Nephelometry and turbidimetry

Principle

• Both methods measure immune complexes (or other particles) formed when antibody reacts with antigen in solution.
• Turbidimetry measures the decrease in intensity of light passing straight through the sample (transmitted light); it can be run on routine spectrophotometric analysers.
• Nephelometry measures light scattered at an angle to the incident beam (commonly 90° or forward angles); it is generally more sensitive at low concentrations.
• Measurements may be endpoint or rate (the maximum rate of complex formation).
• Particle-enhanced assays coat antibody on latex microparticles to increase signal and sensitivity (e.g. high-sensitivity CRP).

Reagents and equipment

• Specific antisera or latex reagents, calibrators, buffer containing polyethylene glycol to speed complex formation.
• Light sources: LEDs or lasers; detectors at 180° (turbidimetry) or at an angle (nephelometry).

Quality control and pitfalls

• Heidelberger curve: signal rises with antigen to the equivalence zone, then falls in antigen excess. Very high antigen can give falsely low results (hook effect); analysers perform antigen-excess checks and retest at higher dilution.
• Lipaemia and particulate matter scatter light and give falsely high results; clear by centrifugation or ultracentrifugation.
• Haemolysis and icterus may affect turbidimetric readings.
• Run at least two control levels; recalibrate on reagent lot change.

Clinical use

• Immunoglobulins IgG, IgA, IgM; complement C3 and C4; CRP and hs-CRP; ASO; rheumatoid factor; transferrin, haptoglobin, prealbumin, ceruloplasmin; serum free light chains; cystatin C; urine microalbumin.