The new generation of allergy testing - Pharmacia CAP System

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Lars Yman, Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden
 
The measurement of total serum IgE and allergen specific IgE antibodies is a well-established component of the investigation of allergic patients. The routine test system, Phadebas RAST, a unique immunoassay invention, has matured into a reliable standard for allergy testing (1). Numerous scientific studies and routine testing of several millions of patients with suspected atopic, IgE-mediated hypersensitivity have taught the medical and clinical laboratory communities that IgE antibody testing using Phadebas RAST is more specific (i.e. fewer false-positive results) but somewhat less sensitive than skin testing. Many investigators have confirmed the relation between the IgE antibody concentration or severity of reaction and natural or voluntary exposure to common allergens. The choice of therapy based on the type and severity of hypersensitivity is of increasing concern. It is obvious that a new test method
  • should have increased sensitivity (without a decrease in specificity)
     
  • have a wider measuring range to better reflect the biological response, and
     
  • permit quantitative measurements (accurate calibration, high precision and reproducibility). How this should be best achieved is less obvious. Increased sensitivity requires increased signal-to-noise ratio, i.e. a better discrimination of low levels of antibody from the non-specific background, (Fig. 1).

How this should be best achieved is less obvious. Increased sensitivity requires increased signal-to-noise ratio, i.e. a better discrimination of low levels of antibody from the non-specific background, (Fig. 1).

Fig. 1. The ideal serological immunoassay.

This ratio is influenced by several factors. Some are listed in Table I. However, the most critical factor is the solid-phase that serves as a support permitting the dual immune reaction (allergen - IgE and IgE - anti-IgE). Regardless of where in the test sequence the solid phase comes in, only a solid-phase with high specific binding capacity and low non-specific interference can provide the basis for a sensitive, wide range assay. Limitations in the performance of the solid-phase can not be compensated for to any significant degree by adjustments of the other factors. Attempts to amplify the second immune reaction and the detection step may even decrease the signal-to-noise ratio and seriously jeopardize the robustness of the assay.

Table I.

Factors determining the signal-to-noise ratio
AllergenConcentration
Composition
Separation deviceSolid-phase capacity
Anti-IgESpecificity
Affinity
Concentration
LabellingDensity
Signal amplification
DetectorSensitivity
 
Based on long experience from developing and manufacturing immunoassays, Pharmacia chose to develop the new generation after Phadebas RAST around a new type of solid-phase. The new test system, Pharmacia CAP System, consists of immunoassay reagents, instrumentation, and information management software developed for the measurement of total and specific IgE in undiluted serum or plasma.
 
The system is designed for sandwich immunoassays and immunometric assays and can be described as the new generation of allergy testing (2). Solid-phase bound allergens are allowed to react with antibodies in the sample. The IgE antibodies are detected by labelled anti-IgE.
 
To minimize handling and increase safety, the system includes instrumentation and computer software that handle the technical manipulations, the measurements and the data management.
 
 
Fig. 2. The structure of the solid-phase.
Solid-Phase
The system is built around a new type of solid-phase, consisting of a flexible hydrophilic carrier polymer encased in a capsule, ImmunoCAP. (Fig 2).
 

 
Fig. 3. Binding capacity.
 
The carrier consists of a CNBr-activated cellulose derivative that can bind at least three times more protein than a corresponding paper disc and about 150 times more than can be adsorbed to the inner surface of a traditional coated tube (Fig. 3). This solid-phase is an excellent carrier of allergens and provides favorable reaction conditions, including short diffusion distances. Equilibrium can be reached in less than 20 min. In fact, most of the IgE antibodies in test samples or standards is bound to the immobilized allergens within two minutes of the initial incubation step. (Fig. 4).
 

Fig. 4. First incubation kinetics.

Allergens
The allergen range is equivalent to that of Phadebas RAST and standardized for maximal reproducibility. The principle behind the quality control of source materials is outlined in Fig. 5.

 

Fig. 5. Basis for allergen source material standardization.

 

Anti-IgE
The system utilizes IgE(Fc) specific combinations of polyclonal and monoclonal anti - IgE antibodies labeled with 125I or a b-galactosidase generating fluorescence. Both reagents show high immunoreactivity and low background noise and allow a wide measuring range. Cross-reactivity with other immunoglobulins is less than one in 2x106. High dose hooks are absent.

 

Calibration
The assay is calibrated against the WHO standard 75/502 for IgE and includes two sets of calibrators: 0.35-100 kUA/l (for specific IgE Ab and low range total IgE) and 2-2000 kUA/l (for wide range total IgE). The design of the new assay is based on three important specifications:

  1. The cut-off (positive/negative) should remain the same as for Phadebas RAST
     
  2. The units and classes should remain
     
  3. A WHO IgE standard previously used for in-house calibration of Phadebas RAST birch reference system should be used directly as the calibrator.

The entire test system is therefore built around the 0.35 kUA/l calibrator, corresponding to Reference D of Phadebas RAST. The anti-IgE is designed to permit a wider measuring range with the same initial slope of the dose response curve (Fig. 6). All solid-phase allergens are then individually optimized for maximum capacity and response and low background noise relative to the measuring range.


 

Fig. 6. Slope of standard curve.

 

The calibrator concentration is consequently constant and constitutes the core around which the system is constructed. The faster and more efficient binding of specific IgE antibodies against the individual allergens leads to increased sensitivity. The radioimmunoassay (RIA) and the fluorescense immunoassay (FEIA) give the same measured values (Fig. 7).

Fig. 7.

Specificity
The quantitative and precise assay effectively discriminates  low IgE Ab levels from the background. The specificity of the system is very high. 28 normal blood donor samples and a cord serum pool tested with 20 common inhalant and food allergens were negative in 577 out of 580 tests. Non-specific binding of high total IgE up to 3000 kUA/l is not significantly different from normal blood donor background when tested with 56 allergens.

Not even 20 000 kUA/l of non-specific IgE interfered in the 6 allergen systems tested up to this level. (Fig. 8).

Fig. 8. Lack of interference by high total IgE.

Sensitivity
Pharmacia CAP System test results correlate closely with positive Phadebas RAST results. The high binding capacity of ImmunoCAP combined, with potent immunoreagents,   permits an improved discrimination, especially in the very low and very high concentration ranges. The more effective presentation of the allergen to the antibody in the test sample improves the capability to detect low concentrations of IgE antibodies against minor allergenic components. The majority of samples tested will,  therefore,   give higher measured values.

No interference from specific IgG antibodies has been observed.

 

Clinical Trials
An extensive international clinical trial program has been initiated for this new generation of IgE antibody assay system. When the first trials in Europe were summarized, it was clear that the increased sensitivity was confirmed (3-12). A typical pattern is shown in Fig. 9. Up to 15% more patients were detected without loss of test specificity.

Fig. 9. Correlation to Phadebas RAST.

A total of 20 allergens were tested at 9 allergy clinics. 1073 pediatric and adult patients were investigated by the allergy specialists according to the principles routinely applied at the clinic. 6549 clinical conclusions were made. In 1454 cases,  the diagnosis was atopic allergy against an identified allergen.

Several interesting observations could be made when analyzing the data.

 

Sensitivity and specificity of different allergens compared to clinical diagnosis
Overall sensitivity and specificity was calculated for all allergens where the total number of clinically positive or negative patients was = 30 (totally 6346 clinical conclusions).

The sensitivity ranged from 78 - 94% (x = 88) and the specificity from 77 - 94% (x = 85)

 

Sensitivity compared to skin test and clinical diagnosis
As should be expected, Pharmacia CAP System results correlate better to the clinical diagnosis made by the allergy specialist than to the isolated skin test results. The analysis of the data (examples in Table II) shows that especially for animal and mold allergens,  a high proportion of positive skin test results were disregarded (i.e. considered as false positive) by the investigators when the clinical diagnosis was made.

Table II.
Pharmacia CAP System sensitivity (%).
Examples of total sensitivity calculated from 9 European trials.

AllergenCompared to
Clinical diagnosisSkin prick test
d19286
e18466
e58879
g39488
t39080
w219187
m67958
aaa
However, also when compared to skin prick test alone,  Pharmacia CAP System shows an increased sensitivity (Table III).

Table III.
Phadebas RAST and Pharmacia CAP System
results in 274 skin prick test positive patients ( House dust mite d1).

 
Sensitivity and specificity in patient groups studied at different clinics
The results of the clinical trials analyzed so far emphasize the fact that there may be considerable differences in the results obtained at different clinics. Differences are likely to be attributed to the patient groups, their exposure to allergens, allergen preparations used for in vivo tests and last,  but probably not least, differences in interpretation and weighting of information derived from clinical history and in vivo tests. Examples of such differences can be seen in Fig. 10, summarizing sensitivity and specificity data obtained for cat (e1) when using the same test on patients from 6 clinics and comparing the results to the clinical diagnoses.
 

Fig. 10. Pharmacia CAP System. Summary of 6 studies on the same allergen.
 
 
Discrimination of cases with true negative and positive diagnoses
Pharmacia CAP System was proven not to give positive reactions in cord serum, normal blood donors, or sera with high non- specific IgE (2). The high specificity was confirmed by blind testing of 20 cord sera together with sera from normal blood donors and allergy patients (9). On the other hand, data strongly suggest that true positive results can also be found  in patients with negative skin prick test and negative clinical diagnosis (10). Thus, in some situations,  Pharmacia CAP System is more sensitive than the in vivo test.
 
The high sensitivity and specificity, combined with a wide measuring range,  result in an efficient discrimination of true negative cases and cases with active atopic disease. Fig. 11 shows the distribution of the measured response of samples divided by the measured response of the 0.35 kUA/l Reference. The data were calculated from 580 tests on healthy blood donors (2) tested with 20 allergens and 442 tests on patients (7, 9) with positive clinical diagnosis to one or several of 8 - 10 allergens.


Fig. 11. Pharmacia CAP System. Discrimination of true negative and positive diagnosis.
 
In summary, Pharmacia CAP System was shown to be a sensitive and specific quantitative assay for allergen-specific IgE antibodies. The clinical investigations proved its diagnostic value and confirmed the correlation between the serum concentration of IgE antibodies and the severity of the allergic disease (7, 12). Its reproducibility and unique measuring range should open the way for wider use in prediction, confirmation and monitoring of atopic disease.
 

References:

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1990