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9 - Development and validation of Hevylite immunoassays

Chapter 9

Hevylite® assays:

  • Are based on polyclonal antisera raised in sheep.
  • Are standardised using International standards for total IgG, IgA and IgM.
  • Undergo rigorous testing during assay manufacture to ensure the quality of the kit components.

9.1. Assay overview

Intact immunoglobulin molecules contain unique junctional epitopes across the heavy chain and light chain constant regions that are the target of immunoglobulin heavy/light chain assays (Hevylite, HLC) (Figure 9.1) [127]. HLC assays quantify the different light chain types of each immunoglobulin class, i.e. IgGκ, IgGλ, IgAκ, IgAλ, IgMκ and IgMλ (Figure 9.2). These molecules are measured in pairs, e.g. IgGκ/IgGλ, to produce ratios in the same manner as serum free light chain (sFLC) κ/λ ratios.

As with Freelite® sFLC immunoassays, polyclonal antibodies raised in sheep provide the most attractive method of recognising polymorphic immunoglobulin molecules. There are theoretically four HLC epitope regions per immunoglobulin molecule - one on each side of each heavy/light chain contact region. Multiple HLC epitopes enable immune complexes to form readily, allowing homogeneous immunoassays to be produced that are suitable for turbidimeters and nephelometers. Latex enhancement is not necessary for IgG and IgA HLC assays, but is required for IgM assays.

9.2. Polyclonal antisera production

To produce polyclonal antisera for HLC assays, sheep are immunised using individual monoclonal immunoglobulins of a single class and light chain type (e.g. IgGκ) [127]. Sheep are simultaneously tolerised with: 1) monoclonal immunoglobulins of the same class but opposite light chain type (i.e. IgGλ); 2) monoclonal immunoglobulins of different classes but the same light chain type (i.e. IgAκ); and, 3) monoclonal free light chains (FLCs) of the same light chain type (i.e. κ FLC). Antigens for immunisation and tolerisation are purified from the serum or urine of patients with monoclonal gammopathies. Antigen purity is assessed using silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.

9.3. Antisera specificity testing

Inevitably, one of the most demanding aspects of HLC assay production is to ensure specificity. A panel of antigens is used to test specificity; this panel includes sera containing monoclonal immunoglobulins of each class, subclass, and light chain type.

Initial immunisation and tolerisation procedures result in antisera that are strongly reactive against each HLC molecule, with a degree of cross-reaction with other immunoglobulin specificities. Cross-reacting antisera are recycled through affinity and adsorption columns until specificity is satisfactory. Antisera with subclass bias are purified against immobilized monoclonal immunoglobulins of other subclass types. Antisera are considered specific when they react in a balanced manner with the appropriate panel of immunoglobulins, and no cross-reactivity occurs with other molecules, including FLCs. The final purified reagents are produced using positive affinity chromatography against the target HLC immunoglobulin. Antisera specificity is the most important aspect of the HLC immunoassays, and is evaluated using several techniques, described below.

To test specificity by radial immunodiffusion (RID), HLC antisera are immobilised in an agarose gel and a panel of antigens are applied to wells cut into the gel. The diameters of the resulting immunoprecipitates are used to determine specificity.

Neat or latex-conjugated HLC antisera are tested for specificity by turbidimetry/nephelometry. Known concentrations of purified potential interfering substances are added to a normal serum containing concentrations of immunoglobulins within the reference interval for each assay. Results for IgG and IgA HLC assays are shown in Figure 9.3. Overall, the specificity assessments show that HLC antisera have minimal reactivity with intact immunoglobulins of the alternate light chain type or class. HLC antisera also have no significant reactivity with FLCs or other potentially interfering substances.

9.4. Accuracy and standardisation

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.

9.5. Maintaining batch-to-batch consistency of polyclonal antisera-based reagents

Maintaining batch-to-batch consistency is essential as HLC assays may be used for monitoring individual patients over many years. The key component of any turbidimetric or nephelometric immunoassay is the polyclonal antisera. Therefore, it is essential to minimise any change in the specificity and performance of the antisera over time.

In order to ensure consistency between batches of polyclonal antisera, a virtual “rolling pool” of antisera has been established. This pool consists of a list of pre-approved antisera (Section 9.3 explains how suitable antisera are identified). During the manufacture of a batch of reagent, an equal portion of each approved antiserum from the list is mixed. As individual antiserum volumes vary, stocks become exhausted at different times. When this occurs, the antiserum is replenished with a new pre-approved antiserum. The pool of polyclonal antisera used in the manufacture of HLC assays always contains at least 90% of the same constituent antisera as the previous batch of reagent. The use of rolling pools of antisera during HLC assay manufacture has minimised batch-to-batch variation whilst ensuring a full range of HLC epitopes are recognised. Batch-to-batch variation is further discussed in Section 9.7. For IgM HLC assays, once a pool of suitable polyclonal antisera is made, sheep antibodies are attached to latex particles (in order to enhance their performance in nephelometric and turbidimetric immunoassays).

9.6. Overview of Hevylite assay validation

During the development and production of Hevylite assays there is a rigorous validation process to ensure that the assays perform correctly and provide the correct diagnostic information. The validation protocols used follow those set out by the Clinical and Laboratory Standards Institute (CLSI), and are outlined in Section 5.6. These include assessment of assay precision, sensitivity, linearity, stability, comparison to predicate devices (serum protein electrophoresis and serum immunofixation electrophoresis) and confirmation of the normal reference range.

9.7. Overview of Hevylite kit manufacture

The manufacture of Hevylite assays follows established, validated protocols, and each batch of reagents undergoes rigorous testing to ensure the quality of the kits (Figure 9.10).

Once all the raw materials for a kit have been manufactured the curve performance and antigen excess capacity of the reagents are evaluated. Values are then assigned to the kit calibrator(s) and controls using the internal reference standard.

Specificity is evaluated by testing panel samples. HLC results for each new batch of antisera are compared with assigned values and results from previous batches. The panel samples include normal sera and patient sera containing polyclonal or monoclonal immunoglobulins (typically from patients with multiple myeloma or Waldenström’s macroglobulinaemia). The results are compared using Passing-Bablok analysis and are considered acceptable when they fall within a defined set of criteria. Typical batch-to-batch comparison data on the Binding Site SPAPLUS is shown in Figure 9.11..

Assay calibration and kit performance are also controlled by analysing large numbers of normal sera. The results are compared with the normal ranges defined in the product insert (Chapter 10). The results are also summated and compared with the relevant total immunoglobulin assay by Passing-Bablok analysis. The linearity of the kit is evaluated by assaying dilutions of a polyclonal fluid, then once a final pre-packaging test is complete, the kit is packaged, ready for release.


9.8. Immunoassay development on different platforms

IgG, IgA and IgM Hevylite assays were initially launched in 2009/2010, and are available on the Binding Site SPAPLUS and Siemens BN™II instruments. Hevylite assays for the Binding Site Optilite® instrument are also in development. A summary of the features of Hevylite IgM assays is shown in Table 9.2. A full description of Binding Site instruments is present in Chapter 37. A general discussion of Hevylite assay implementation and interpretation is in Chapter 11.

The Binding Site SPAPLUSSiemens BNII
IgMκ IgMλ IgMκ IgMλ
Sensitivity (g/L)
[Dilution]
0.020
[1/1]
0.018
[1/1]
0.010
[1/5]
0.009
[1/5]
Precision (within run, %CV)1.5 - 2.4% 1.7 - 2.0% 2.8 - 5.6% 1.7 - 4.2%
Precision (between run, %CV)1.3 - 4.9% 0.5 - 5.4% 2.7 - 4.8% 1.1 - 9.3%
Prozone parametersYes Yes Yes Yes
Analytical time (min)15 15 12 12
Higher dilutions1 Automatic
then off-line
1 Automatic
then off-line
Automatic Automatic

Table 9.2. Comparison of features for IgM HLC assays on the BNII and SPAPLUS.

Questions

  1. How is the specificity of HLC antisera tested?
  2. How are internal reference preparations for HLC assays standardised?

Answers

  1. Using a panel of antigens (including monoclonal immunoglobulins of each class, subclass, and light chain type) using RID and turbidimetric/nephelometric techniques (Section 9.3).
  2. Using international serum protein reference materials (CRM470 and DA 470k; Section 9.4).
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