Immunoassays for free light chain measurement

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4.1. Introduction

There are numerous assays for monoclonal FLCs in urine (Table 4.1). Some, such as protein precipitation, are simple screening tests but are insensitive and non-specific, while others such as IFE are more sensitive but laborious and may be difficult to interpret.

Based upon these requirements, some commonly used tests for FLCs are analysed in Table 4.1. Each assay has features that are satisfactory, to some degree or other, but all fail to detect FLCs in serum at concentrations within the normal range (3-25mg/L); or even at concentrations that are several times higher than the normal range. Dipstick assays, based upon dye uptake, are particularly unreliable for measuring cationic proteins such as FLCs and should not be used.

One modern solution to the problems of inadequate assay specificity and sensitivity is to use antibody-based methods. The following chapter describes the development of serum FLC immunoassays and their validation on a variety of laboratory analysers.

4.2. Immunoassays for free light chains

A. Polyclonal antisera versus monoclonal antibodies

It is essential that FLC antibodies have high specificity and affinity. Early FLC immunoassays (Chapter 5) used polyclonal antisera but good specificity was difficult to obtain. Monoclonal antibodies (Mabs) seemed to be the obvious solution to the problem. However, considerable effort by my colleagues in the Immunology Department at The University of Birmingham, failed to produce antibodies that reliably recognised a full range of monoclonal FLCs. Other reported Mabs have also failed to have clinical impact, possibly for similar reasons. Furthermore, Mabs cannot be used in nephelometric or turbidimetric assays because they do not form immunoprecipitates, so enzyme immunoassays (or similar types) are required. These 3-stage assays need high serum dilutions and take considerable time to perform. Hence, they do not readily form part of routine clinical testing alongside immunoglobulin assays.

Figure 4.1 Immunoelectrophoresis showing the specificity of κ FLC antisera.

Figure 4.3 Haemagglutination assays showing the specificity of κ FLC antisera against red blood cells (RBC) coated with purified FLCs and IgG.

Figure 4.2 Western blots showing the specificity of the polyclonal FLC antisera compared with monoclonal antibodies and the reaction of polyclonal antisera against FLC monomers and dimers separated by non-reducing SDS-PAGE. (Lane 1, molecular weight markers. Lanes 2 & 3, urine containing κ FLCs and 4 & 5, normal serum all probed with mono- and polyclonal anti-κ. Lanes 6 & 7, urine containing λ FLCs and 8 & 9, normal serum probed with mono- and polyclonal anti-λ. Lanes 10 & 11, polyclonal FLC antisera reacting with monomers and dimers of κ and λ).

Figure 4.4 Specificity of (A) κ and (B) λ FLC antisera assessed by interference with the results of typical nephelometric assays upon the addition of various substances. Mean and 95% confidence limits for each added substance are shown.

To be practical, we therefore focussed on optimising polyclonal FLC antisera. The following description is an outline of the successful procedures involved in their development. Details of the many steps are proprietry and are not available. However, in summary, sheep were immunised with κ or λ molecules that had been purified from urines containing Bence Jones proteins. The resultant antisera were adsorbed against purified IgG, A and M 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 until satisfactory.

B. Antisera specificity

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

1. Immunoelectrophoresis

The antibodies were purified until they showed no cross-reactions by immunoelectrophoresis with the alternate FLC and intact immunoglobulin molecules (Figure 4.1).

2. Western blot analysis

This sensitive technique was 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 25-30kDa 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 4.2).

3. Haemagglutination assays

These 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 4.3).

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 4.4). Overall, the specificity assessments showed that FLC antisera had minimal reactivitywith 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 independent specificity analyses of the nephelometric Freelite latex reagents. Nakano et al.^[15], reported an evaluation but, in error, only tested FLC antisera that were manufactured for IFE, where specificity requirements are less demanding ^[16].

C. Accuracy and standardisation

Accuracy is defined as the closeness of achieved results compared with their absolute values. Unfortunately, international standards do not exist for FLC measurements, so there are no reference points from which to assess the accuracy of results. Furthermore, each monoclonal light chain is unique, with its own special set of surface epitopes, so accurate measurements are difficult to obtain. Nevertheless, the light chain constant region domains have little structural variability, so they are good antibody targets.

In order to ensure accurate FLC 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 ^[17].

Figure 4.5 Flow chart for the production of standards for serum FLC immunoassays.

This was achieved in the following manner (Figure 4.5):-

1. Production and accurate quantification of pure polyclonal, “primary” FLC standards.

2. Production of secondary and “working” reference materials. Comparison with the primary standards.

3. Production of calibration materials for use in the FLC assays that were referenced against the “working” standards.

4. Analysis of a variety of normal and abnormal samples using a reference nephelometric method.

5. Comparison of results from other instruments with the reference method.

The various materials were manufactured and purified in accordance with established procedures ^[18]. Each primary FLC preparation was found to be greater than 99% pure by silver-stained SDS-PAGE while 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 reference materials were prepared from pools of different monoclonal κ and λ proteins. These were not considered ideal for use as working calibrators, so additional reference materials were prepared from sera that contained elevated polyclonal FLCs (Figure 4.5).

The pure FLC preparations were used to assign κ and λ concentrations to the secondary reference preparations. Subsequently, κ and λ values were assigned to the serum pool by nephelometry. Each stage of the value transfer was completed at three dilutions and repeated three times. All protein preparations were stabilised and stored at -80°C until required. The final FLC values for the “working” reference preparations were 46mg/L for κ and 71.4mg/L for λ. These calibration values were used for all subsequent laboratory and clinical studies.

An alternative reference material based upon polyclonal FLCs extracted from urine has been proposed ^[15]. However, serum FLCs may differ chemically from urine FLCs and concentration measurements in the latter study were based upon inappropriate dye uptake comparisons with albumin rather than amino acid analysis. As yet, these two preparations have not been compared. Clearly, there is need for international agreement on standardisation with wide availability of a reference material.

D. Assay sensitivity

Assay sensitivity (the lowest limit of antigen detection) is somewhat dependent upon the types of sample measured. FLC assays can detect less than 0.1mg/L of κ and λ FLCs in undiluted, clear urines and CSF. Serum sensitivity is typically 5-fold worse because of interference from lipids and other light-scattering particles (Table 4.2 and Chapter 6). Concentrations at 1-2mg/L are below the normal serum range so the FLC immunoassays allow extreme κ/λ concentration ratios to be established with considerable accuracy. This is important for monitoring patients under treatment because FLC concentrations may be below the normal range. Additional sensitivity for urine and CSF measurements (Chapter 22) can be obtained by adjusting concentrations of the reactants.

Table 4.2. Representative routine sensitivity levels of frequently used FLC assays.

E. Assay ranges

The measuring ranges of the FLC assays are dependent upon two factors: the slope of the respective calibration curve and the portion selected for the assay (Figures 4.6 and 4.7). The latter should be chosen to allow the maximum number of normal and abnormal clinical samples to be measured at the initial sample dilution. A typical analytical range for κ is 3-150 mg/L and for λ, 5-200 mg/L (Chapter 27). Samples containing higher concentrations require further dilution (see Antigen excess, below).

Figure 4.6 Calibration curve for κ FLC using the Roche Modular P.

Figure 4.8 Diagram illustrating the mechanism of antigen excess. The light scattering signal falls in antigen excess because of smaller immune complexes.

Figure 4.7 Calibration curve for λ FLC using the Roche Modular P.

Figure 4.9 Demonstration of antigen excess capacity for FLC assays using monoclonal proteins. Addition of high concentrations of the patients’ sera caused the assays results (in light scattering units) to plateau.

Patients with bone marrow suppression may have low concentrations of the alternate FLC, where the precision of the calibration curves is relatively poor. Consequently, some consideration should be given to the accuracy of κ/λ ratios that include low FLC concentrations.

F. Antigen excess

Antigen excess causes immunoassays to underestimate very high concentrations of protein (Figure 4.8). Some instruments will identify antigen excess situations and recommend sample redilution. In the case of FLC measurements, sample concentrations can range from <1mg/L to >100,000mg/L. This is a greater range than almost any other serum protein test. Consequently, very high levels may be under-estimated on some instruments because of antigen excess. Historically, it was suggested, although incorrectly, that this problem precluded accurate FLC measurements ^[19].

Antigen excess is an important issue. During the development of the FLC assays on the Behring BNII analyser, results indicated linear quantification at up to 200 mg/L with antigen excess at higher levels (Figure 4.9). Subsequently, monoclonal FLCs from 304 patients were assessed. At a 1:20 serum dilution, 6 high samples were incorrectly identified as moderately elevated and a further two were identified as normal. When the samples were tested at a dilution of 1:100, all of the 8 troublesome samples were correctly identified as highly elevated. Later assays were modified to use a starting serum dilution of 1:100 ^[20].

Larger sample studies on the BNII have determined the incidence of antigen excess more accurately. Clark RJ. et al.^[21], at the Mayo Clinic, studied 7,538 consecutive samples over a 4 month period using 1:100 and 1:400 sample dilutions. There were 9 samples with κ antigen excess but no samples with λ antigen excess giving an incidence of 1/840 (0.12%). Of importance, all the κ and λ samples had elevated FLC concentrations or abnormal κ/λ ratios at the initial dilution of 1/100.

Hence, a simple policy of retesting any sera that have abnormal levels, at a higher dilution, identifies all antigen excess samples ^[21][22][23]. These recommendations will be included in the manufacturer’s instructions for the sFLC assays on the BNII and other instruments. However, samples with large FLC structural variations could theoretically still be problematical and show antigen excess at fairly low concentrations (see Amyloid disease, Chapter 15).

G. Precision

The precision of the sFLC assays is good, with percentage coefficients of variation typically less than 8%. However, samples measured at low concentrations, the use of poorly maintained instruments, and operation by inexperienced staff will produce worse precision data. Results from the manufacturer are produced by expert staff under ideal conditions so that minor variations between production batches can be identified and corrected. Similar results are achievable in busy, routine laboratories. If they are less good, it is appropriate to seek assistance so that the causes can be identified. Some results of precision studies assessed at different parts of the measuring ranges are shown in Table 4.4. Further details are available for individual instruments in Chapter 27.

Figure 4.10 κ/λ log plot of approximately 4,000 abnormal serum FLC samples showing “gaps” at the lower and upper ends of the instrument’s assay range. Samples in the normal range were excluded.

Figure 4.12 Linearity of (A) κ and (B) λ assays on an Hitachi 911.

Figure 4.11 As for Figure 4.10 but showing κ sFLC results alone. The “steps” in the plot occur at the ends of the instrument’s assay range, where there is a change in sample dilution. Similar results occured for λ.

Figure 4.13 Stability of FLCs over different times and at different temperatures. (κ [square]; λ [triangle]. Standard deviations of results are mostly within the symbols).

Some years after the introduction of the FLC assay it became apparent that samples with results just outside the lower and upper ends of the instrument ranges were slightly lower and higher respectively, when repeated at the new dilutions. This is due to an uncharacterised substance(s) that “interferes” with the latex assays. Since the concentrations of the interfering substance are different at the second dilution, the results are altered. This gives a “gap” in results of 2-3mg/L at the lower end of the curve (at 4-7mg/L) and 8-9mg/L at the upper end of the curve (at 62-70mg/L) on, for example, the Roche Modular P (Figures 4.10 and 4.11).

In order to reduce the frequency of samples falling at the ends of the curves, the assay ranges will be extended downwards and upwards so that fewer normal samples require re-dilution. This has improved the precision of results at this borderline region of the analytical range.

H. Linearity

Assays are considered to be linear when the results obtained from measurement of a sample at a series of dilutions are equivalent to the results expected from the original measurement. Non-linearity is due to a number of factors including: poor antibody specificity; inability of the antibody to recognise different forms of the antigen; nonspecific assay interference from lipids or fibrin; unsuitable materials for the calibrators or standards etc. Assessment of linearity forms an important part of immunoassay development.

FLC assays are potentially prone to non-linearity. Light chains are monoclonal proteins which exist as a number of sub-groups, in polymeric forms and contain unique combinations of hypervariable regions. Inevitably, these features cause non-linearity in some samples. Examples of good linearity are shown in Figure 4.12.

I. Free light chain polymerisation

FLC molecules are usually monomers or dimers but higher polymeric forms frequently occur ^{[24][25][26][27][28][29][30]}. They act as multi-antigenic targets in mmunoprecipitation assays which accelerates the formation of aggregates and leads to over-estimation of antigen concentrations. This occurs in patients with NSMM who have undetectable concentrations of serum FLCs by IFE but high concentrations by nephelometry (Chapter 9) . Sölling ^[31][32] indicated that with his assays, monomers and dimers were detected equally using antibodies against whole light chains, but antibodies directed only against FLC epitopes may preferentially detect dimers ^[29].

In order to determine the effect of polymerisation on FLC quantification, FLC monomers, dimers and polymers were purified from myeloma sera and concentrations compared with total protein measurements. It was apparent that purified dimers were over-estimated by 1.5-fold and higher polymers by 1.5 to 3.5-fold ^[33]. However, in a study by Émond et al.^[30], a greater than 7-fold over-estimation was observed in two samples (when compared with CZE) in association with polymers of up to 200kDa.

An additional factor is that SPE tests can underestimate FLC concentrations. Variable polymerisation may cause “smearing” of monoclonal bands on the gels so that only a proportion of the monoclonal protein is measured (Figure 9.2). FLCs also take up less protein stain than albumin so their concentration is underestimated ^[2]. It is likely that a combination of these factors causes the ten-fold or more over-estimations seen in the sera of some patients.

Whatever the explanation for the unexpected high results, it will be difficult to develop FLC assays that measure all molecular forms equally. Perfect quantification will be elusive.

J. Stability

FLC molecules are very stable in serum and urine (Figure 4.13). Tencer et al. ^[34][35] , showed that there was little variation in the concentrations of FLCs in samples stored at -20°C over a two year period. We have found similar concentrations of FLCs in both fresh and old normal sera stored for 20 years at -20°C. Clearly, these results indicate that FLCs do not dissociate from intact immunoglobulins, or fragment, over prolonged periods.

Stability of the FLC antisera is also important. “Open-vial stability” refers to the shelf-life of the antisera after their first use. This may be short because pipetting procedures can introduce FLC molecules into the vials that react with the remaining antisera and reduce its activity. Care should be taken not to contaminate antisera vials with sera from previously pipetted samples.

K. Serum and plasma comparisons for free light chain assays

Figure 4.14 Comparison of serum and acid citrate dextrose plasma samples from 50 blood donors and 20 MM patients using a Beckman Immage.

Figure 4.15 Comparison of serum samples on different platforms for kappa (left) and lambda (right).

Figure 4.16 Comparison of results obtained from two batches of kits with the mean results from previous analyses. (The mean results were obtained from an average of 12 assays over 6 batches of reagent. The samples included 30 normal sera and 30 myeloma sera of various immunoglobulin types).

Some laboratories prefer to analyse blood proteins in plasma rather than sera. A study was performed to directly compare the concentrations of FLCs in plasma and serum. 50 paired serum and plasma samples from blood donors and 20 paired samples from patients with MM were studied on a Beckman Immage, a BNII and a Hitachi 911. Plasma samples were collected in acid citrate dextrose or heparin and the sera were prepared by adding fibrin. Table 4.3 and Figure 4.14 show comparison data obtained on different instruments ^[36].

Table 4.3. Comparison of serum and ACD plasma FLC concentrations from 50 blood donors and 20 MM patients.

4.3. Comparison of immunoassays on different instruments

sFLC kits are made for many laboratory instruments. A list of those currently available is given in Chapter 27. Comparison of FLC results using an Hitachi 911 and a Behring BNII are shown in Figure 4.15. and five different instruments are shown in Table 4.4. Comparisons of normal ranges are discussed in Chapter 5 . There are no significant differences between the results on different instruments ^[37].

Table 4.4. Summary of the characteristics of FLC assays on different instruments. (C.V. = coefficient of variation in precision studies. Numbers in brackets refer to sample dilutions). See Chapter 27 for more details.

4.4. Comparison of different serum FLC immunoassays

Figure 4.17 Analysis of outcome of customer enquiries.

The Binding Site serum FLC assay (Freelite™) is the only one validated for serum measurements. New Scientific Company (NSC), from Italy, sells a urine assay for FLCs that they claim can be used for serum measurements. However, since the antisera are not latex-conjugated, there is insufficient sensitivity to measure normal sFLC concentrations. Furthermore, the antisera cross-react with intact immunoglobulins so that normal samples produce higher results than the Freelite assays. Consequently, sera containing elevated monoclonal FLCs may be classified as normal. In a study of 24 patients with monoclonal gammopathies, 15 were abnormal by Freelite but only 5 using NSC reagents. Of the 7 patients with AL amyloidosis in the cohort, all were positive by the Freelite assay but only 2 using NSC reagents. Furthermore, fluctuations of FLC concentrations were observed with Freelite reagents during monitoring that were undetectable by NSC reagents ^[38][39]. Thus, by scientific and clinical critera, such unvalidated reagents should not be used for serum FLC measurements.

4.5. Quality control of free light chain antisera and kits

Maintaining batch-to-batch consistency is essential as the assays may be used for monitoring individual patients over many years. Effective quality control is ensured using a variety of techniques, two of which are briefly described here. External quality assurance schemes are described in Chapter 28.

Figure 4.18 Numbers of units sold per customer enquiry.

Specificity is controlled by comparing a set of test results from each new batch of antiserum with results from previous batches. Typically, the panel of samples includes normal sera, sera with elevated polyclonal FLCs and myeloma sera. The results are compared using regression analysis and are considered acceptable when they fall within a defined set of criteria (Figure 4.16).

Figure 4.19 AKTA biopilot system for controlling column chromatography.

Once suitable antisera have been selected and attached to latex particles, the other kit components are selected and assembled. Similar materials are used for calibrators and controls and comprise sera containing high concentrations of polyclonal FLCs. The first step is to assign a value to the kit calibrators using the working reference material. This is achieved using 100 separate assays and 10 separate calibration curves. The second step is to assign values to the control reagents using similar procedures.

Analytical comparisons are also made using normal sera. A typical evaluation on 30 normal samples produced the following results: mean κ = 10.8mg/L (range 4.5 - 17.1mg/L), mean λ = 18.0mg/L (range 8.2 - 31.7mg/L), mean κ/λ ratio = 0.58. Interinstrument agreement is also important with results from 4 instruments shown in Table 4.4.

The long-term effect of technical improvements, together with more experience of kit production, customer feedback (Figure 4.17) and larger batch production, has improved quality. This is apparent from the considerable reduction in customer enquiry rates, per unit sold, since the kits were first introduced 8 years ago (Figure 4.18) ^[40]. Upward trends typically occur as larger reagent batches are manufactured. These are advantageous because there is more material that can be used for assignment of reference values, stability checks, calibrator checks, specificity and sensitivity analysis, etc. Downward trends are associated with the introduction of new kits such as for the SPA_PLUS instrument, in Autumn 2007. Newly introduced assays always have teething problems so it is of note that issues with the early kits have been largely resolved.

4.6. Scale-up of antiserum production

Scale-up involves producing larger batches, both by pooling antisera from more sheep and by using larger and more automated laboratory equipment. The polyclonal reagents have to be adsorbed to complete specificity and then affinity purified by passage through large chromatography columns. This is controlled using an AKTA biopilot system that can automatically load, wash, elute and collect antibody peaks (Figure 4.19). Batch sizes have increased a thousand fold, from a few hundred millilitres to hundreds of litres. In the life-cycle of diagnostic tests, however, the FLC immunoassays are still in their youth. Improvements will be seen in sensitivity, specificity, reactivity, precision, utility, costs, etc.

References