Fast and specific determination of mouse, rabbit or human IgG or IgM concentration.

Easy-Titer Antibody Assay Kits include antibody-sensitized microspheres to measure the specific concentration of mouse, rabbit and human antibodies by an easy and rapid microagglutination technique using standard microplates and UV-Vis plate reader (spectrophotometer). Each kit is specific for a particular species and class of immunoglobulin and, unlike total protein assays, can specifically measure the concentration of target antibody in samples (e.g., serum, plasma, culture supernatant) that contain other proteins. The kits are extremely sensitive, requiring very small sample volumes. Antibody concentration is determined from the assay response (absorbance) by comparison to a standard curve prepared using dilutions of a known antibody sample (sold separately).

Figure 1. Easy-Titer Assay Kits use antibody-sensitized microspheres to specifically assay target antibody species based on microagglutination.	Figure 2. Generalized assay standard curve for Easy-Titer Antibody Assay Kits. The typical assay range is 8-500 ng/ml.

How Easy-Titer Antibody Assays Work

Easy-Titer Assay Kits detect and measure specific target antibodies using agglutination of microspheres that are coated ("sensitized") with the specific anti-IgG or IgM polyclonal antibodies. In the appropriate aqueous buffer (supplied in kit), the monodispersed antibody-coated microspheres (> 1 µm diameter) have highest absorptivity (l-max) to incident light having a wavelength (340 nm) that is equal to approximately half their diameter. When sample is added, two or more microspheres bind to each antibody target via their coated specific polyclonal antibodies, and this agglutination into effectively larger apparent spheres results in proportional decrease in absorptivity (lower absorbance).

Typical microagglutination assays depend on a change in light-scattering and corresponding change in transmittance, to which absorbance is inversely related. Easy-Titer Assay Kits use a special dilution buffer whose refractive index eliminates the effect of light-scattering on the monodispersed microspheres for the measurement wavelength used. Because of this, the final 10- to 20-fold dilution of the sample for use in the assay must be done using the Dilution Buffer supplied in the kit.

Figure 3 (right). Human IgG-Gamma Kit (#23325) specifically detects whole IgG (blue) or Fc fragments (red). Fab fragments are not detected. Because IgG has a larger mass than the other fragments assayed, its line appears relatively shifted upward (or right) in this plot of mass-per-volume concentrations. Figure 4 (bottom left). Human IgG Kit (#23310) detects human IgG and does not cross-react with human IgM. Figure 5 (bottom right). Human IgM Kit (#23315) detects human IgM and does not cross-react with human IgG.

Easy-Titer Antibody Assays are Faster and Easier than ELISA

1. Prepare standards (5-500 ng/ml) by diluting purified antibody in Kit Dilution Buffer
2. Prepare samples by diluting at least 20-fold in Dilution Buffer to within assay range (8-500 ng/ml).
3. Vortex vial of microsphere beads to create homogeneous suspension.
4. Pipette 20 µl of bead suspension and 20 µl of each sample and standard into 96-well microplate wells.
5. Incubate microplate for 5 minutes with vigorous mixing.
6. Add 100 µl of Kit Blocking Reagent.
7. Incubate microplate for 5 minutes with vigorous mixing.
8. Measure absorbance on standard plate reader (340 nm or 405 nm).
9. Plot standard curve and interpolate samples to determine concentration.

Highlights

• Kits for five popular targets – choose a kit specific for a particular species and class of immunoglobulin; no cross-reaction with other species and classes of the target antibody
• Antibody-based specificity – measure concentration of target antibody in a sample, not just total protein; no need to purify antibody to assess its concentration
• Faster and easier than ELISA – three-component, homogenous assay; 10 minutes total incubation time
• No special equipment needed – uses standard vortex mixer, pipetter, 96-well microplate, plate shaker and reader (measure absorbance at 340 nm or 405 nm)
• Sensitive – assay range (standard curve) 8-500 ng/ml; requires starting sample of at least 300 ng/ml
• Reproducible – coefficient of variation < 5%; error depends on dilution and pipetting technique
• Antibody standards sold separately – see product list for suggested products; use any antibody standard with proper target identity and known concentration (greater than 10 µg/ml)