a rapid enhanced chemiluminescent immunoassay for allergen-specific ige antibodies

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JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 1 53-58 (1986) A Rapid Enhanced Chemiluminescent lmmunoassay for Allergen-specific IgE Antibodies Heather J. Stiles, Helen J. Bicknell and J a p e A. Matthews' Wolfson Research Laboratories, Department of Clinical Chemistry, Queen Elizabeth Medical Centre, Birmingham, 815 2TH. UK An enhanced chemiluminescent imrnunoassay is described for the measurement of allergen-specific IgE antibodies. The assay is demonstrated for pollen from four grass species. A comparison was made between results obtained by this method and those obtained by the radioallergosorbent (RAST) procedure; a high degree of correlation (r = 0.95) was found for D. glomerata specific IgE. The assay is rapid and can be carried out in under 1 hour. The advantages of the luminescent assay as compared with the RAST procedure are discussed. Keywords: enhanced chemiluminescence; immunoassay; allergen; IgE INTRODUCTION The radioallergosorbent assay (RAST) has been used for many years for the detection of allergen-specific IgE antibodies in serum from individuals with atopic diseases. Results of Rast correlate well with clinical history (Stenius et af., 1971) and with diagnostic skin tests (Norman et af., 1973). RAST tests are frequently carried out for the investigation of hayfever, in order to detect IgE antibodies to a variety of grass, weed and tree pollens. The RAST assay involves the binding of IgE from human serum to allergens bound to a solid support, and the subsequent detection of the bound IgE with a labelled anti-IgE antibody. The label may be iodine'2s or, more recently, a colorimetrically detected en- zyme. Several variations of the procedure have been reported, for example, the use of biotiny- lated antibodies and their detection with enzyme- avidin complexes (Plebani et al., 1984). The *Author for correspondence 0884-3996/86/020053-06$05 .OO @ 1986 by John Wiley 6t Sons, Ltd. methods used currently are slow procedures, often having an overnight incubation step, or four incubation steps of up to two hours each. In this paper we describe a rapid enhanced chemilu- minescent assay for allergen-specific IgE. The assay is carried out on microtitre plates and specific IgE antibodies are detected using horse- radish peroxidase-labelled anti IgE (HRP-anti IgE), with an enhanced chemiluminescent end- point. Light emission from the enhanced chemilu- minescent reaction continues .for at least 15 minutes (Thorpe et af., 1985a), and either quantitative or photographic results can be obtained. MATERIALS AND METHODS Materials Pollen from cocksfoot grass (Dactylis gfomerata L.), cultivated rye (Secale cereafe L.), brome Received I4 March 1986 Revised 28 April 1986

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JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 1 53-58 (1986)

A Rapid Enhanced Chemiluminescent lmmunoassay for Allergen-specific IgE Anti bodies

Heather J. Stiles, Helen J. Bicknell and J a p e A. Matthews' Wolfson Research Laboratories, Department of Clinical Chemistry, Queen Elizabeth Medical Centre, Birmingham, 815 2TH. UK

An enhanced chemiluminescent imrnunoassay is described for the measurement of allergen-specific IgE antibodies. The assay is demonstrated for pollen from four grass species. A comparison was made between results obtained by this method and those obtained by the radioallergosorbent (RAST) procedure; a high degree of correlation (r = 0.95) was found for D. glomerata specific IgE. The assay is rapid and can be carried out in under 1 hour. The advantages of the luminescent assay as compared with the RAST procedure are discussed.

Keywords: enhanced chemiluminescence; immunoassay; allergen; IgE

INTRODUCTION

The radioallergosorbent assay (RAST) has been used for many years for the detection of allergen-specific IgE antibodies in serum from individuals with atopic diseases. Results of Rast correlate well with clinical history (Stenius et a f . , 1971) and with diagnostic skin tests (Norman et a f . , 1973). RAST tests are frequently carried out for the investigation of hayfever, in order to detect IgE antibodies to a variety of grass, weed and tree pollens. The RAST assay involves the binding of IgE from human serum to allergens bound to a solid support, and the subsequent detection of the bound IgE with a labelled anti-IgE antibody. The label may be iodine'2s or, more recently, a colorimetrically detected en- zyme. Several variations of the procedure have been reported, for example, the use of biotiny- lated antibodies and their detection with enzyme- avidin complexes (Plebani et al., 1984). The

*Author for correspondence

0884-3996/86/020053-06$05 .OO @ 1986 by John Wiley 6t Sons, Ltd.

methods used currently are slow procedures, often having an overnight incubation step, or four incubation steps of up to two hours each. In this paper we describe a rapid enhanced chemilu- minescent assay for allergen-specific IgE. The assay is carried out on microtitre plates and specific IgE antibodies are detected using horse- radish peroxidase-labelled anti IgE (HRP-anti IgE), with an enhanced chemiluminescent end- point. Light emission from the enhanced chemilu- minescent reaction continues .for at least 15 minutes (Thorpe et a f . , 1985a), and either quantitative or photographic results can be obtained.

MATERIALS AND METHODS

Materials

Pollen from cocksfoot grass (Dactylis gfomerata L.), cultivated rye (Secale cereafe L.), brome

Received I4 March 1986 Revised 28 April 1986

54 H. J. STILES, H. J. BICKNELLAND J. A. MATTHEWS

grass (Bromus inermirs L.), and Kentucky blue grass (Poa praterisis L.) were obtained from Sigma Chemical Company Ltd. (Poole, Dorset, UK). Phadezym RAST kits for the measurement of allergen-specific IgE, and pre-coated allergen discs were obtained from Pharmacia Diagnostics Ltd. (Milton Keynes, UK). Horseradish perox- idase-labelled anti-human IgE (HRP-anti IgE) was obtained from Dako, (Denmark). Standard Cocksfoot allergenic extract was kindly supplied by the National Institute of Biological Standards and Control. Human serum was obtained from volunteers showing symptoms of hayfever varying from severe through moderate. Control sera were obtained from volunteers showing no symptoms of hayfever.

Phadezym RAST procedure

Assay of allergen-specific IgE was carried out exactly as described in the manufcturers instruc- tions (Pharmacia Diagnostics Ltd., Milton Keynes, UK). Briefly, human serum samples were incubated for 3 hours with allergen coated discs after which the discs were .thoroughly washed. An anti-human IgE-enzyme conjugate was then added and incubated overnight. After further washing, the colour development solution was added and development allowed to proceed for 2 hours. Results were measured spectropho- tometrically. Using the reference reagents sup- plied with the kit as standards, the amount of allergen-specific IgE in the serum samples was expressed in terms of PRUiml (Phadezym RAST units/ml).

Preparation of pollen extracts

Pollen allergens were extracted essentially as described by Howlett and Clark (1981). A 10% suspension of pollen in 0.15 molil NaCI, 0.1 mol/l Tris pH 8.0 was stirred on ice for 60 min. The suspension was filtered and the filtrate centri- fuged as 16OOOg for 15 min at 4°C. The supernatant was made 80% with respect to ammonium sulfate and stirred on ice for 60 min. Precipitated protein was collected by centrifuga- tion at 16000g for 15 min at 4"C, and resus- pended in 10 mmolil sodium phosphate buffer pH 6.4. After dialysis against the same buffer (24 hours with 3 changes) the extract was stored in

small aliquots at - 20°C. The protein concentra- tion was determined by the Ponceau S dye- binding method (Pesce and Strande, 1973).

Enhanced chemiluminescent immunoassay

This assay was carried out in the wells of microtitre plates. For quantitative results black MICROFLUORTM plates (Dynatech Laborator- ies, Billingshurst, UK) were used, but flexible, transparent PVC microtitre plates were selected when photographic results were required. Allergen extracts were diluted in 0.1 molil carbonate buffer, to a concentration of 9 pg/ml, pH 9.5, and 150 p1 pipetted into each well of a microtitre plate. Plates were incubated overnight at 4°C and washed extensively with phosphate- buffered saline (Oxoid, UK) containing 0.05% Tween 20 (PBS-Tween). Serum specimens (100 pi, diluted 1 in 2 to 1 in 5 with PBS) were added to the wells and incubated at room temperature for 15-60 min. Plates were again washed and subsequently 100 pl HRP-labelled anti-human IgE (diluted 1 in 400 to 1 in 1000 in PBS-Tween) was added to each well. After incubation for 15-60 min. at room temperature, the plates were washed and the presence of HRP bound to the wells was detected luminescently. The amount of HRP bound to the well is proportional to the concentration of allergen-specific IgE in the serum specimen.

Chemiluminescent detection

Chemiluminescent reagent (Kricka et al., 1983) contains luminol (1.25 mmolil) and hydrogen peroxide (2.7 mmolil) in 0.1 mmoM Tris-HC1 buffer, pH 8.6. To this solution was added 0.136 mmol/l p-iodophenol as an enhancer. Luminol is purified before use by charcoal decolorization and recrystallization from hot sodium hydroxide as described by Ham et al. (1979). Quantitative measurements of light emission were made using a luminescent microtitre plate reader based on an end-window photomultiplier tube (992414, Thorn EM1 Electron Tubes Ltd., Runslip, UK) in a photocurrent mode (Thorpe et al., 1985a). Photographic results were obtained using a camera luminometer as described by Bunce et al. (1985), in which a contact print of the glowing microtitre plate is taken on high-speed instant

IMMUNOASSAY OF ALLERGEN-SPECIFIC IgE ANTIBODIES 55

photographic film (Polaroid Type 612, ASA 20,000).

RESULTS

Precision Studies

Pools of serum containing high or low levels of allergen-specific IgE antibodies, directed against various grass pollens, were prepared on the basis of results obtained for individual serum speci- mens by the RAST test procedure. These serum pools, and five individual serum specimens, were assayed several times each, and the mean result and coefficient of variation were calculated (Table 1). Those serum samples giving high readings (i.e. 2, 3, 5 ) were from individuals with severe symptoms of hay-fever. For all but one sample, the coefficients of variation fell into the range, 2-10%. The results obtained for D. glornerutu pollen-specific antibodies were similar when the plate was coated with either the extract prepared in our laboratory or with the standard extract obtained from the National Institute of Biological Standards and Control.

Relatlve concentratlon

Figure 1. Linearity of the chemiluminescent immunoas- say. Serial dilutions of a serum specimen were assayed in order to determine linearity. The dilutions are expressed in terms of relative concentration. The assay was carried out using 15 min. incubation times and 1 in 400 dilution of HRP-anti IgE

Table 1. Precision of allergen-specific IgE measurement by the chemiluminescent immunoassay. The pools of serum, classified as high and low, were prepared on the basis of results obtained on individual serum samples by the appropriate RAST assay

Grass Specimen emission of variation determinations Serum Mean light Coefficient Number of

INTRA-ASSA Y D . glornerata D . glornerutu D. glornerata D. glornerata D. glornerata S . cereale S. cereale P . pratensis P. pratensis B. inerrnus B. inerrnus

1 2 3 4 5

High pool Low pool High pool Low pool High pool Low pool

85 4168 3530 388

1197 2173 27 1

2917 319

1640 187

10.0 5.1 5.0 5.5 8.0 3.0

12.5 3.7 9.6 2.3 9.4

18 18 18 18 18 5 5 5 5 5 5

INTER-ASSAY D. glornerata High pool 1954 8.0 24 D. glornerata Low pool 510 7.0 24

56 H. J. STILES, H. J. BICKNELLAND J. A. MATTHEWS

Linearity of the chemiluminescent assay

A sample of serum giving a high reading on both the RAST and the chemiluminescent assay for IgE agains D. glomerutn was assigned an arbitary concentration of 1. This sample was diluted 1 in 2 (relative concentration = 0.5) then serially diluted to give a range of relative concentrations. These dilutions were all assayed, at least in duplicate, by the chemiluminescent method (Fig. 1). For this experiment 15 min. incubation times were used in combination with a 1 in 400 dilution of HRP-anti IgE. Over the range tested, the assay showed a linear relationship of light emission with the concentration of allergen-specific IgE. The total assay time in this experiment was approx- imately 45 minutes.

Comparison of chemiluminescent assay with RAST

Ten individual serum specimens were assayed by RAST for D.glomerata pollen-specific IgE anti-

bodies and were then assayed in duplicate by the chemiluminescent procedure. The results are described in Fig. 2. The line of best fit was determined by linear regression analysis and was found to be y = 0 . 0 7 3 ~ + 0.12. A significant level of correlation was found between the two methods, the coefficient of correlation being 0.95. Similar analyses were carried out for allergen- specific IgE antibodies against two other grasses, and the two methods again were found to be significantly correlated. For S. cereale and P. prutensis allergens, the correlation coefficients were 0.78 and 0.81 respectively. In addition to the results shown in Fig. 2 a further 14 serum specirncns were tested for D. glomerata-specific IgE, and a similar level of correlation was found.

In order to investigate the suitability of photographic detection of the chemiluminescent assay for the diagnosis of hayfever, photographic results were obtained for 12 individual serum specimens and compared with results obtained by RAST (Fig. 3). By visual interpretation, the light emission recorded photographically correlates well the PRU/ml measured by RAST. The serum

0 5 10 15

Phadetym RAST units per ml

Figure 2. Correlation of the chemiluminescent immunoassay with RAST assay. Ten individual serum specimens were assayed by the RAST assay and then in duplicate by the chemiluminescent assay. Linear regression analysis gives the equation y = 0.073~ + 0.12. The correlation coefficients ( r ) is 0.95. The chemiluminescent assay was carried out using 60 min. incubation times and a 1 in 1000 dilution of HRP-anti IgE

IMMUNOASSAY OF ALLERGEN-SPECIFIC IgE ANTIBODIES 57

Figure 3. Photographic determination of allergen-specific IgE levels. The assay was carried out in a transport microtitre plate and the results recorded on instant photgraphic film. 12 serum specimens, whose RAST results were known, were assayed. The left-hand panel gives RAST results in PRUlml; the right-hand panel shows the photographic results for the same serum specimens

specimens having PRU/ml greater than 5 were from individuals showing severe symptoms of hayfever, those with PRU/ml between 1 and 5 had mild symptoms of hayfever and those with PRU/ml equal to zero were symptom free.

DISCUSSION

The immunoassay for allergen-specific IgE anti- bodies using an enhanced-luminescent end point

has been shown to give reliable results, which correlate well with measurements made by the commercially available RAST assay ( r = 0.78 -0.05) and with clinical symptoms. Four major grass pollen allergens have been investigated in this way. The assay is flexible in that either quantitative results or permanent photographic results can be obtained.

Enhanced chemiluminescence provides an ex- tremely sensitive and very rapid means of measuring peroxidase activity (Thorpe et al., 1985b). Therefore, it is possible to reduce the time needed by carrying out the enzyme im- munoassay under non-equilibrium immunological conditions. Reliable results were obtained, both quantitatively and photographically using 15 min. incubation times for both steps. Allowing 5 min. for the washing steps, and one to two minutes for chemiluminescent detection, the total time is only 45 min. By comparison, the RAST kit used has an overnight incubation step and a total assay time of approximately 24 hours. A rapid assay could be of great value as, by sensitizing a microtitre plate with a panel of allergens, a patient's allergy profile could be recorded within a very short time of a blood specimen being taken, while the patient is waiting.

The assay of allergen-specific IgE has been previously shown to be valuable in the diagnosis of atopic diseases. The method described in this paper gives results which correlate well with previously reported methods and is both reliable and rapid.

Acknowledgements

The authors would like to thank Dr L. J. Kricka for advice concerning enhanced chemilu'minescence. The financial support of the Department of Health and Social Security, and the Medical Research Council are gratefully acknowledged. Aspects of this work are the subject of patents and patent applications which have been assigned to the British Technology Group (NRDC).

REFERENCES

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H . J. STILES, H. J. BICKNELL AND J. A. MATTHEWS

Ham, G., Belcher, R., Kricka, L. J . and Carter, T. J . N. (1979). Stability of trace iodine solutions. Anal. Letf., 12, 535-541.

Howlett, B. and Clark, A. (1981). Isolation and partial characterisation of two antigenic glycoproteins from rye-grass (Lolium perenne) pollen. Biochem. J . 197,

Kricka, L. J., Thorpe, G. H . G . and Whitehead, T. P. (1983). Enhanced luminescent or luminometric assay. European Patent Publication 116454.

Norman, P. S., Lichtenstein, L. M. and Ishizaka, K. (1973). Diagnostic tests in ragweed hay fever: a comparison of direct skin tests, IgE antibody measurements and basophil histamine release. 1. Allergy. Clin. Immunol., 52, 510.

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