Chapter 9

Assay accuracy is defined as the degree of closeness of achieved results relative to their actual (true) values. International standards exist for total IgG, IgA and IgM (CRM 470 and DA470k) [127][224]. Therefore, these standards were used to assign values to HLC assay reference materials to ensure accurate results. An overview of the process of calibrator assignment for HLC assays is described below, using the IgA assays as an example.

9.4.1. Primary standards and internal reference standards

For IgA HLC assays, polyclonal IgA was purified from pooled normal human sera by ammonium-sulfate precipitation, anion-exchange chromatography, and immunoaffinity chromatography. Low-level contamination with alternate classes of immunoglobulins was removed by immunoaffinity adsorption and/or protein G affinity chromatography. Fractionation of κ from λ light chain forms was achieved using both protein L and anti–light chain immunoaffinity matrices.

Purified IgAκ and IgAλ were >99% pure by silver-stained SDS-PAGE, (Figure 9.4) and light chain specific as indicated by enzyme immunoassays and Western blot analysis (Figure 9.5 and 9.6). Contamination with other immunoglobulin and light chain types was <0.5% by enzyme immunoassay. Finally, the total protein concentration of the primary standards (determined using the bicinchoninic acid method) and CRM 470 plus DA470k were used to assign IgAκ and IgAλ values to the IgA HLC internal reference standard (Figure 9.7). Similar methods were used for the calibration of the IgG and IgM HLC assays.

9.4.2. Kit calibrators and controls

HLC kit calibrators are prepared from pooled normal human sera, and kit controls from human sera containing high concentrations of polyclonal immunoglobulins. Values are assigned to kit calibrators using the internal reference materials (Figure 9.7). This is achieved by turbidimetry/nephelometry over five calibration curves, with the internal reference material assayed at four different dilutions with values across the curve range.

9.4.3. Calibration curves

The measuring ranges of HLC assays are dependent upon two factors: the slope of the respective calibration curve and the portion selected for the assay. The latter should be chosen to allow the maximum number of normal and abnormal clinical samples to be measured at the initial sample dilution.

Typical analytical ranges (at the standard dilution) on the Binding Site SPAPLUS® are shown in Table 9.1, and typical calibration curves are shown in Figure 9.8. Samples containing higher concentrations require further dilution. Samples containing lower concentrations can be re-assayed at a lower dilution (or neat on the SPAPLUS).
Specificity Dilution Approximate measuring range (g/L)
IgGκ1/20 1.9 – 40.0
IgGλ1/20 0.92 – 29.5
IgAκ1/10 0.18 – 11.2
IgAλ1/10 0.16 – 10.4
IgMκ1/10 0.20 – 5.00
IgMλ1/10 0.18 – 4.50

Table 9.1. Measuring ranges for HLC assays on the Binding Site SPAPLUS.

Calibration curves are validated by the measurement of high and low control samples. It is also recommended that all laboratories take part in external quality assurance schemes, to allow comparison of performance and results among different test sites (Chapter 39).

9.4.4. Correlation with total immunoglobulin measurements

During validation, a detailed comparison study is performed for each assay: summated concentrations of HLC immunoglobulin pairs (e.g. IgAκ + IgAλ) are compared with total immunoglobulin measurements (e.g. IgA) as a test of assay accuracy. All specificities demonstrate a high degree of correlation, and the results are summarised in each product insert. An example is shown in Figure 9.9.