immunologytoday,January I981
13
The radioallergosorbent test
(aAST) T. G. Merrett RAST Allergy and Research Unit, Benenden Chest Hospital, Cranbrook, Kent, TN17 4AX, U.K.
T. G. Merrett reviews the devel@ment of the R A S T test as a quantitative assay procedure for IgE and discusses its advantages and disadvantages in the clinical diagnosis of allergy. Allergy a n d i m m u n o g l o b u l l n E Allergy is a hypersensitive reaction with an immunological basis. Although humoral and cellular mechanisms may be involved, it is the Type-I reaction mediated by humoral antibodies (reagins) which is most frequently recognized as contributing to the symptoms of obstructive airways disease, rhino-conjunctivitis and eczema. The most important reagin in man is immunoglobulin E (IgE) and nowadays any person with IgE antibodies directed against c o m m o n inhalant allergens is classified as atopic and is likely to have inherited a tendency to get asthma, hay fever or eczema. Atopics also tend to be allergic to food and drink, and may have gastrointestinal as well as more usual symptoms. Atopic allergy affects about 15% of the population in Europe and North America, usually starts before 30 years of age and occurs at a well-defined season of the year or in a particular place - commonly within 10-20 rain of exposure to allergen. Families with atopic children undergo much stress and anxiety, and the twin objectives of providing them with an earlier diagnosis and better treatment are being achieved only gradually. In the mid-1960s a minor class of immunoglobulin (IgE 1 and its abnormal counterpart (E myeloma protein - IgND) 2 were discovered, almost simultaneously, in the U.S.A. and Sweden. Until then the Prausnitz--K/.istner (P-K) test was the basic assay used to quantify atopic allergy; it had demonstrated the passive transfer of several allergies from atopic to non-atopic persons via circulating reagins and that native reagin was firmly bound to h u m a n skin tissue. Risk of hepatitis has been responsible for its demise, but this test demonstrated the class identity of IgE and IgND because a positive test could be completely inhibited when allergic sera were pre-incubated with the myeloma serum 3.
The possibility of replacing allergy tests in vivo by absolutely safe in-vitro ones was very exciting, and the Swedish workers soon published methods for the quantification of (1) serum lgE (by conventional solid phase radioimmunoassay) 4 and (2) allergen-specific IgE (by a sandwich radioimmunoassay - the RAST) 5. The group was doubly fortunate because it had the technology to measure the nanogram amounts of IgE present in normal serum (Table I) and, the milligrams of IgND from which to prepare specific anti-IgE sera (rabbit antisera to Fc fragment of papain-cleared h u m a n LgE) and radiolabelled IgE. Radioimmunoassay
(RIA)
This technique is ideal for clinical application when a specific assay of high sensitivity is needed for the routine estimation of an antigen in a large number of test specimens. There are two main variations of RIA, bI.SANDWICH
a). CONVENTIONAL RIA
RIA
T~
Brnax~Q~- . . . . . . . . . . . . . . .
Bmax
B0'5 ~
0
IGE ( u/ml )
Bn.s. 0
i
IGE ( u/m[ )
Fig. 1 Conventional RIA (a) and sandwich RIA (b) calibration curves.
Control points are denoted as follows:T=total counts added per assay tube; 13ns =minimum (non-specific) binding of labelled . tracer; Bmax=mammum binding of tracer; and B I> g> g>
:D.
E3-;D~.-
I>=)->
~
All +
I>:D--
(b) Weak test extract 00
.. I:>
+
~__ :D-:D--
~
E3-- + 2:3-"
>
*
00 OO O
IgE (specific)
solidphase
anti-lgE tracer
Fig. 4 RAST inhibition is used to standardize the allergenic extract (~>).
(a) When the extract is similar to the reference preparation attached to the solid support (,) the binding of a RAST-positive serum to the solid phase is prevented. (b) If the extract contains only a small concentration of"reference allergens the binding is relatively unaffected and the normally positive RAST result is obtained.
immunology today,January 1981
17
Alternatively, the non-specific binding might be caused by a labelled-anti-IgE whose specificity was directed against an i n a p p r o p r i a t e region of the epsilon chain. In future, the incidence of false results will be reduced by using less porous supports, better allergens, and more specific anti-IgE tracers, but meanwhile all sera with very high total IgE levels should be checked for non-specific binding to a nonallergen, such as alligator serum 2°.
DIRECT RAST (a) Potent test extract
Z)--
(b) Weak test extract
Z~._
~"
>
23-IgE (specific)
solidphase
4- O O OO 00
'>
anti-lgE tracer
Fig. 5 T h e direct RAST. The allergenic extract to be tested is linked to the solid-phase and then reacted with clinically hypersensitive sera. When a potent extract is discovered the test sequence in (a) can be preceded by preincubation of the RAST-pnsitive serum with the reference extract. Normally RAST inhibition will be observed, but if it is not then previously unrecognized allergen(s) is present in the test extract.
tions if the total serum IgE is above the u p p e r limit of the normal range. This is an efficient way to guide the investigation of atopic persons and their relevant allergies. The R A S T screen has been m a d e into a single-tube test Is and its efficiency has been shown to be such that 95% of U K adults with any positive R A S T result to a panel of 19 inhalant and ingestant allergens were positive to at least one of the three screening allergens 17. R A S T results must always be interpreted in conjunction with the case history, because there are 'clinical false-positives' - i.e. positive R A S T results for which there is no corresponding history of hypersensitivity - and 'clinical false-negatives' which are the reverse.
Clinical false-positives The ' t r u e ' results may indicate a past sensitivity, a cross-reactivity with another allergen (e.g. perhaps as m a n y as 20% of 'grass-pollen only' sufferers give positive results to wheat-flour), or some m e c h a n i s m which prevents mast-cell degranulation by interfering with the a t t a c h m e n t of IgE antibodies to either mast-cells or allergens. Methodologically false results are usually found when the total serum IgE in the test specimen is exceptionall high - over 5000 units per mP 9. T h e y may be due to a non-specific t r a p p i n g of IgE in the pores of the particles, or p a p e r discs, used to support the allergen, or to a non-specific binding of IgE to substances - such as lectins - in the allergenic extracts.
Clinical false-negatives These results indicate that non-allergic or, at least, n o n - I g E m e c h a n i s m s (e.g. involving s h o r t - t e r m sensitizing IgG 2122 and IgD 23) or even a failing in current R A S T methodology, should be considered. There is the possibility that some IgE reactions can be sufficiently localized within the gut 24, nasal mucosa or lungs, so as to remain undetected by skin-testing 25 or, when the symptoms are of recent origin, by the R A S T 26. In food allergic disease, failure of the R A S T has been ascribed to the use of undigested rather than digested food p r o t e i n S , but it is also possible that an excess of food antigen could interfere with R A S T detection, especially since i m m u n e complexes involving IgE have been detected in the circulation 28. R A S T methods m a y be failing to measure low, but significant, levels of specific IgE. A n y test operating at its limits of sensitivity is subject to imprecision and inaccuracy, and the P h a d e b a s R A S T is no exception 29 - coefficients of between-assay variation may be as high as 25% at the level of 1 P.R.U., even in optimized and quality-controlled assays. Therefore, modifications which introduce 'significant' new scores below the ' 2 ' leveP ° must be treated with caution, since the numbers of clinical false-positives can only increase, and will confuse less experienced investigators. Nevertheless, this and other attempts (such as the m i n i - R A S T 3~) to increase the sensitivity of the test, m a y help to clarify borderline diagnoses and to confirm preliminary data suggesting that IgEmediated food allergy is a major contributor to migraine 32. F o o d a l l e r g i c d i s e a s e 33
Care should be taken to avoid confusing this syndrome with adverse reactions to food (or drink) which have no i m m u n e basis. However, although there are immediate hypersensitivity reactions to foods which can usually be confirmed by R A S T and skintesting 34.,3s, the symptoms which occur one hour, or more, after eating can be very difficult to diagnose except by keeping a diary card of all ingestants and following tedious elimination diets. Clearly any test which can short-circuit these irksome procedures would be very welcome, but although the R A S T is often helpful its results are too frequently categorized as 'clinical false-positives '36,37, especially those for cereals, pea and almond. T h e recognition of cow's
18
milk e n t e r o p a t h y in infants as a s e p a r a t e s y n d r o m e ~8 has helped to r e d u c e the i n c i d e n c e of 'clinically falsen e g a t i v e ' R A S T results substantially. It seems best to suspect I g E - m e d i a t e d food allergy w h e n the atopic p a t i e n t ' s s y m p t o m s p r e s e n t a comb i n a t i o n of a s t h m a and e c z e m a which o c c u r r e d before 30 years of age and are associated w i t h a definite adverse r e a c t i o n to at least one food. In such cases we use the R A S T to screen for I g E antibodies against egg, milk, fish, wheat-flour, p e a n u t and brazil nut - but always m e a s u r e any non-specific binding, w h i c h can occur w h e n the total s e r u m I g E level is over 5000 units p e r ml, w i t h insolubilized alligator serum. Positive food R A S T results indicate a diagnosis of food allergic disease, a l t h o u g h the p r i n c i p a l i n g e s t a n t allergens m a y still have to be identified.
Alternative IgE antibody assays T h e R A S T uses r a d i o a c t i v e l y - l a b e l l e d a n t i - I g E w h i c h has a l i m i t e d shelf-life, a n d can only be used in laboratories licensed to h a n d l e radi-oactive isotopes. R e p l a c i n g the radioactive label with an e n z y m i c one will b r o a d e n the a p p e a l of the test, but this c h a n g e alone is unlikely to offer sufficient a d v a n t a g e s to cause laboratories a l r e a d y well e q u i p p e d w i t h a u t o m a t i c g a m m a - s c i n t i l l a t i o n counters to switch f r o m a radioi m m u n o a s s a y to an e n z y m e i m m u n o a s s a y . R a d i o i m m u n o a s s a y s u s i n g labelled allergen 39,4° instead of labelled a n t i - I g E are r a t h e r m o r e interesting, b e c a u s e t h e y suffer no interference f r o m blocking I g G a n t i b o d i e s a n d the a m o u n t s of I g E available for b i n d i n g labelled allergen can be quantified. T h e t e c h n i q u e is also m o r e readily a d a p t e d to m e a s u r i n g specific I g G a n t i b o d i e s t h a n is the I g G - R A S T . H o w e v e r , in these labelled allergen t e c h n i q u e s it is not always possible to label all the m a j o r a n d m i n o r allergens in a single extract, a n d the difficulty of prep a r i n g the labels for m a n y different I g E specificities makes the t e c h n i q u e m u c h better suited to specific r e s e a r c h projects t h a n to r o u t i n e clinical diagnoses. It follows t h a t even after 13 years of use the R A S T p r i n c i p l e is difficult to b e t t e r but, in the future, a u t o m a t e d or at least c h e a p e r s e m i - a u t o m a t e d p r o c e d u r e s m u s t be sought in o r d e r to cope w i t h the r o u t i n e d e m a n d s for the test at a r e a s o n a b l e cost. P e r h a p s the p a r t i c l e - c o u n t i n g i m m u n o a s s a y ( P A C I A )
13
The radioallergosorbent test
(aAST) T. G. Merrett RAST Allergy and Research Unit, Benenden Chest Hospital, Cranbrook, Kent, TN17 4AX, U.K.
T. G. Merrett reviews the devel@ment of the R A S T test as a quantitative assay procedure for IgE and discusses its advantages and disadvantages in the clinical diagnosis of allergy. Allergy a n d i m m u n o g l o b u l l n E Allergy is a hypersensitive reaction with an immunological basis. Although humoral and cellular mechanisms may be involved, it is the Type-I reaction mediated by humoral antibodies (reagins) which is most frequently recognized as contributing to the symptoms of obstructive airways disease, rhino-conjunctivitis and eczema. The most important reagin in man is immunoglobulin E (IgE) and nowadays any person with IgE antibodies directed against c o m m o n inhalant allergens is classified as atopic and is likely to have inherited a tendency to get asthma, hay fever or eczema. Atopics also tend to be allergic to food and drink, and may have gastrointestinal as well as more usual symptoms. Atopic allergy affects about 15% of the population in Europe and North America, usually starts before 30 years of age and occurs at a well-defined season of the year or in a particular place - commonly within 10-20 rain of exposure to allergen. Families with atopic children undergo much stress and anxiety, and the twin objectives of providing them with an earlier diagnosis and better treatment are being achieved only gradually. In the mid-1960s a minor class of immunoglobulin (IgE 1 and its abnormal counterpart (E myeloma protein - IgND) 2 were discovered, almost simultaneously, in the U.S.A. and Sweden. Until then the Prausnitz--K/.istner (P-K) test was the basic assay used to quantify atopic allergy; it had demonstrated the passive transfer of several allergies from atopic to non-atopic persons via circulating reagins and that native reagin was firmly bound to h u m a n skin tissue. Risk of hepatitis has been responsible for its demise, but this test demonstrated the class identity of IgE and IgND because a positive test could be completely inhibited when allergic sera were pre-incubated with the myeloma serum 3.
The possibility of replacing allergy tests in vivo by absolutely safe in-vitro ones was very exciting, and the Swedish workers soon published methods for the quantification of (1) serum lgE (by conventional solid phase radioimmunoassay) 4 and (2) allergen-specific IgE (by a sandwich radioimmunoassay - the RAST) 5. The group was doubly fortunate because it had the technology to measure the nanogram amounts of IgE present in normal serum (Table I) and, the milligrams of IgND from which to prepare specific anti-IgE sera (rabbit antisera to Fc fragment of papain-cleared h u m a n LgE) and radiolabelled IgE. Radioimmunoassay
(RIA)
This technique is ideal for clinical application when a specific assay of high sensitivity is needed for the routine estimation of an antigen in a large number of test specimens. There are two main variations of RIA, bI.SANDWICH
a). CONVENTIONAL RIA
RIA
T~
Brnax~Q~- . . . . . . . . . . . . . . .
Bmax
B0'5 ~
0
IGE ( u/ml )
Bn.s. 0
i
IGE ( u/m[ )
Fig. 1 Conventional RIA (a) and sandwich RIA (b) calibration curves.
Control points are denoted as follows:T=total counts added per assay tube; 13ns =minimum (non-specific) binding of labelled . tracer; Bmax=mammum binding of tracer; and B I> g> g>
:D.
E3-;D~.-
I>=)->
~
All +
I>:D--
(b) Weak test extract 00
.. I:>
+
~__ :D-:D--
~
E3-- + 2:3-"
>
*
00 OO O
IgE (specific)
solidphase
anti-lgE tracer
Fig. 4 RAST inhibition is used to standardize the allergenic extract (~>).
(a) When the extract is similar to the reference preparation attached to the solid support (,) the binding of a RAST-positive serum to the solid phase is prevented. (b) If the extract contains only a small concentration of"reference allergens the binding is relatively unaffected and the normally positive RAST result is obtained.
immunology today,January 1981
17
Alternatively, the non-specific binding might be caused by a labelled-anti-IgE whose specificity was directed against an i n a p p r o p r i a t e region of the epsilon chain. In future, the incidence of false results will be reduced by using less porous supports, better allergens, and more specific anti-IgE tracers, but meanwhile all sera with very high total IgE levels should be checked for non-specific binding to a nonallergen, such as alligator serum 2°.
DIRECT RAST (a) Potent test extract
Z)--
(b) Weak test extract
Z~._
~"
>
23-IgE (specific)
solidphase
4- O O OO 00
'>
anti-lgE tracer
Fig. 5 T h e direct RAST. The allergenic extract to be tested is linked to the solid-phase and then reacted with clinically hypersensitive sera. When a potent extract is discovered the test sequence in (a) can be preceded by preincubation of the RAST-pnsitive serum with the reference extract. Normally RAST inhibition will be observed, but if it is not then previously unrecognized allergen(s) is present in the test extract.
tions if the total serum IgE is above the u p p e r limit of the normal range. This is an efficient way to guide the investigation of atopic persons and their relevant allergies. The R A S T screen has been m a d e into a single-tube test Is and its efficiency has been shown to be such that 95% of U K adults with any positive R A S T result to a panel of 19 inhalant and ingestant allergens were positive to at least one of the three screening allergens 17. R A S T results must always be interpreted in conjunction with the case history, because there are 'clinical false-positives' - i.e. positive R A S T results for which there is no corresponding history of hypersensitivity - and 'clinical false-negatives' which are the reverse.
Clinical false-positives The ' t r u e ' results may indicate a past sensitivity, a cross-reactivity with another allergen (e.g. perhaps as m a n y as 20% of 'grass-pollen only' sufferers give positive results to wheat-flour), or some m e c h a n i s m which prevents mast-cell degranulation by interfering with the a t t a c h m e n t of IgE antibodies to either mast-cells or allergens. Methodologically false results are usually found when the total serum IgE in the test specimen is exceptionall high - over 5000 units per mP 9. T h e y may be due to a non-specific t r a p p i n g of IgE in the pores of the particles, or p a p e r discs, used to support the allergen, or to a non-specific binding of IgE to substances - such as lectins - in the allergenic extracts.
Clinical false-negatives These results indicate that non-allergic or, at least, n o n - I g E m e c h a n i s m s (e.g. involving s h o r t - t e r m sensitizing IgG 2122 and IgD 23) or even a failing in current R A S T methodology, should be considered. There is the possibility that some IgE reactions can be sufficiently localized within the gut 24, nasal mucosa or lungs, so as to remain undetected by skin-testing 25 or, when the symptoms are of recent origin, by the R A S T 26. In food allergic disease, failure of the R A S T has been ascribed to the use of undigested rather than digested food p r o t e i n S , but it is also possible that an excess of food antigen could interfere with R A S T detection, especially since i m m u n e complexes involving IgE have been detected in the circulation 28. R A S T methods m a y be failing to measure low, but significant, levels of specific IgE. A n y test operating at its limits of sensitivity is subject to imprecision and inaccuracy, and the P h a d e b a s R A S T is no exception 29 - coefficients of between-assay variation may be as high as 25% at the level of 1 P.R.U., even in optimized and quality-controlled assays. Therefore, modifications which introduce 'significant' new scores below the ' 2 ' leveP ° must be treated with caution, since the numbers of clinical false-positives can only increase, and will confuse less experienced investigators. Nevertheless, this and other attempts (such as the m i n i - R A S T 3~) to increase the sensitivity of the test, m a y help to clarify borderline diagnoses and to confirm preliminary data suggesting that IgEmediated food allergy is a major contributor to migraine 32. F o o d a l l e r g i c d i s e a s e 33
Care should be taken to avoid confusing this syndrome with adverse reactions to food (or drink) which have no i m m u n e basis. However, although there are immediate hypersensitivity reactions to foods which can usually be confirmed by R A S T and skintesting 34.,3s, the symptoms which occur one hour, or more, after eating can be very difficult to diagnose except by keeping a diary card of all ingestants and following tedious elimination diets. Clearly any test which can short-circuit these irksome procedures would be very welcome, but although the R A S T is often helpful its results are too frequently categorized as 'clinical false-positives '36,37, especially those for cereals, pea and almond. T h e recognition of cow's
18
milk e n t e r o p a t h y in infants as a s e p a r a t e s y n d r o m e ~8 has helped to r e d u c e the i n c i d e n c e of 'clinically falsen e g a t i v e ' R A S T results substantially. It seems best to suspect I g E - m e d i a t e d food allergy w h e n the atopic p a t i e n t ' s s y m p t o m s p r e s e n t a comb i n a t i o n of a s t h m a and e c z e m a which o c c u r r e d before 30 years of age and are associated w i t h a definite adverse r e a c t i o n to at least one food. In such cases we use the R A S T to screen for I g E antibodies against egg, milk, fish, wheat-flour, p e a n u t and brazil nut - but always m e a s u r e any non-specific binding, w h i c h can occur w h e n the total s e r u m I g E level is over 5000 units p e r ml, w i t h insolubilized alligator serum. Positive food R A S T results indicate a diagnosis of food allergic disease, a l t h o u g h the p r i n c i p a l i n g e s t a n t allergens m a y still have to be identified.
Alternative IgE antibody assays T h e R A S T uses r a d i o a c t i v e l y - l a b e l l e d a n t i - I g E w h i c h has a l i m i t e d shelf-life, a n d can only be used in laboratories licensed to h a n d l e radi-oactive isotopes. R e p l a c i n g the radioactive label with an e n z y m i c one will b r o a d e n the a p p e a l of the test, but this c h a n g e alone is unlikely to offer sufficient a d v a n t a g e s to cause laboratories a l r e a d y well e q u i p p e d w i t h a u t o m a t i c g a m m a - s c i n t i l l a t i o n counters to switch f r o m a radioi m m u n o a s s a y to an e n z y m e i m m u n o a s s a y . R a d i o i m m u n o a s s a y s u s i n g labelled allergen 39,4° instead of labelled a n t i - I g E are r a t h e r m o r e interesting, b e c a u s e t h e y suffer no interference f r o m blocking I g G a n t i b o d i e s a n d the a m o u n t s of I g E available for b i n d i n g labelled allergen can be quantified. T h e t e c h n i q u e is also m o r e readily a d a p t e d to m e a s u r i n g specific I g G a n t i b o d i e s t h a n is the I g G - R A S T . H o w e v e r , in these labelled allergen t e c h n i q u e s it is not always possible to label all the m a j o r a n d m i n o r allergens in a single extract, a n d the difficulty of prep a r i n g the labels for m a n y different I g E specificities makes the t e c h n i q u e m u c h better suited to specific r e s e a r c h projects t h a n to r o u t i n e clinical diagnoses. It follows t h a t even after 13 years of use the R A S T p r i n c i p l e is difficult to b e t t e r but, in the future, a u t o m a t e d or at least c h e a p e r s e m i - a u t o m a t e d p r o c e d u r e s m u s t be sought in o r d e r to cope w i t h the r o u t i n e d e m a n d s for the test at a r e a s o n a b l e cost. P e r h a p s the p a r t i c l e - c o u n t i n g i m m u n o a s s a y ( P A C I A )
