1. Standard curve generated for quantification of rabbit IgG in serum. Cytodiagnostics Zepto™ Ultra Express Green Microspheres were conjugated to an anti-rabbit IgG antibody according to the protocol above followed by incubation with various concentrations of rabbit IgG. Detection was performed using a PE-labeled anti-rabbit antibody followed by analysis with flow cytometry.