1. BACKGROUND
In Europe, RAST (Radioallergosorbent test)
is a laboratory method commonly used in the diagnosis of type I allergy. RAST measures
allergen-specific IgE antibodies in the patient's serum, e.g., latex specific
IgE in the serum of a patient with allergy to natural rubber latex (NRL).
RAST inhibition, on the other hand, is a method to measure allergen
activity of an extract (1) Helsinki University Central Hospital has
regulatory control over all allergen preparations intended to be sold in
Finland and used for the diagnosis and/or treatment of allergic diseases
(2) In this control work, RAST inhibition method has been utilized for
more than 10 years. We also have used RAST inhibition method to
evaluate latex allergen activity in various medical and consumer
products made of NRL.
2. RAST INHIBITION PROCEDURE
In our RAST inhibition, an optimal amount of latex allergens is bound to
activated paper discs, which are used as solid phase for bound allergens.
The same NRL also is used as the reference with a given arbitrary activity of 100.000 relative latex units (RLU/ml) to which all samples are
compared. From the samples a 1:5 weight per volume extract is prepared.
The source for latex specific IgE antibodies is a pool of sera from more
than 30 patients with a confirmed allergy to latex (diagnosis made by
Dr. Kristiina Turjanmaa, Tampere, Finland), and with a high latex specific IgE test result. Patients include children and adults, healthcare workers
and lay people. The IgE antibodies bound to the solid-phase allergens are
detected with a radio-labeled anti-IgE (Pharmacia, Uppsala, Sweden).
Several serial dilutions of the test extract and the reference are prepared
and incubated first with same amount of the pooled IgE serum. Immunological reaction takes place between the allergen and the antibody, and
some IgE is partly or completely blocked by the allergens. After incubation, one latex RAST disk is added to each dilution. Specific IgE
antibodies which were left free in the first incubation step bind to the
allergens on the disc and can be measured using a radio-labeled anti-IgE
and a gamma-counter.
The percentage of the inhibition of the latex RAST is calculated using
the control disk (with no allergen inhibitor), and the background binding
deleted. In order to get reliable results, a dose-response curve must be
obtained. The relative potency of the sample in relation to the reference
is calculated using parallel line assay method (Anderson et al 1981).
Our method shows a sensitivity of O.I ug/ml protein as measured using
the Lowry method; the interassay coefficient of variation is 20%. A good
correlation has been obtained between the results from in vitro RAST
inhibition and the results from in vivo using skin prick testing performed
by Dr. K. Turjanmaa (R=0.95, p<0.001). On the other hand, our RAST
inhibition results did not correlate with the results from the LEAP assay
method (R=0.29, p>0.5). So far, we have evaluated nearly 100 different
NRL samples for their latex allergen activity in vitro.
3. RESULTS
When various NRL products available on the Finnish market in 1992
and 1994 were evaluated, a 400-fold difference in the latex allergen
activity was demonstrated (Table I). From the gloves, highest activity
was found in Triflex (Travenol) and the lowest in Biogel (Regent). In
Gammex (Ansell), a six-fold reduction was demonstrated.
High latex allergen activity also was demonstrated in a self-adherent
wrap sometimes used on leg-ulcer, and in a peelable mould making
material, which caused asthma in a patient. In one brand of baby pacifiers,
the latex allergen activity was about 200-fold higher than in
another. In condoms, the activity varied 20-fold and was at about the
same level as in toy balloons.
4. CONCLUSION
RAST inhibition is a valuable method to measure allergen activity in
vitro in order to compare products already on the market or to control the
product development. In addition, our RAST inhibition method correlates
extremely well with the results from in-vivo skin prick testing.
So far, results are expressed in arbitrary units calculated from an inhouse reference which may be different in various institutes since no
international or WHO-approved NRL reference is available. Therefore,
exact figures from various centers may differ, but the products can be
ranked equally.
The sensitivity and specificity of the method depend on several factors.
Solid-phase allergens must include all potential latex allergens and especially
those of the test extract. In addition, in order to measure the total
allergen activity, the pool of sera must contain IgE antibodies against all
these allergens.
