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5 - Development and validation of Freelite immunoassays

Chapter 5

Summary:

  • Freelite® assays use sheep polyclonal antisera directed against the hidden epitopes of FLC molecules located at the interface between the light and heavy chains of immunoglobulins.
  • Batch-to-batch consistency of Freelite reagents is maintained using a rolling pool of polyclonal antisera.
  • Freelite assays are validated according to protocols set out by the Clinical and Laboratory Standards Institute, including precision, linearity, interference and stability.

5.1. Assay overview

Freelite κ and λ serum free light chain (sFLC) assays use polyclonal antisera directed against the “hidden” epitopes of FLC molecules that are located at the interface between the light and heavy chains of intact immunoglobulins (Figure 5.1). These epitopes are only accessible when light chain molecules are not associated with the immunoglobulin heavy chain. κ and λ FLCs are measured in pairs to produce κ/λ sFLC ratios, or calculate the difference between the involved and uninvolved sFLC concentrations (Section 7.2.2). Polyclonal antibodies raised in sheep provide the most attractive method of recognising the highly polymorphic FLC molecules (Chapter 3). Latex enhancement increases the sensitivity of Freelite assays, to a few mg/L, and the assays are performed by turbidimetry or nephelometry on a number of automated laboratory instruments (Chapter 37).

5.2. Polyclonal antisera versus monoclonal antibodies

It is essential that FLC immunoassays utilise antibodies that have high specificity and affinity. Early FLC immunoassays, from other groups, used polyclonal antisera but good specificity was difficult to obtain (Chapter 2). Monoclonal antibodies seemed to be the obvious solution to the problem. However, considerable effort failed to produce antibodies that reliably recognised a full range of monoclonal FLCs (Chapter 3). Similar findings have been reported for other monoclonal antibody-based FLC immunoassays and are discussed further in Chapter 8.

In contrast, an assay based on polyclonal antisera can recognise a wide variety of FLC epitopes, including the diverse range of pathological monoclonal FLC produced by patients with monoclonal gammopathies. Therefore, research focussed on optimising polyclonal FLC antisera to ensure the reliable detection of the huge variety of monoclonal FLCs. The following description is an outline of the successful procedures involved in the development of Freelite sFLC assays. In summary, sheep were immunised with κ or λ molecules that had been purified from urine samples containing Bence Jones proteins. The resultant antisera were adsorbed against purified IgG, IgA and monoclonal proteins and then affinity purified against mixtures of the respective FLCs that had been immobilised onto Sepharose. Antisera requiring further adsorption, as judged by the tests described below, were recycled through the adsorption and testing procedures, resulting in antisera highly specific for FLCs and deemed satisfactory for assay use.

5.3. Antisera specificity testing

Specificity is the most important aspect of the immunoassays and was evaluated using several techniques.


5.3.1. Immunoelectrophoresis

Polyclonal antiserum was purified until it showed no cross-reactions by immunoelectrophoresis with the alternate FLC and intact immunoglobulin molecules (Figure 5.2)

5.3.2. Western blot analysis

Western blot analysis is a sensitive technique used to assess the reactivity of the antisera against immunoglobulin fragments and FLC polymers. The results showed that both κ and λ FLC antisera reacted strongly, with two closely migrating bands at 25 - 30 kDa, and weakly with several larger and smaller molecular weight fragments. Similar staining patterns were observed using monoclonal antibodies. The FLC antisera were readily able to detect monomers and dimers of both κ and λ molecules (Figure 5.3).

5.3.3. Haemagglutination assays

Haemagglutination assays are far more sensitive than immunoelectrophoresis, and provide better assessment of specificity. Sheep red blood cells were sensitised with individual FLCs and purified IgG, IgA and IgM, and tested against the FLC antisera. The results showed that κ and λ FLC antibodies reacted with the appropriately labelled cells at >1:16,000 dilution and at <1:2 against cells coated with the alternate FLCs or intact immunoglobulins (Figure 5.4).

5.3.4. Nephelometry

Latex-conjugated FLC antisera were tested for specificity by nephelometry. Potentially interfering substances were added to serum containing known concentrations of FLCs and the changes in values indicated the effect on the assays (Figure 5.5). Nephelometric assays demonstrated that FLC antisera had minimal reactivity with light chains on intact immunoglobulins and other potentially interfering substances (0.2 - 0.01%). These values are within the purity specification for FLC contamination in the tested interfering materials.

There have been no published independent specificity analyses of the nephelometric Freelite latex reagents. Nakano et al. [148] reported an evaluation but, in error, only tested FLC antisera that were manufactured for immunofixation electrophoresis (IFE), where specificity requirements are less demanding.

5.4. Accuracy and standardisation

Assay accuracy is defined as the degree of closeness of achieved results relative to their actual (true) values. Unfortunately, international standards do not exist for FLC measurements, so there are no reference points from which to assess the accuracy of results. In order to ensure accuracy in Freelite immunoassays, a suitable basis for standardisation and calibration was required. It was considered that polyclonal FLCs should be used in order to minimise any potential problems that might arise from the use of unique monoclonal proteins.

The production of assay calibrators was achieved in the following manner: 1) production and accurate quantification of pure polyclonal, primary κ and λ FLC standards; 2) production of secondary internal reference standards; and 3) production of calibration materials for use in the FLC kits (calibrated to the internal reference) (Figure 5.6).

Each primary standard was found to be greater than 99% pure by silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), whilst the alternate FLC was not detected by haemagglutination-inhibition and dot blot assays. The amino acid content of each primary standard was then determined in order to produce an accurate estimation of the protein content. Secondary standards were initially prepared from pools of different monoclonal κ and λ proteins. These were not considered ideal for use as working calibrators, so additional polyclonal standards were prepared from a pool of sera that contained elevated polyclonal sFLCs. These standards are known as κ or λ Internal Reference standards. The primary standards were used to assign κ and λ FLC concentrations to the Internal Reference standards by nephelometry. Each stage of the value transfer was completed at three dilutions and repeated three times. The final FLC values for the Internal Reference standards were 46.0 mg/L and 71.4 mg/L for κ and λ sFLCs, respectively. These values were used for all subsequent laboratory and clinical studies and used to assign values to kit calibrators and controls.

The measuring ranges of Freelite nephelometric/turbidimetric 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® for κ and λ sFLCs are 4.0 - 180 mg/L and 4.5 - 165 mg/L, respectively (Figure 5.7). Samples containing higher concentrations require further dilution. 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).

5.5. Maintaining batch-to-batch consistency of polyclonal antisera-based latex reagents

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

In order to ensure consistency between production batches of polyclonal antisera, a virtual “rolling pool” of antisera has been established. This pool consists of a list of pre-approved antisera (Section 5.3 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 Freelite assays will always contain at least 90% of the same constituent antisera as the previous batch of reagent. The use of rolling pools of antisera during FLC assay manufacture has minimised batch-to-batch variation whilst ensuring a full range of FLC epitopes are recognised [149].

Once a pool of suitable polyclonal antisera has been created, the sheep antibodies are attached to latex particles (in order to enhance their performance in nephelometric and turbidimetric immunoassays).

5.6. Overview of Freelite assay validation

During the development of Freelite assays there is a rigorous validation process to ensure that the assays perform correctly and provide the correct diagnostic information. The validation protocols follow those set out by the Clinical and Laboratory Standards Institute, and are outlined in Table 5.1.

Validation Comments Validation protocol
PrecisionAt multiple levels across the measuring range Within run, between analyser, between batch, and total precision
Analytical sensitivityAt the lower end of the measuring range Limit of detection, limit of quantitation, and limit of blank
LinearityAcross the measuring range Disease state sera
InterferenceBy the most common assay interferents Haemoglobin, bilirubin, lipid, relevant drugs etc.
StabilityOf kit to determine kit expiry Real time
Of open reagents On board
Of reagents that are removed from the analyser
and refrigerated when not in use
Open vial
Of kits that are heated or frozen to mimic
worst case conditions during shipment to customers
Extremes of temperature
Comparison to predicate deviceUsing a range of samples relevant to the utility of the assay Healthy blood donors and disease state sera
Confirmation of normal reference rangeQuoted by the manufacturer Healthy blood donor sera

Table 5.1. Summary of Freelite assay validation protocols.

5.7. Overview of Freelite kit manufacture

The manufacture of Freelite assays follows established validated protocols. Each batch of reagents undergoes rigorous testing to ensure the quality of the kit components (Figure 5.8). Once the latex and supplementary reagent has been manufactured, the precision and linearity of the assay is tested. Next, a value is assigned to the kit calibrators using the Internal Reference standard. This is achieved using 100 separate assays and 10 separate calibration curves. Values are assigned to control samples using similar protocols.

For each new batch of antisera, specificity is controlled by comparing sFLC results for panels of samples with results from previous batches. The panel samples include normal sera and patient sera containing polyclonal or monoclonal sFLCs (typically from multiple myeloma patients). 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 is shown in Figure 5.9.

Analytical comparisons are also made using a large number of normal sera. A typical evaluation of a panel of 90 normal samples on the Binding Site SPAPLUS produced the following results: mean κ = 8.86 mg/L (range 4.32 - 20.6 mg/L), mean λ = 11.85 mg/L (range 3.77 - 28.77 mg/L). Freelite normal ranges are further discussed in Chapter 6. Once a final pre-packaging test is complete, the kit is packaged ready for release.

5.8. Immunoassay development on different platforms

Freelite assays were initially developed for the Siemens BN™II nephelometer, and following their successful launch in the year 2000, the range of platforms has expanded and the assays are currently available on a total of 10 different instruments. Developing Freelite assays across different platforms poses a challenge as each instrument has unique features (summarised in Table 5.2). These include differences in the optical detection systems and the methods for reagent and sample handling. Therefore, assays developed for each platform may vary slightly in terms of sensitivity, measuring range, precision and antigen excess detection. However, there is good agreement between the sFLC results obtained with the different instruments. An example of κ and λ sFLC results obtained for BNII and SPAPLUS instruments is shown in Figure 5.10.

System feature
Examples
Sampling Cuvettes (disposable/semi-disposable/non-disposable), cuvette cleaning method, probes/pipettes
System liquids Wash solutions, sample diluent, tubing and pumps
Reagent storage Compartments and carousels, temperature control (refrigerated/non-refrigerated)
Sample dilution Automatic on-board/manual off-line
Detection system Light source, light detector (nephelometric/turbidimetric)
Channels Open/closed
Software and parameters Sample flags, automatic antigen excess detection
Integration Tracked system/stand-alone, laboratory information system integration

Table 5.2. Features that may vary between different analytical platforms.

Questions

  1. How are Freelite assays routinely standardised?

Answers

  1. As no international standards exist for FLC measurements, Freelite assays are standardised against κ and λ internal reference materials, which, in turn, are calibrated to highly purified primary standards (Section 5.4).
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