IMMUNOASSAY

Abstract

The invention provides a method of quantifying multiple antigen-specific immunoglobulins in a test sample, the method comprising utilising a serial dilution of anti-immunoglobulin antibodies, fragments or derivatives thereof, immobilised on a solid support in combination with a serial dilution of a reference sample of immunoglobulin to generate multiple binding capacity curves. Such binding capacity curves are matched to specific dose response curves generated for each specific antigen to be tested using serum samples of known reactivity to those antigens to provide a calibration system that enables more accurate analysis of antigen-specific immunoglobulin in a sample. The invention also provides methods for calibrating a device suitable for assaying multiple antigen-specific immunoglobulins binding to multiple antigens or fragments thereof immobilised on a solid support. A multi-allergen test system and kits for use in the methods are also provided.

Claims

1. A method of quantifying multiple antigen-specific immunoglobulins in a test sample, the method comprising the steps of; (i) assaying binding of a series of samples containing immunoglobulin of known antigen reactivity to multiple antigen components or fragments thereof immobilised on a first solid support; (ii) comparing the level of binding in step (i) with the known reactivity to produce a dose response curve for each antigen component or fragment thereof; (iii) assaying binding of a serial dilution of a reference immunoglobulin sample of the same immunoglobulin subtype as that used in part (i) with a known total amount of immunoglobulin to a serial dilution of anti-immunoglobulin antibodies, fragments or derivatives thereof, immobilised on the first, or a second, solid support; (iv) comparing the level of binding in step (iii) with the known total amount of reference immunoglobulin to produce a binding capacity curve for each anti-immunoglobulin antibody, fragment or derivative dilution; (v) comparing the dose response curves produced in step (ii) with the binding capacity curves produced in step (iv), identifying the binding capacity curve that most closely matches the dose-response curve for each antigen or fragment thereof, and assigning a binding capacity curve to each antigen or fragment thereof on this basis; (vi) assaying binding of antigen-specific immunoglobulin in the test sample to the antigen components or fragments thereof immobilised on the first, second, or a third solid support; and (vii) comparing the level of binding in step (vi), with respect to each individual antigen or fragment thereof, to the binding capacity curve assigned to that antigen or fragment thereof in step (v) and quantifying the level of antigen-specific immunoglobulin present in the test sample.

2. The method of claim 1, wherein the binding capacity curves produced in step (iv) are clustered into representative binding capacity curves to represent different levels of binding capacity and wherein the comparing in step (v) is carried out with respect to the dose-response curves produced in step (ii) and the representative binding capacity curves, rather than the binding capacity curves produced in step (iv).

3. A method of calibrating a device suitable for assaying binding of multiple antigen-specific immunoglobulins to multiple antigens or fragments thereof immobilised on a solid support, the method comprising the steps of; (i) assaying binding of a series of samples containing immunoglobulin of known antigen reactivity to multiple antigen components or fragments thereof immobilised on a first solid support; (ii) comparing the level of binding in step (i) with the known reactivity to produce a dose response curve for each antigen component or fragment thereof; (iii) assaying binding of a serial dilution of a reference immunoglobulin sample of the same subtype as that used in part (i) with a known total amount of immunoglobulin to a serial dilution of anti-immunoglobulin antibodies, fragments or derivatives thereof, immobilised on the first, or a second solid support; (iv) comparing the level of binding in step (iii) with the known total amount of reference immunoglobulin to produce a binding capacity curve for each anti-immunoglobulin antibody, fragment or derivative dilution; (v) comparing the dose response curves produced in step (ii) with the binding capacity curves produced in step (iv), identifying the binding capacity curve that most closely matches the dose-response curve for each antigen or fragment thereof, and assigning a binding capacity curve to each antigen or fragment thereof on this basis; and (vi) inputting the binding capacity curves generated in step (v) into the device such that the binding capacity curves for each antigen can be interpolated with signals produced from samples containing unknown amounts of immunoglobulin that specifically binds that antigen.

4. The method of claim 3, wherein the binding capacity curves produced in step (iv) are clustered into representative binding capacity curves to represent different levels of binding capacity and wherein the comparing in step (v) is carried out with respect to the dose-response curves produced in step (ii) and the representative binding capacity curves, rather than the binding capacity curves produced in step (iv).

5. The method of claim 1, wherein the antigens are recombinant or derived from a natural extract, or a combination thereof, and optionally, wherein the antigens are purified.

6-7. (canceled)

8. A kit of parts comprising: a) a multi-allergen test system comprising a serial dilution of anti-lgE antibodies, fragments or derivatives thereof immobilised on a first solid support, and optionally further comprising allergen components or fragments thereof immobilised on the first solid support, or a second solid support; and b) one or more of the following: i) a reference IgE sample; ii) a first antibody preparation comprising first antibodies that bind IgE; iii) a second antibody preparation comprising second antibodies that specifically bind the first antibodies; iv) a third antibody preparation comprising third antibodies that specifically bind the second antibodies; and wherein either the second antibodies or the third antibodies are conjugated to a detectable marker.

9. The kit of claim 8, wherein the detectable marker is an enzyme.

10. The kit of claim 8, wherein the detectable marker is a chemiluminescent moiety, a radioactive moiety, or a fluorescent moiety.

11. The method of claim 1, wherein the first, second, or third solid support is a microarray chip.

12. The method of claim 1, wherein the antigens are allergens, the immunoglobulin is lgE and the anti-immunoglobulin antibodies are anti-lgE antiobodies.

13. The kit of claim 9, wherein the enzyme is Horseradish Peroxidase.

14. The kit of claim 8, wherein the antigens are recombinant or derived from a natural extract, or a combination thereof, and optionally, wherein the antigens are purified.

15. The method of claim 2, wherein the different levels of binding capacity are very high binding, high binding, medium binding and low binding.

16. The method of claim 4, wherein the different levels of binding capacity are very high binding, high binding, medium binding and low binding.

17. The method of claim 3, wherein the antigens are recombinant or derived from a natural extract, or a combination thereof, and optionally, wherein the antigens are purified.

18. The method of claim 3, wherein the antigens are allergens, the immunoglobulin is lgE and the anti-immunoglobulin antibodies are anti-lgE antiobodies.

19. The method of claim 3, wherein the first, second, or third solid support is a microarray chip.

20. The kit of claim 8, wherein the first or second solid support is a microarray chip.

Description

[0071] Embodiments of the invention will now be described, by way of example only with reference to the Figures in which:

[0072] FIG. 1 is a graphical representation of multiple allergen dose-response curves;

[0073] FIG. 2A is a graphical representation of multiple binding-capacity curves; and

[0074] FIG. 2B is a graphical representation of consolidated binding-capacity curves according to the invention.

EXAMPLE 1: IMMUNOASSAY WITH BINDING OPACITY CALIBRATION SYSTEM

[0075] Described is a calibration system suitable for precisely quantifying serum allergen-specific IgE, using a microarray-based immunoassay as a platform. The described immunoassay contains approximately 100 different allergenic extracts that cover a panel of approximately 100 different allergies.

[0076] The described calibration system can reliably describe the dose-response behaviour of all 100 allergen extracts. Each allergen extract is a unique compound with a different IgE binding capacity, i.e. different dose-response steepness. The present calibration system takes account of these different binding capacities to provide an accurate system for measuring allergen-specific IgE levels in a sample.

Example Microarray Chip

[0077] The herein described system is a microarray-based test using miniaturized immunoassays designed for the measurement of up to approximately 103 allergens. Allergen extracts are immobilized onto chemically activated glass slides to generate the arrays. Each natural allergen extract is spotted onto the microarray in its optimal protein concentration and buffer (previously selected). Additionally, the microarray comprises positive controls (e.g. goat anti-mouse IgG) and negative controls (e.g. non-specific protein, such as bovine serum albumin), and capture anti-human IgE (polyclonal goat is anti-human IgE) spotted in serial dilutions.

Example Antibody Visualisation Protocol

[0078] The following is an example of a protocol that may be used to visualise binding of IgE, either in a serum sample or a reference sample (the assays are carried out at the same time, with the same reagents where appropriate, such that potential environmental variations are controlled for), to the herein described microarray:

[0079] Separate arrays are first incubated with IgE samples (either serum or reference) and subsequently with monoclonal anti-human (or other appropriate antibody, depending on the assay samples) IgE antibody (for example, anti-human IgE mouse IgG), which will bind the human IgE from the serum or reference sample, if IgE is present and bound to the spotted allergens. Then a goat polyclonal anti-mouse IgG antibody conjugated with Horseradish peroxidase (HRP) is added to the array, followed by Alexa555 fluorophore labelled Tyramide. Appropriate washing steps are carried out between each antibody incubation step.

[0080] In the presence of hydrogen peroxide (H.sub.2O.sub.2), HRP enzyme converts Tyramide-Alexa555 into highly reactive, short-lived tyramide-Alexa555 radicals that react with nucleophilic residues in the vicinity of the HRP-target interaction site. This produces an emission of fluorescence at a specific wavelength (555 nm) of intensity proportional to the amount of bound HRP enzyme.

[0081] Use of the polyclonal antibody and of the HRP-Tyramide system (a non-liner signal amplification system) greatly increases the sensitivity of the microarray immunoassay test.

[0082] The above protocol provides a fluorescence intensity for each allergen spot that is plotted on a graph with serum sample concentration to provide a curve that is interpolated with a reference curve to quantify IgE level.

Calibration Method of Invention

[0083] The herein described calibration method is exemplified by the following steps ng the microarray chip of the invention:

1 Identification of dose-response curve for each allergen. A number of serum samples with known IgE reactivity are tested on the microarray chip. The signal intensity obtained from the allergens is collected and used to generate allergen dose-response curves.
2. Production of a panel of binding capacity curves. Serial dilutions of WHO/NIBSC International Reference IgE Preparation (from OA to 100 International Unit/ml) are incubated onto the chips. IgEs are bound by the spotted capture-anti-human IgE and the signal intensity generated is measured and used to build a panel of binding capacity curves. Each curve corresponds to one of the different concentrations of capture anti-human IgE and is obtained by plotting the WHO/NIBSC IgE concentrations used for the incubation of the chip versus the corresponding obtained signal intensity. A number of binding curves is produced according to the number of different spots of capture anti-human IgE present on the chip (see FIG. 2A, where each curve corresponds to one of the different concentrations of capture anti-human IgE spotted onto the arrays and was obtained by plotting the WHO/NIBSC IgE concentrations versus the correspondingly obtained signal intensity).
3. Clustering of binding capacity curves. Binding-capacity curves are then clustered in groups to represent different binding capacity (for example, Very high, High, Medium and Low binding capacity). A regression curve is produced for each group and stored in the software of the analyzer instrument (see FIG. 2B, which shows binding capacity curves clustered into groups).
4. Allergens assigned binding capacity curve. According to the slope and shape of the allergen dose-response curves obtained as described in point 1, one of the binding-capacity curves (Master Curves) is assigned to each allergen.
5. Quantification of allergen-specific IgE. Using this calibration system, allergen-specific IgEs of an unknown patient serum are measured by interpolation of the signal intensity obtained from a particular allergen spotted onto the microarray chip to the specifically assigned binding capacity curve. Allergen reactivity is expressed in International Unit/ml and/or Class Score.
6. Using internal controls (adjuster) in each chip, the system can build the dose-response Internal Curve taking into account the storage and environment conditions of the slide adjusting the Master Curves obtained at point No. 4 accordingly. The internal calibration ID consists of running an algorithm to move the Master Calibration Curve based on the signal of the adjusters. For example, if the signal of the adjuster, whose expected value is 1000 units gives 950, the algorithm may lead to a shift in the Master Calibration Curve of 5%.

[0084] Unlike typical allergen immunoassays, which use a single calibration curve, the immunoassay system of the invention takes into account the differences in the binding capacity of each allergen. This provides a much more accurate assay for quantification of allergen-specific IgE in a sample.