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Allergy to latex, avocado pear, and banana: Evidence for a 30 kd antigen in immunoblotting
Allergens, IgE, mediators, inflammatory mechanisms Allergy to latex, avocado pear, and banana: Evidence for a 30 kd antigen in immunoblotting Frangois Lavaud, MD, Alain Prevost, MD, Claude Cossart, MD, Laurence Guerin, PhD, Jacky Bernard, PhD, and Serge Kochman, MD Reims and Varennes, France Allergens of natural latex, latex gloves, avocado pear, and banana extracts were investigated by an immunoblotting technique in sera of patients experiencing associated latex and fruit allergies. Extracts were separated by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. After incubation with patients' sera, lgE antibodies were revealed by a goat anti-human IgE alkaline-phosphatase conjugate. Seventeen serum samples from patients with well-documented latex allergy were studied. Among these patients, 10 demonstrated an allergy to avocado pear sometimes associated with banana. In sera from patients with latex and fruit allergy, prominent IgE binding was revealed at about 30 kd with latex and fruit extracts. Serum controls remained negative. Cross-inhibition of immunoblotting confirmed that this main allergen is linked to a common epitope present in latex and fruits. This must be related to clinical findings and previous observations of cross-reactivity. (J ALLERGY CLIN IMMUNOL 1995;95:557-64.) Key words: Late~ immediate hypersensitivity, fruit, immunoblot, cross-reactivity, allergens
Case reports of allergic reactions to latex proteins have been described with various products since 1979.1 The allergenicity of latex has been demonstrated by immunologic techniques such as immunoblotting. Recent studies revealed antigenic constituents with molecular weights (MWs) from 10 to 110 kd. 2-5 Occasionally, latex allergy is associated with allergy to fruit, especially banana, 6-9 but it may also be associated with allergy to avocado pear 7, s or other fruits belonging to phytogenetically dissimilar classes. 9-12 Cross-reactivity has been shown between latex and banana. 6 We have also recently described cross-reactivity among latex, banana, and avocado pear. 7 From the Department of RespiratoryDiseases and Allergology, University and Regional Medical Center, Reims; and Allerbio Laboratory, Varennes. Received for publication June 10, 1993; revised Oct. 22, 1993; accepted for publication Feb. 4, 1994. Reprint requests: Francois Lavaud, MD, CHR Reims, Unite d'Allergologie, H6pital Maison Blanche, Reims, F 51092, France. Copyright © 1995 by Mosby-Year Book, Inc. 0091-6749/95 $3.00 + 0 1/1/55249
Abbreviations used: MW: Molecular weight NTE: NaC1 1 mol/L, Tris 0.1 mol/L, ethylenediaminetetraacetic acid 0.01 mol/L SDS-PAGE: Sodium dodecylsulfate-polyacrylamide gel electrophoresis
In this study we have performed immunoblotting analysis and inhibition of immunoblotting to further investigate allergenic components in natural latex from Hevea brasiliensis, latex gloves, banana, and avocado pear. Sera from patients with latex allergy were studied to assess a common profile among these allergens. Two groups of sera were isolated according to their association with a proven fruit allergy.
METHODS Preparation of allergenic extracts Natural latex. Drops of fresh latex serum, drained after being cut from leaf stalks of the rubber tree 557
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T A B L E I. Clinical, skin test, and RAST data Patient No.
Sex
Age (yr)
Symptoms
Skin tests
RAST score
11
F
29
L:RC A:None B:None
L: + + + A:Neg B:Neg
L:I A:<0.35 B:<0.35
12
F
28
L:RC A:None B:None
L: + + + A:Neg B:Neg
L:0.52 A:<0.35 B:<0.35
13
F
34
L:U A:None B:None
L: + + + + A:Neg B:Neg
L:2.55 A: <0.35 B: <0.35
14
F
37
L:AS A:None B:None
L:+ + + + A:Neg B:Neg
L:2.65 A: <0.35 B:<0.35
15
F
35
L:U A:None B:None
L:+ + + + A:Neg B:Neg
L:3.7 A: <0.35 B:<0.35
16
F
25
L:U A:None B:None
L:+ + + + A:Neg B:Neg
L:1.26 A: <0.35 B: <0.35
17
F
29
L:U A:None B:None
L:+ + + A:Neg B:Neg
L:3.6 A: <0.35 B: <0.35
21
F
50
L:U A:G B:None
L: + + + A: + + B:Neg
L:1.06 A:1.46 B:<0.35
22
M
27
L:U + R A:U+R B:None
L: + + + A: + + B: + +
L:7.7 A:2.28 B:<0.35
23
F
32
L:U A:G B:None
L: + + + A: + + B:Neg
L:3.2 A:2.4 B:0.6
24
F
29
L:S A:P+AE B:None
L: + + + + A:+ + B:Neg
L:2.7 A:3.8 B:0.62
25
F
24
L:U+R A:U+R B:None
L: + + + A:+ + B:Neg
L:15.5 A:2.6 B: <0.35
31
F
37
L:U A:U B:U
L:+ + + + A:+ + + B:+ + +
L:7.4 A:3.5 B:1.88
32
F
34
L:U+R A:U+S B:U
L:+ + + + A:+ + + + B:+ + +
L:13.7 A:4.4 B:4.7
33
F
33
L:U A:U+AE B:U + A E
L:+ + + + A: + + + B: + + +
L:13.35 A:17.07 B:3.6
Results of skin prick tests are given as: + < 1/z positive reference, + + > 1/2 positive reference, + + + > positive reference, and + + + + > twice positive reference. RAST score is given in PRU/ml of serum. L, Latex; RC, rhinoconjunctivitis; .4, avocado pear; Neg, negative; B, banana; U, urticaria; AS, asthma; G, glossitis; R, rhinitis; S, shock; P, lip pruritus; AE, angioedema.
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559
TABLE I. Cont'd Patient No.
Sex
Age (yr)
Symptoms
Skin tests
34
M
33
L:U A:U + S +AS B:L+U
L: + + + + A: + + + B:+ + +
L:9.75 A:3.8 B:1.18
35
F
43
L:U A:U +AS B:U+AS
L: + + + A:+ + + B:+ + +
L:4.5 A:ll.1 B:3.2
(H. brasiliensis) were immediately collected in 1 ml of NTE IN solution (NaC1 1 mol/L, Tris 0.1 mol/L, ethylenediaminetetraacetic acid 0.01 mol/L). After centrifugation (500 g for 10 minutes), the supernatant from the middle aqueous phase was collected. Latex surgical gloves. Two sterile gloves (Triflex; Baxter Healthcare Corp., Hospital Supply, McGaw Park, IlL) were washed, then cut into 2 cm 2 bits. These were incubated in 50 ml of NTE 1N solution, at 37° C for 24 hours. After centrifugation at 500 g for 10 minutes, the supernatant was isolated. Fruits. Twenty grams of crushed avocado pear pulp was incubated in 10 ml of NTE 1N solution at 37 ° C for 6 hours. The resultant mixture was centrifugated (750 g for 15 minutes), then the supernatant was collected. Twenty grams of crushed banana pulp was incubated in 5 ml of NTE 1N solution at 37°C for 6 hours. After centrifugation (500 g for 10 minutes), the supernatant was isolated. Protein adjustment. For each extract, the protein concentration was determined by Lowry's method. 13 The appropriate volume of supernatant was determined in order to obtain the same protein concentration of 300 ~g for each acrylamide gel. SDS-PAGE followed by electroblotting and immunoblotting To detect specific antigen components, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis of fruit, latex, and glove extracts was performed by loading 300 Ixg of protein from each fresh extract onto gels of 15% acrylamide, as described by Laemmli. 14 One part of the gel was stained with silver nitrate (Bio-Rad Laboratories, Life Science Group, Hercules, Calif.), and the other part was blotted onto a nitrocellulose sheet by applying 60 V across the membrane for 6 hours in a Bio-Rad transblotting cell with a 200/2.0 power supply. These latter strips were quenched with 3% skimmed milk for 1 hour at room temperature in buffer containing 2 mml of Tris base, and were washed in Tris-buffered saline containing 0.2% Tween 20. Then membrane strips were incubated overnight with a 1:100 dilution of patients' or control subjects' sera at room temperature with gentle agitation. Membranes were
RAST score
then washed twice with Tris-buffered saline containing Tween 20 and incubated with a 1:100 dilution of goat anti-human IgE alkaline-phosphatase conjugate (Incstar Corp., Stillwater, Minn.). Color was developed with phosphatase-conjugate substrate (Bio-Rad Laboratories).
Cross-inhibition of immunoblotting Serum samples were preincubated with continuous shaking overnight at 4 ° C with 5, 10, 20, 40, 50, and 70 t~g/ml of natural latex, avocado, and banana extracts. Thereafter, each serum was tested against each antigen in immunoblotting, 15 as described above. Preincubation controls were performed with honeybee venom and ryegrass extracts at the same concentrations.
Human sera Seventeen patients (Table I), 15 women and 2 men ranging in age from 24 to 67 years (average age, 34 years), provided serum for this study. Allergy to latex was documented by a clinical history of urticaria and itching (9 patients), urticaria with rhinitis (3 patients), rhinoconjunctivitis (2 patients), anaphylactic shock (2 patients), and asthma (1 patient) after contact with latex gloves. Two patients also experienced lip edema when blowing up latex balloons. Among the patients, five also had a symptomatic food allergy to avocado pear and banana, and five other patients had a food allergy to avocado pear only. The clinical history was suggestive: urticaria and lip pruritus or glossitis were observed in all patients and were sometimes associated with rhinitis (3 patients), facial angioedema (2 patients), immediate shock after eating (2 patients), or wheezing (2 patients). Symptoms of fruit allergy occurred soon after ingestion. No other food was involved. Symptoms disappeared when allergens were removed and recurred after a new contact (3 cases). In all patients, skin prick tests were performed with a premoistened latex glove and with crude avocado pear and fresh banana extracts. The positive results of the tests were designated as follows: + < 1/e positive surface reference (histamine control: 10 mg/ml), + + > V2 positive reference, + + + > positive reference, and + + + + > twice reference.
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69.
46. 30,
21,5-
14,$,
MW
1
2
3
4
FIG. 1. SDS-PAGE, 15%, stained with silver nitrate. Lane I, Latex glove extract; lane 2, fresh serum latex extract; lane 3, avocado pear extract; lane 4, banana extract. Each extract contains approximately 300 Ixg of protein. MW, Protein molecular weight markers.
Specific IgE values for latex, avocado pear, and banana were determined by the Phadebas RAST (Pharmacia, Uppsala, Sweden). According to the clinical findings and laboratory data, sera were isolated in two groups. Group 1 comprised serum samples sera 11 to 17 from seven patients with only latex allergy (clinical history of latex allergy, no clinical evidence for a fruit allergy, positive prick test and RAST responses to latex, negative responses to fruits). Group 2 comprised serum samples 21 to 25 and 31 to 35 from 10 patients with a latex allergy and a fruit allergy (suggestive symptoms, positive prick test and RAST responses to latex and avocado pear) including eight patients who also reacted to banana. Serum samples from 10 nonallergic subjects were collected as controls: the subjects were non-atopic with no history of allergy and negative prick test and RAST responses to latex and fruits. In addition, serum samples from three atopic nurses who had negative skin test responses to latex but who had been exposed to latex were tested. RESULTS SDS-PAGE
The stained gel electrophoresis of concentrated natural latex and glove extracts revealed multiple protein bands ranging in relative MWs from 14 to 100 kd (Fig. 1). The most abundant bands had MWs of 14 kd and 30 to 35 kd, respectively. For avocado pear extracts, more than 20 protein bands were scattered between 10 and 120 kd,
mainly at 14, 21, 25, 30 to 35, 46, and 50 to 55 kd; and for banana extracts, protein bands were scattered from 10 to 100 kd, mainly at 14, 25, 30 to 35, and 68 to 75 kd. IgE i m m u n o b l o t t i n g
In IgE immunoblotting, all patients' sera from group 2 (fruit- and latex-associated allergy) reacted to natural latex proteins and also to avocado pear and banana. The results are shown in Table II and Fig. 2. The most important allergenic c o m p o n e n t in these extracts was at about 30 kd, and this band was found for the three extracts. With latex glove extracts, this 30 kd band was also revealed in five serum samples (nos. 21, 24, 31, 32, and 33). Minor bands were also isolated with latex glove extracts at 70 kd (2 sera), 50 kd (2 sera), 28 kd (1 serum), 21 kd (3 sera). For natural latex extract, there were also minor bands at 45 kd (1 serum), 21 kd (6 sera), 18 kd (1 serum), 14 kd (1 serum), and 10 kd (1 serum). A minor band was found with avocado pear extracts at 21 kd in one serum; for banana extracts a minor band was revealed at 35 kd in 1 serum (no. 34). All sera from the group 1 (monoallergy to latex) reacted to latex proteins with glove extracts. A band was present for the 7 sera at 21 kd. However, only one serum sample reacted with a 30 kd
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J ALLERGY CLIN IMMUNOL VOLUME 95, NUMBER 2
15
33
32
561
35
34
kD
%
69
~i
"
46
:!
=
}~
B
A
L
.30 :
5
ii
_21,5_
14,3
o
iS
A
L
B
A
L
B
A
L
II
A
L
FIG. 2. IgE immunoblotting with patients' sera. Selected patterns of sera from patients with latex allergy without (no. 15) or with an associated fruit allergy (nos. 32 to 35). /5', Banana; A, avocado pear; L, natural latex.
T A B L E II. IgE i m m u n o b l o t t i n g with patients' sera
Serum Sample No. 11 12 13 14 15 16 17 21 22 23 24 25 31 32 33 34 35 Control
Gloves: M W (kd) 21
28
30
35
45
Natural latex: M W (kd) 50
70
10
14
18
21
30
35
45
+
+ + + + + + +
4-
+ + +
+ + + +
+ +
+ + +
+
4+
21
30
30
+
+
+
+
+ + + + + + + + + +
+
+ +
+ 4+
+
+
+ + + +
35
4+ +
4-
+
+
4-
+
Banana: MW (kd)
+
+
+ +
50
Avocado pear: MW (kd)
+ + + + + + + +
+
+
+ + + + + + + +
Seven samples were obtained from 10 patients with latex allergy without (nos. 11 to 17) and with an associated fruit allergy (nos. 21 to 25 and 31 to 35). Control sera was obtained from 10 nonallergic subjects and three atopic subjects with negative response to latex who had been exposed to latex.
562
Lavaud et al.
J ALLERGYCLINIMMUNOL FEBRUARY 1995
kD
AVOCADO
BANANA
PEAR
p,
69
:
__46
~!
=~
i:
:i
"
LATEX
!il
i !:: ii:! 7: i!=~ .....
C
~. . . .
a
A
L
i~
,f ,:~
~
~
~
C
A
B
L
C
L
B
A
FIG. 3. Representative immunoblot patterns of reactivity observed in inhibition studies. Serum from patient 35 was preincubated with 50 I~g each of banana (first panel), avocado pear (second panel), and latex extract (third panel), then tested in immunoblot versus banana (lane B), avocado pear (lane A), and latex (lane L). Lane C shows the initial pattern without preincubation.
protein. Minor bands were revealed at 50 kd (2 sera), 45 kd (3 sera), and 35 kd (1 serum). For natural latex extract, only two sera (nos. 13 and 16) reacted with a 30 kd antigen, five reacted with a 21 kd antigen, one with 35 kd and 45 kd antigens, and two with a 50 kd antigen. Serum samples 13 and 16 also expressed bands in response to banana or avocado pear for a 30 kd antigen. These could represent an asymptomatic sensitization. All of the subjects in the control group showed negative results in the IgE immunoblotting analysis. Cross-inhibition of immunoblotting
Immunoblot inhibition studies were performed in the 10 serum samples from group 2 (fruit and latex allergy) to determine whether 30 kd proteins from latex, avocado pear, and banana are related. Patients' sera were preincubated with latex and fruit extracts before they were tested in immunoblotting against each antigen. For all sera, immunoblots were inhibited in a dose-dependent manner. As shown in Fig. 3, a complete inhibition of all 30 kd bands was observed after preincubation with 50 Ixg of avocado and latex extracts. A partial inhibition was obtained with this concentration of banana extract. On the contrary, preincubation with other allergens (honeybee venom, ryegrass pollen) did not inhibit any 30 kd immunoblot band. DISCUSSION
Latex proteins are numerous, and allergy to latex is related to a complex mixture of pro-
teins.4,16,17 The major protein bands are usually observed by SDS-PAGE from 10 to 14 kd, from 30 to 45 kd, and at about 67 kd,4,18,19, z0 with a doublet at 27 and 28 kd as demonstrated by Slater and Chhabra. 19 Turjanmaa et al. 3 showed that allergenic fractions from latex gloves had distinct peaks at 2, 5, and 30 kd in high-pressure chromatography. Many studies have pointed out the importance of a 30 KD protein present in latex extracts. Carillo et al. 2 showed that allergens from natural latex had an MW of 30 kd and greater. In a recent study Jaeger et al. 2° revealed several bands with a similar reaction pattern in sera from 15 patients with latex IgE-positive sera. The strongest bands were found not only at about 14 kd but also between 30 and 45 kd. Zacharisen et al.21 also found a protein fraction with an approximate MW of 30 kd. Bands of 27 and 30 kd were also found by Alenius et al.2z in sera from patients in the United States and Finland. In these reports, as in the work of Turjanmaa et al.,3 it is not clear whether patients were also allergic to fruits. In this study we have shown epitopes that are recognized by immunoblotting at MW of approximately 30 kd. They were present in fresh latex serum, latex gloves, avocado pear, and banana. The allergenic components present in latex gloves are usually also found in fresh Hevea latex serum,3, 5, z3 and we suggest that the use of fresh latex extracts, which is easier because of a higher protein concentration, should be preferred for studying latex allergy. Furthermore, the protein content of rubber gloves has been shown to be highly vari-
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able.3,17,19 This could explain why, in our study, some sera did not recognize a 30 kd band in glove extracts. Previously, we have shown, by R A S T inhibition, cross-reactivity among latex, avocado pear, and banana. 7 This was also reported by M'Raihi et al. 6 with latex proteins and banana, and more recently with latex, chestnut, and b a n a n a ? a, 25 Evidence for coexistent IgE antibodies or allergenic similarity between latex and banana was also suggested by immunospot inhibition and crossed-line radioimmunoelectrophoresis. 26 This suspected cross-reactivity among latex, avocado, and banana was proved by immunoblot-inhibition studies. Preincubation of patients' sera with each extract totally or partially inhibited the 30 kd protein reactivity, suggesting the presence of a c o m m o n allergen in the three extracts. Recent studies of immunoblotinhibition 27 have also detected such epitopes. This is important because patients with allergy to fruit, undergoing surgery or medical care in which latex may be used, are at risk for anaphylactic reactions. 7,12 For latex, the protein we detected around 30 kd could not be hevein, which is quantitatively the most important latex anionic protein, but it has a very low MW, first estimated to be 10 kd zs and later reduced to 5 kd. 29 Hydrolytic enzymes are fairly numerous, and their location varies within latex. 3° Thus hydrolases, which are associated with sedimentable particles, are located in lutoids, a particular type of intracellular organelle, which are today interpreted as polydispersed vacuomes. A m o n g hydrolases, a lysozyme activity is associated with two proteins, the hevamines, 3°"31 with an o p t i m u m p H of 4.0 and an M W of approximately 26 kd. Although the chemical characterization of the 30 kd band is not yet complete, the M W and its basic nature ( o p t i m u m p H , 4.0 to 5.6) (to be published) allow us to think that it could b e hevamine. Moreover, latex hevamine composition is very close to that of fig and papaya, 3°, 32 and similar lutoids were also r e p o r t e d in the latex of 20 plants f r o m the A p o c y n a c e a e , Caricacae, Convolvulaceae, E u p h o r b i a c e a e , M o r a ceae, Amaryllidae, A r e c a c e a e , and M u s a c e a e families. 33 Anyway, epitopes, or the proteins themselves, must be widespread in fruits, as correlated with clinical observations. We have only studied avocado pear and banana, but many authors have made an association between latex allergy and other fruit allergies: chestnuts, 22'25 figs, passion fruit, and celery12; nuts and citrus fruits34; kiwi fruit11; tomato, grape, and pineapple9; and pea-
563
nuts. 1° The study of Wadee et al.35 demonstrated that IgE antibodies could be detected for a 30 kd protein present in several fruits (strawberry, banana, guava, citrus fruits, and peach). Latex was not tested, but this observation must be acknowledged in our findings. In conclusion, we observed two groups of patients with different immunoblotting profiles. In sera from patients with an allergy to latex and fruits (avocado pear and banana) a constant common band was found, linked to common epitopes from 30 kd proteins. In the group of patients who only reacted to latex, this band was usually not found, even if other bands were present, especially at 21 kd. It is possible that a 30 kd protein band is a sign of a latex- and fruit-associated allergy. Its detection may prove useful in treating patients when a single allergy to latex or to fruit is clinically suspected. REFERENCES
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35.
zation from chestnuts and banana in patients with urticaria and anaphylaxis from contact with latex. Ann Allergy 1993;70:35-9. Makinen-Kiljunen S, Turjanmaa K, Palosuo T, Reunala T. Latex and banana: structurally similar antigens and allergens. XVth Congress of the European Academy of Allergology and Clinical Immunology. Paris, France, 10-15 May 1992 [Abstract]. Allergy 1992;47(Suppl):56. Makinen-Kiljunen S, Alenius H, Ahlroth M, Turjanmaa K, Palosuo T, Reunala T. Immunoblot inhibition detects several common allergens in rubber latex and banana [Abstract]. J ALLERGYCLIN IMMUNOL1993;91:242. Archer BL. The proteins of Hevea brasiliensis. 4. Isolation and characterisation of crystallin hevein. Biochem J 1960; 75:236-40. Tata SJ. Hevein: its isolation, purification and some structural aspects. In: Malaysian Rubber Producers Research Assoication, ed. Proceedings of International Rubber Conference 1975. Kuala Lumpur: Rubber Research Institute Malaysia, 1976:441-7. D'Auzac J, Jacob JL, Chrestin H. Physiology of rubber tree latex. Boca Raton, Florida: CRC Press, 1989. Tara SJ, Boyce AN, Archer BL, Audley BG. Lysosomes: major components of the sedimentable phase of Hevea brasiliensis. J Rubber Res Inst Malays 1976;24:233-6. Glazer AN, Barel AO, Howard JB, Brown DB. Isolation and characterization of fig lysozyme. J Biol Chem 1969;244: 3583-9. Southorn WA. A complex subcellular component of widespread occurrence in plant lactices. J Exp Botany 1964;15: 616-21. Fabro L, Muhlethaler K, Wutrich B. Anaphylaktische Reaktion auf Latex, ein Soforttypallergen von zunehmender Bedeutung. Hautartzt 1989;40:208-11. Wadee AA, Boring LA, Rabson AR. Fruit allergy: demonstration of IgE antibodies to a 30 kd protein present in several fruits. J ALLERGY CLIN IMMUNOL 1990;85:801-7.
Case reports of allergic reactions to latex proteins have been described with various products since 1979.1 The allergenicity of latex has been demonstrated by immunologic techniques such as immunoblotting. Recent studies revealed antigenic constituents with molecular weights (MWs) from 10 to 110 kd. 2-5 Occasionally, latex allergy is associated with allergy to fruit, especially banana, 6-9 but it may also be associated with allergy to avocado pear 7, s or other fruits belonging to phytogenetically dissimilar classes. 9-12 Cross-reactivity has been shown between latex and banana. 6 We have also recently described cross-reactivity among latex, banana, and avocado pear. 7 From the Department of RespiratoryDiseases and Allergology, University and Regional Medical Center, Reims; and Allerbio Laboratory, Varennes. Received for publication June 10, 1993; revised Oct. 22, 1993; accepted for publication Feb. 4, 1994. Reprint requests: Francois Lavaud, MD, CHR Reims, Unite d'Allergologie, H6pital Maison Blanche, Reims, F 51092, France. Copyright © 1995 by Mosby-Year Book, Inc. 0091-6749/95 $3.00 + 0 1/1/55249
Abbreviations used: MW: Molecular weight NTE: NaC1 1 mol/L, Tris 0.1 mol/L, ethylenediaminetetraacetic acid 0.01 mol/L SDS-PAGE: Sodium dodecylsulfate-polyacrylamide gel electrophoresis
In this study we have performed immunoblotting analysis and inhibition of immunoblotting to further investigate allergenic components in natural latex from Hevea brasiliensis, latex gloves, banana, and avocado pear. Sera from patients with latex allergy were studied to assess a common profile among these allergens. Two groups of sera were isolated according to their association with a proven fruit allergy.
METHODS Preparation of allergenic extracts Natural latex. Drops of fresh latex serum, drained after being cut from leaf stalks of the rubber tree 557
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T A B L E I. Clinical, skin test, and RAST data Patient No.
Sex
Age (yr)
Symptoms
Skin tests
RAST score
11
F
29
L:RC A:None B:None
L: + + + A:Neg B:Neg
L:I A:<0.35 B:<0.35
12
F
28
L:RC A:None B:None
L: + + + A:Neg B:Neg
L:0.52 A:<0.35 B:<0.35
13
F
34
L:U A:None B:None
L: + + + + A:Neg B:Neg
L:2.55 A: <0.35 B: <0.35
14
F
37
L:AS A:None B:None
L:+ + + + A:Neg B:Neg
L:2.65 A: <0.35 B:<0.35
15
F
35
L:U A:None B:None
L:+ + + + A:Neg B:Neg
L:3.7 A: <0.35 B:<0.35
16
F
25
L:U A:None B:None
L:+ + + + A:Neg B:Neg
L:1.26 A: <0.35 B: <0.35
17
F
29
L:U A:None B:None
L:+ + + A:Neg B:Neg
L:3.6 A: <0.35 B: <0.35
21
F
50
L:U A:G B:None
L: + + + A: + + B:Neg
L:1.06 A:1.46 B:<0.35
22
M
27
L:U + R A:U+R B:None
L: + + + A: + + B: + +
L:7.7 A:2.28 B:<0.35
23
F
32
L:U A:G B:None
L: + + + A: + + B:Neg
L:3.2 A:2.4 B:0.6
24
F
29
L:S A:P+AE B:None
L: + + + + A:+ + B:Neg
L:2.7 A:3.8 B:0.62
25
F
24
L:U+R A:U+R B:None
L: + + + A:+ + B:Neg
L:15.5 A:2.6 B: <0.35
31
F
37
L:U A:U B:U
L:+ + + + A:+ + + B:+ + +
L:7.4 A:3.5 B:1.88
32
F
34
L:U+R A:U+S B:U
L:+ + + + A:+ + + + B:+ + +
L:13.7 A:4.4 B:4.7
33
F
33
L:U A:U+AE B:U + A E
L:+ + + + A: + + + B: + + +
L:13.35 A:17.07 B:3.6
Results of skin prick tests are given as: + < 1/z positive reference, + + > 1/2 positive reference, + + + > positive reference, and + + + + > twice positive reference. RAST score is given in PRU/ml of serum. L, Latex; RC, rhinoconjunctivitis; .4, avocado pear; Neg, negative; B, banana; U, urticaria; AS, asthma; G, glossitis; R, rhinitis; S, shock; P, lip pruritus; AE, angioedema.
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559
TABLE I. Cont'd Patient No.
Sex
Age (yr)
Symptoms
Skin tests
34
M
33
L:U A:U + S +AS B:L+U
L: + + + + A: + + + B:+ + +
L:9.75 A:3.8 B:1.18
35
F
43
L:U A:U +AS B:U+AS
L: + + + A:+ + + B:+ + +
L:4.5 A:ll.1 B:3.2
(H. brasiliensis) were immediately collected in 1 ml of NTE IN solution (NaC1 1 mol/L, Tris 0.1 mol/L, ethylenediaminetetraacetic acid 0.01 mol/L). After centrifugation (500 g for 10 minutes), the supernatant from the middle aqueous phase was collected. Latex surgical gloves. Two sterile gloves (Triflex; Baxter Healthcare Corp., Hospital Supply, McGaw Park, IlL) were washed, then cut into 2 cm 2 bits. These were incubated in 50 ml of NTE 1N solution, at 37° C for 24 hours. After centrifugation at 500 g for 10 minutes, the supernatant was isolated. Fruits. Twenty grams of crushed avocado pear pulp was incubated in 10 ml of NTE 1N solution at 37 ° C for 6 hours. The resultant mixture was centrifugated (750 g for 15 minutes), then the supernatant was collected. Twenty grams of crushed banana pulp was incubated in 5 ml of NTE 1N solution at 37°C for 6 hours. After centrifugation (500 g for 10 minutes), the supernatant was isolated. Protein adjustment. For each extract, the protein concentration was determined by Lowry's method. 13 The appropriate volume of supernatant was determined in order to obtain the same protein concentration of 300 ~g for each acrylamide gel. SDS-PAGE followed by electroblotting and immunoblotting To detect specific antigen components, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis of fruit, latex, and glove extracts was performed by loading 300 Ixg of protein from each fresh extract onto gels of 15% acrylamide, as described by Laemmli. 14 One part of the gel was stained with silver nitrate (Bio-Rad Laboratories, Life Science Group, Hercules, Calif.), and the other part was blotted onto a nitrocellulose sheet by applying 60 V across the membrane for 6 hours in a Bio-Rad transblotting cell with a 200/2.0 power supply. These latter strips were quenched with 3% skimmed milk for 1 hour at room temperature in buffer containing 2 mml of Tris base, and were washed in Tris-buffered saline containing 0.2% Tween 20. Then membrane strips were incubated overnight with a 1:100 dilution of patients' or control subjects' sera at room temperature with gentle agitation. Membranes were
RAST score
then washed twice with Tris-buffered saline containing Tween 20 and incubated with a 1:100 dilution of goat anti-human IgE alkaline-phosphatase conjugate (Incstar Corp., Stillwater, Minn.). Color was developed with phosphatase-conjugate substrate (Bio-Rad Laboratories).
Cross-inhibition of immunoblotting Serum samples were preincubated with continuous shaking overnight at 4 ° C with 5, 10, 20, 40, 50, and 70 t~g/ml of natural latex, avocado, and banana extracts. Thereafter, each serum was tested against each antigen in immunoblotting, 15 as described above. Preincubation controls were performed with honeybee venom and ryegrass extracts at the same concentrations.
Human sera Seventeen patients (Table I), 15 women and 2 men ranging in age from 24 to 67 years (average age, 34 years), provided serum for this study. Allergy to latex was documented by a clinical history of urticaria and itching (9 patients), urticaria with rhinitis (3 patients), rhinoconjunctivitis (2 patients), anaphylactic shock (2 patients), and asthma (1 patient) after contact with latex gloves. Two patients also experienced lip edema when blowing up latex balloons. Among the patients, five also had a symptomatic food allergy to avocado pear and banana, and five other patients had a food allergy to avocado pear only. The clinical history was suggestive: urticaria and lip pruritus or glossitis were observed in all patients and were sometimes associated with rhinitis (3 patients), facial angioedema (2 patients), immediate shock after eating (2 patients), or wheezing (2 patients). Symptoms of fruit allergy occurred soon after ingestion. No other food was involved. Symptoms disappeared when allergens were removed and recurred after a new contact (3 cases). In all patients, skin prick tests were performed with a premoistened latex glove and with crude avocado pear and fresh banana extracts. The positive results of the tests were designated as follows: + < 1/e positive surface reference (histamine control: 10 mg/ml), + + > V2 positive reference, + + + > positive reference, and + + + + > twice reference.
560 Lavaud et al.
J ALLERGYCLINIMMUNOL FEBRUARY 1995
69.
46. 30,
21,5-
14,$,
MW
1
2
3
4
FIG. 1. SDS-PAGE, 15%, stained with silver nitrate. Lane I, Latex glove extract; lane 2, fresh serum latex extract; lane 3, avocado pear extract; lane 4, banana extract. Each extract contains approximately 300 Ixg of protein. MW, Protein molecular weight markers.
Specific IgE values for latex, avocado pear, and banana were determined by the Phadebas RAST (Pharmacia, Uppsala, Sweden). According to the clinical findings and laboratory data, sera were isolated in two groups. Group 1 comprised serum samples sera 11 to 17 from seven patients with only latex allergy (clinical history of latex allergy, no clinical evidence for a fruit allergy, positive prick test and RAST responses to latex, negative responses to fruits). Group 2 comprised serum samples 21 to 25 and 31 to 35 from 10 patients with a latex allergy and a fruit allergy (suggestive symptoms, positive prick test and RAST responses to latex and avocado pear) including eight patients who also reacted to banana. Serum samples from 10 nonallergic subjects were collected as controls: the subjects were non-atopic with no history of allergy and negative prick test and RAST responses to latex and fruits. In addition, serum samples from three atopic nurses who had negative skin test responses to latex but who had been exposed to latex were tested. RESULTS SDS-PAGE
The stained gel electrophoresis of concentrated natural latex and glove extracts revealed multiple protein bands ranging in relative MWs from 14 to 100 kd (Fig. 1). The most abundant bands had MWs of 14 kd and 30 to 35 kd, respectively. For avocado pear extracts, more than 20 protein bands were scattered between 10 and 120 kd,
mainly at 14, 21, 25, 30 to 35, 46, and 50 to 55 kd; and for banana extracts, protein bands were scattered from 10 to 100 kd, mainly at 14, 25, 30 to 35, and 68 to 75 kd. IgE i m m u n o b l o t t i n g
In IgE immunoblotting, all patients' sera from group 2 (fruit- and latex-associated allergy) reacted to natural latex proteins and also to avocado pear and banana. The results are shown in Table II and Fig. 2. The most important allergenic c o m p o n e n t in these extracts was at about 30 kd, and this band was found for the three extracts. With latex glove extracts, this 30 kd band was also revealed in five serum samples (nos. 21, 24, 31, 32, and 33). Minor bands were also isolated with latex glove extracts at 70 kd (2 sera), 50 kd (2 sera), 28 kd (1 serum), 21 kd (3 sera). For natural latex extract, there were also minor bands at 45 kd (1 serum), 21 kd (6 sera), 18 kd (1 serum), 14 kd (1 serum), and 10 kd (1 serum). A minor band was found with avocado pear extracts at 21 kd in one serum; for banana extracts a minor band was revealed at 35 kd in 1 serum (no. 34). All sera from the group 1 (monoallergy to latex) reacted to latex proteins with glove extracts. A band was present for the 7 sera at 21 kd. However, only one serum sample reacted with a 30 kd
Lavaud et al.
J ALLERGY CLIN IMMUNOL VOLUME 95, NUMBER 2
15
33
32
561
35
34
kD
%
69
~i
"
46
:!
=
}~
B
A
L
.30 :
5
ii
_21,5_
14,3
o
iS
A
L
B
A
L
B
A
L
II
A
L
FIG. 2. IgE immunoblotting with patients' sera. Selected patterns of sera from patients with latex allergy without (no. 15) or with an associated fruit allergy (nos. 32 to 35). /5', Banana; A, avocado pear; L, natural latex.
T A B L E II. IgE i m m u n o b l o t t i n g with patients' sera
Serum Sample No. 11 12 13 14 15 16 17 21 22 23 24 25 31 32 33 34 35 Control
Gloves: M W (kd) 21
28
30
35
45
Natural latex: M W (kd) 50
70
10
14
18
21
30
35
45
+
+ + + + + + +
4-
+ + +
+ + + +
+ +
+ + +
+
4+
21
30
30
+
+
+
+
+ + + + + + + + + +
+
+ +
+ 4+
+
+
+ + + +
35
4+ +
4-
+
+
4-
+
Banana: MW (kd)
+
+
+ +
50
Avocado pear: MW (kd)
+ + + + + + + +
+
+
+ + + + + + + +
Seven samples were obtained from 10 patients with latex allergy without (nos. 11 to 17) and with an associated fruit allergy (nos. 21 to 25 and 31 to 35). Control sera was obtained from 10 nonallergic subjects and three atopic subjects with negative response to latex who had been exposed to latex.
562
Lavaud et al.
J ALLERGYCLINIMMUNOL FEBRUARY 1995
kD
AVOCADO
BANANA
PEAR
p,
69
:
__46
~!
=~
i:
:i
"
LATEX
!il
i !:: ii:! 7: i!=~ .....
C
~. . . .
a
A
L
i~
,f ,:~
~
~
~
C
A
B
L
C
L
B
A
FIG. 3. Representative immunoblot patterns of reactivity observed in inhibition studies. Serum from patient 35 was preincubated with 50 I~g each of banana (first panel), avocado pear (second panel), and latex extract (third panel), then tested in immunoblot versus banana (lane B), avocado pear (lane A), and latex (lane L). Lane C shows the initial pattern without preincubation.
protein. Minor bands were revealed at 50 kd (2 sera), 45 kd (3 sera), and 35 kd (1 serum). For natural latex extract, only two sera (nos. 13 and 16) reacted with a 30 kd antigen, five reacted with a 21 kd antigen, one with 35 kd and 45 kd antigens, and two with a 50 kd antigen. Serum samples 13 and 16 also expressed bands in response to banana or avocado pear for a 30 kd antigen. These could represent an asymptomatic sensitization. All of the subjects in the control group showed negative results in the IgE immunoblotting analysis. Cross-inhibition of immunoblotting
Immunoblot inhibition studies were performed in the 10 serum samples from group 2 (fruit and latex allergy) to determine whether 30 kd proteins from latex, avocado pear, and banana are related. Patients' sera were preincubated with latex and fruit extracts before they were tested in immunoblotting against each antigen. For all sera, immunoblots were inhibited in a dose-dependent manner. As shown in Fig. 3, a complete inhibition of all 30 kd bands was observed after preincubation with 50 Ixg of avocado and latex extracts. A partial inhibition was obtained with this concentration of banana extract. On the contrary, preincubation with other allergens (honeybee venom, ryegrass pollen) did not inhibit any 30 kd immunoblot band. DISCUSSION
Latex proteins are numerous, and allergy to latex is related to a complex mixture of pro-
teins.4,16,17 The major protein bands are usually observed by SDS-PAGE from 10 to 14 kd, from 30 to 45 kd, and at about 67 kd,4,18,19, z0 with a doublet at 27 and 28 kd as demonstrated by Slater and Chhabra. 19 Turjanmaa et al. 3 showed that allergenic fractions from latex gloves had distinct peaks at 2, 5, and 30 kd in high-pressure chromatography. Many studies have pointed out the importance of a 30 KD protein present in latex extracts. Carillo et al. 2 showed that allergens from natural latex had an MW of 30 kd and greater. In a recent study Jaeger et al. 2° revealed several bands with a similar reaction pattern in sera from 15 patients with latex IgE-positive sera. The strongest bands were found not only at about 14 kd but also between 30 and 45 kd. Zacharisen et al.21 also found a protein fraction with an approximate MW of 30 kd. Bands of 27 and 30 kd were also found by Alenius et al.2z in sera from patients in the United States and Finland. In these reports, as in the work of Turjanmaa et al.,3 it is not clear whether patients were also allergic to fruits. In this study we have shown epitopes that are recognized by immunoblotting at MW of approximately 30 kd. They were present in fresh latex serum, latex gloves, avocado pear, and banana. The allergenic components present in latex gloves are usually also found in fresh Hevea latex serum,3, 5, z3 and we suggest that the use of fresh latex extracts, which is easier because of a higher protein concentration, should be preferred for studying latex allergy. Furthermore, the protein content of rubber gloves has been shown to be highly vari-
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J ALLERGY CLIN IMMUNOL VOLUME 95, NUMBER 2
able.3,17,19 This could explain why, in our study, some sera did not recognize a 30 kd band in glove extracts. Previously, we have shown, by R A S T inhibition, cross-reactivity among latex, avocado pear, and banana. 7 This was also reported by M'Raihi et al. 6 with latex proteins and banana, and more recently with latex, chestnut, and b a n a n a ? a, 25 Evidence for coexistent IgE antibodies or allergenic similarity between latex and banana was also suggested by immunospot inhibition and crossed-line radioimmunoelectrophoresis. 26 This suspected cross-reactivity among latex, avocado, and banana was proved by immunoblot-inhibition studies. Preincubation of patients' sera with each extract totally or partially inhibited the 30 kd protein reactivity, suggesting the presence of a c o m m o n allergen in the three extracts. Recent studies of immunoblotinhibition 27 have also detected such epitopes. This is important because patients with allergy to fruit, undergoing surgery or medical care in which latex may be used, are at risk for anaphylactic reactions. 7,12 For latex, the protein we detected around 30 kd could not be hevein, which is quantitatively the most important latex anionic protein, but it has a very low MW, first estimated to be 10 kd zs and later reduced to 5 kd. 29 Hydrolytic enzymes are fairly numerous, and their location varies within latex. 3° Thus hydrolases, which are associated with sedimentable particles, are located in lutoids, a particular type of intracellular organelle, which are today interpreted as polydispersed vacuomes. A m o n g hydrolases, a lysozyme activity is associated with two proteins, the hevamines, 3°"31 with an o p t i m u m p H of 4.0 and an M W of approximately 26 kd. Although the chemical characterization of the 30 kd band is not yet complete, the M W and its basic nature ( o p t i m u m p H , 4.0 to 5.6) (to be published) allow us to think that it could b e hevamine. Moreover, latex hevamine composition is very close to that of fig and papaya, 3°, 32 and similar lutoids were also r e p o r t e d in the latex of 20 plants f r o m the A p o c y n a c e a e , Caricacae, Convolvulaceae, E u p h o r b i a c e a e , M o r a ceae, Amaryllidae, A r e c a c e a e , and M u s a c e a e families. 33 Anyway, epitopes, or the proteins themselves, must be widespread in fruits, as correlated with clinical observations. We have only studied avocado pear and banana, but many authors have made an association between latex allergy and other fruit allergies: chestnuts, 22'25 figs, passion fruit, and celery12; nuts and citrus fruits34; kiwi fruit11; tomato, grape, and pineapple9; and pea-
563
nuts. 1° The study of Wadee et al.35 demonstrated that IgE antibodies could be detected for a 30 kd protein present in several fruits (strawberry, banana, guava, citrus fruits, and peach). Latex was not tested, but this observation must be acknowledged in our findings. In conclusion, we observed two groups of patients with different immunoblotting profiles. In sera from patients with an allergy to latex and fruits (avocado pear and banana) a constant common band was found, linked to common epitopes from 30 kd proteins. In the group of patients who only reacted to latex, this band was usually not found, even if other bands were present, especially at 21 kd. It is possible that a 30 kd protein band is a sign of a latex- and fruit-associated allergy. Its detection may prove useful in treating patients when a single allergy to latex or to fruit is clinically suspected. REFERENCES
1. Nutter AF. Contact urticaria to rubber. Br J Dermatol 1979;101:597-8. 2. Carillo T, Cuevas M, Munoz T, Hinojosa M, Moneo I. Contact urticaria and rhinitis from latex surgical gloves. Contact Dermatitis 1986;15:69-72. 3. Turjanmaa K, Laurila K, Makinen-Kiljunen S, Reunala T. Rubber contact urticaria: allergenic properties of 19 brands of latex gloves. Contact Dermatitis 1988;19:362-7. 4. Morales C, Basomba A, Carreira J, Sastre A. Anaphylaxis produced by rubber glove contact. Case reports and immunological identification of the antigens involved. Clin Exp Allergy 1989;19:425-30. 5. Turjanmaa K, Reunala T, Alenius H, Brummer-Korvenkotio H, Palosuo T. Allergens in latex surgical gloves and glove powder. Lancet 1990;337:1588. 6. M'Raihi L, Charpin D, Pons A, Bongrand P, Vervloet D. Cross-reactivitybetween latex and banana. J ALLERGYCLIN IMMUNOL1991;87:129-30.
7. Lavaud F, Cossart C, Reiter V, Bernard J, Deltour G, Holmquist I. Latex allergy in patient with allergy to fruit. Lancet 1992;339:492-3. 8. Borries M, Vieluf D, Abeck D, Ring J. Food anaphylaxisin two patients with latex allergy. XVth Congress of the European Academy of Allergology and Clinical Immunology. Paris, France, 10-15 May 1992 [Abstract]. Allergy 1992;47(Suppl):57. 9. Leynadier F, Pecquet C, Dry J. Anaphylaxisto latex during surgery. Anaesthesia 1989;44:547-50. 10. Axelsson GK, Eriksson M. Anaphylaxis and angioedema due to rubber allergy in children. Acta Paediatr Scand 1988;77:314-6. 11. Gaignon I, VeyckemansF, Gribomont BF. Latex allergy in a child: report of a case. Acta Anaesthesiol Belg 1991;42: 219-23. 12. Ceuppens JL, Van Durme P, Dooms-Goossens A. Latex allergy in patient with allergy to fruit. Lancet 1992;339: 492-3. 13. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75.
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14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 1970; 227:680-5. 15. Johansson E, Borga A, Johansson SGO, Van Hage-Hamsten M. Immunoblot multi-allergen inhibition studies of allergenic cross-reactivity of the dust mites Lepidoglyphus destructor and Dermatophagoides pteronyssinus. Clin Exp Allergy 1991;21:511-8. 16. Levy DA, Charpin D, Pecquet C, Leynadier F, Vervloet D. Allergy to latex. Allergy 1992;47:579-87. 17. Tomazic VJ, Withrow TJ, Fisher BR, Dillard F. Latexassociated allergies and anaphylactic reactions. Clin Immunol Immunopathol 1992;64:89-97. 18. Mathew SN, Melton A, Wagner W, Battisto JR. Latex hypersensitivity: prevalence among children with spina bifida and immunoblotting identification of latex proteins [Abstract]. J ALLERGYCLIN IMMUNOL1992;89:225. 19. Slater JE, Chhabra SK. Latex antigens. J ALLERGY CLIN IMMUNOL1992;89:673-8. 20. Jaeger D, Kleinhans D, Czuppon AB, Baur X. Latexspecific proteins causing immediate-type cutaneous, nasal, bronchial, and systemic reactions. J ALLERGYCLIN IMMUNOL 1992;89:759-68. 21. Zacharisen MC, Kurup VP, Resnick A, et al. Characterization of latex antigens reacting with IgE antibodies in the sera of the patients with allergy to latex [Abstract]. J ALLERGYCLIN IMMUNOL1992;89:225. 22. Alenius H, Kelly KJ, Kurup VP, et al. Differences IgE reactivity to latex antigens in sera from patients from the US and Finland [Abstract]. J .AllERGYCLINIMMUNOL1993;91: 240. 23. Alenius H, Turjanmaa K, Palosuo T, Makinen-Kiljunen S, Reunala T. Surgical latex glove allergy: characterization of rubber protein allergens by immunoblotting. Int Arch Appl Immunol 1991;96:376-80. 24. Rodriguez M, Vega F, Garcia MT, et al. Hypersensitivity to latex, chestnut and banana. Ann Allergy 1993;70:31-4. 25. Fernandez de Corres L, Moneo I, Munoz D, et al. Sensiti-
26.
27.
28.
29.
30. 31.
32.
33.
34.
35.
zation from chestnuts and banana in patients with urticaria and anaphylaxis from contact with latex. Ann Allergy 1993;70:35-9. Makinen-Kiljunen S, Turjanmaa K, Palosuo T, Reunala T. Latex and banana: structurally similar antigens and allergens. XVth Congress of the European Academy of Allergology and Clinical Immunology. Paris, France, 10-15 May 1992 [Abstract]. Allergy 1992;47(Suppl):56. Makinen-Kiljunen S, Alenius H, Ahlroth M, Turjanmaa K, Palosuo T, Reunala T. Immunoblot inhibition detects several common allergens in rubber latex and banana [Abstract]. J ALLERGYCLIN IMMUNOL1993;91:242. Archer BL. The proteins of Hevea brasiliensis. 4. Isolation and characterisation of crystallin hevein. Biochem J 1960; 75:236-40. Tata SJ. Hevein: its isolation, purification and some structural aspects. In: Malaysian Rubber Producers Research Assoication, ed. Proceedings of International Rubber Conference 1975. Kuala Lumpur: Rubber Research Institute Malaysia, 1976:441-7. D'Auzac J, Jacob JL, Chrestin H. Physiology of rubber tree latex. Boca Raton, Florida: CRC Press, 1989. Tara SJ, Boyce AN, Archer BL, Audley BG. Lysosomes: major components of the sedimentable phase of Hevea brasiliensis. J Rubber Res Inst Malays 1976;24:233-6. Glazer AN, Barel AO, Howard JB, Brown DB. Isolation and characterization of fig lysozyme. J Biol Chem 1969;244: 3583-9. Southorn WA. A complex subcellular component of widespread occurrence in plant lactices. J Exp Botany 1964;15: 616-21. Fabro L, Muhlethaler K, Wutrich B. Anaphylaktische Reaktion auf Latex, ein Soforttypallergen von zunehmender Bedeutung. Hautartzt 1989;40:208-11. Wadee AA, Boring LA, Rabson AR. Fruit allergy: demonstration of IgE antibodies to a 30 kd protein present in several fruits. J ALLERGY CLIN IMMUNOL 1990;85:801-7.