Comparison of latex–specific IgE binding among nonammoniated latex, ammoniated latex, and latex glove allergenic extracts by ELISA and immunoblot inhibition

Comparison of latex-specific IgE binding among nonammoniated latex, ammoniated latex, and latex glove allergenic extracts by ELISA and immunoblot inhibition Akira Akasawa, MD, PhD, Li-Shan Hsieh, PhD, and Yuan Lin, PhD Rockville, Md. Background: Nonammoniated latex, ammoniated latex, and latex glove extracts' have been used as source materials for the preparation of allergenic extracts for the diagnosis of" latex alle~. These materials showed different patterns of protein bands' and immunoreactive bands'. ttowever, their IgE-reactive repertoires were not compared. Objective: The goals of this study were to compare the IgE reactivity and to define the common IgE-reactive epitopes among three latex allergenic extracts. Methods: Two serum pools' were obtained from adults" and children with latex allergy to evaluate the lgE reactivity among three latex extracts'. IgE reactivity and IgE-reactive proteins were compared by inhibition ELISA and inhibition immunoblot methods, respectively. Results: In this study inhibition curves were similar for nonammoniated latex and ammoniated latex but were different when the latex glove extracts were used. Several protein bands of" ammoniated latex and latex glove extracts' could not be inhibited by the nonammoniated latex. The ammoniated latex and latex glove extracts were able to remove all the latex-specific IgE from the serum. Conclusion: The IgE-reactive proteins differ among different latex extracts'. Ammoniated latex and latex glove extracts contain more complete immunoreactive repertoires for detecting IgE antibodies. Our study provides useful information for selecting the latex extract. (J Allergy Clin lmmunol 1996;97.'1116-20.) Key words: Latex allergens, latex proteins, latex glove

Three different latex source materials are often used to prePare reagents to evaluate latex allergy: nonammoniated latex sap, ammoniated latex sap, and latex gloves. Latex glove extract (LG) is commonly used for skin testing. Nonammoniated latex extract (NAL) and ammoniated latex extract (AL) are used for in vitro diagnostic testing. Proteins from both gloves and ammoniated latex sap are subjected to hydrolysis and denaturation during processing.r, 2 Nonammoniated latex sap was chosen as an alternative source material for the preparation of reference reagents. A recent study demonstrated that LGs contain a repertoire of From The Laboratory of lmmunobiochemistry, Division of Allergenic Products and Parasitology, Center for Biologics Evaluation and Research, Food and Drug Administration. Received for publication Apr. 24, 1995; revised June 23, 1995; accepted for publication June 26, 1995. Reprint requests: Yuan Lin, PhD, FD/CBER HFM-422, 1401 Rockville Pike, Rockville, MD 20852-1441. 1/1/67369

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Abbreviations used AL: Ammoniated latex extract LG: Latex glove extract NAL: Nonammoniated latex extract PBS: Phosphate-buffered saline

allergens as complete as that of N A L and AL, whereas N A L and L G provide the most sensitive and greatest consistency? The purpose of this study was to compare the proteins and their serum reactivities among NAL, AL, and L G by using both E L I S A and immunoblotting inhibition technique.

METHODS Nonammoniated latex extract Preparation of NAL was described previously? This extract contained C-serum and particle proteins from a rubber tree (Hevea brasiIiensis, clone 600, Malaysia), obtained by ultracentrifugation, The protein concentra-

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tion was 2.89 lng/ml as measured by BCA Protein Assay (Pierce Chemical Co., Rockford, Ill.).

Ammoniated latex extract Ammoniated latex was collected from the same source as the nonammoniated latex. Fresh latex sap was mixed with ammonia (final concentration, 200 mmol/L) with the same procedure used for the commercial rubber products. Ammoniated latex was centrifuged, and Cserum was collected. The buffer solution of A L was exchanged to phosphate-buffered (PBS) through PD-10 Columns (Pharmacia, Piscataway, N.J.). The protein concentration of A L was 17.5 mg/ml.

Latex glove extract LG 1 was extracted from Bantex latex gloves (American Bantax, Burlingame, Calif.). Eight grams of latex glove material was cut into 1 cmz pieces and shaken with 50 ml of PBS, pH 7.4, by horizontal rotation for 24 hours at 4° C. The extract was centrifuged at 50,000 g at 4° C for 1 hour to remove any powder and was sterile-filtered through a 0.22 Fm filter unit, lyophilized, and reconstituted in 2.5 ml of deionized distilled water. LG 1 concentrates were then exchanged to lX PBS through PD-10 columns. Protein concentration of LG 1 was 1.40 mg/ml. LG 2, which was prepared in a manner similar to that for L G 1, was provided by Dr. Robert Hamilton (Johns .Hopkins University, Baltimore, Md.). LG 2 was extracted from Triflex Latex Gloves (Baxter Healthcare Corp., Valencia, Calif.)? Protein concentration of LG 2 was 1.72 mg/ml.

concentrations of extracts in micrograms were calculated from dilutions and initial concentrations of the extracts. Coated plates were washed with B114 (PBS with 0.1% BRIJ 35 [Sigma Diagnostics, St. Louis, Mo.]) with an Automated Washer (Bio-Tek Instruments, Inc., Winooski, Vt.) and blocked with 1% bovine serum albumin (Boehringer Mannheim, Indianapolis, Ind.) in Bl14 for 1 hour and washed with Bl14. The treated sera were diluted 1:50 with B114 solution, added to wells, and incubated for 2 hours; and wells were washed with B114. Adsorbed IgE was detected with peroxidase-labeled goat IgG anti-human IgE(e) antibody (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted 1:1000. Plates were washed with B114 and developed with TMB solution (Kirkegaard & Perry Laboratories). Absorbance of 450 nm was measured by a MR5000 Microplate Reader (Dynatech Laboratories Inc., Chantilly, Va.). Percent inhibition was calculated as follows: %Inhibition = ( OD 0 - OD ) + OD o × 100, where OD o is absorbance with use of noninhibited serum and OD is absorbance with use of inhibited serum.

Inhibition immunoblot Latex extracts were separated and blotted as previously described] except that the strips were incubated overnight with the treated sera diluted 1:10 in BlI4.

RESULTS Inhibition ELISA

Two serum pools were generously provided by Dr. Robert Hamilton (Johns Hopkins University). The adult serum pool was collected from 184 health care workers with latex allergy. The pediatric serum pool was collected from 101 children with spina bifida and latex allergy.

W h e n the a d u l t s e r u m p o o l was used, similar i n h i b i t i o n curves w e r e o b t a i n e d for N A L a n d A L , r e g a r d l e s s of which extract was u s e d to c o a t the plates. H o w e v e r , it r e q u i r e d only o n e t e n t h o f the a m o u n t o f L G to achieve 50% i n h i b i t i o n as c o m p a r e d with t h a t o f N A L a n d A L (Fig. 1, A , B, a n d C). W h e n t h e p e d i a t r i c s e r u m p o o l was used, the p a t t e r n s o f i n h i b i t i o n s e e m e d to b e different, dep e n d i n g on the m a t e r i a l u s e d to c o a t t h e plates. L G s i m p a r t m o r e inhibitory activity t h a n N A L and A L (Fig. 1, D a n d F); however, it is i n t e r e s t i n g to n o t e that w h e n A L was u s e d to coat t h e plates, the i n h i b i t o r y activity o f N A L was m u c h r e d u c e d as c o m p a r e d with that of A L a n d L G 1 (Fig. 1, E l .

Inhibition ELISA

Inhibition immunoblot

Optimal dilutions of coating extract and serum pool were determined by performing a checkerboard titration with serial dilutions of extract and serum pool on microtiter plates (E.I.A./R.I.A. plates; Costar, Cambridge, Mass.). Optimal dilutions for extract (1:225) and for serum pool (1:50) were chosen by the intensity of the color development. Microtiter plates were coated with the latex extracts diluted 1:225 with PBS and incubated overnight at 4 ° C. The serum pools were incubated with equal volumes of serially diluted latex extracts (l:10 °, 10 l, 10 2, 10 3, and 10 .4 ) for 2 hours at 4° C. The

I n h i b i t i o n i m m u n o b l o t s w e r e p e r f o r m e d to detect t h e c r o s s - r e a c t i v i t y o f p r o t e i n s p r e p a r e d f r o m N A L , A L , a n d L G . Fig. 2 (A, B, a n d C) shows the inhibition i m m u n o b l o t s to N A L , A L , a n d L G 1 with t h e a d u l t s e r u m pool, which was first t r e a t e d with N A L , A L , a n d L G 1. T h e d i s a p p e a r a n c e of m o s t I g E - r e a c t i v e b a n d s was directly c o r r e l a t e d to t h e i n c r e a s e in c o n c e n t r a t i o n of inhibitors. N A L did n o t c o m p l e t e l y inhibit p r o t e i n b a n d s of 10, 18, 29, a n d 46 kd o f N A L w h e n N A L was a n a l y z e d by

Heat-treated NAL To denature latex proteins, a vial of NAL was heated at 250 ° F under 18 psi for 20 minutes, and the buffer solution was exchanged to PBS through PD-10 columns.

Serum pools

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0 25

25

..E 50

50

O .13 c

o~

75

100

75 i

i

i

i

i

0 0.001 0.1 1 0.0001 0.01

100

i

10 100

i

I

0 0.001 0.1 0.0001 0.01

a

1

10 100

0 25

25

:E 50 t-

50

75

75

c-

E

o c~

100

100 0.001 0.1 0.0001 0.01

1

0 0.001 0.1 0.0001 0.01

10 100

1

10 100

0 25

25

:E 50 ¢-

50

¢O c~

o~

75

75

100

100 0 0.001 0.1 1 10 100 0.0001 0.01 Amount of inhibitor ( gg )

0.001 0.1 1 10 100 0.0001 0.01 Amount of inhibitor ( gg )

Nonammoniated latex extract A

---o---- Glove extract

Ammoniated latex extract

FIG. 1. Inhibition ELISA. A t o C, Inhibition of adult ser um pool; s y m b o l s indicate extracts. D t o F, Inhibition of pediatric serum pool; s y m b o l s indicate extracts. Plates w e r e coated with extracts as follows: A and D, NAL; B and E, AL; and C and F, LG.

sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to a ProBlott membrane (Applied Biosystems, Foster City, Calif.) (Fig. 2, A). NAL also failed to inhibit a 46 kd protein band of AL (Fig. 2, B) and 6.5, 29, and 46 kd protein bands of LG 1 (Fig. 2, C). However, reactive intensity of these protein bands became diminished compared with that of noninhibited serum. AL and LG I could inhibit all IgE-reactive bands of all three extracts when higher concentrations of inhibitors were used. When the serum pool from children was used, NAL did not inhibit a 29 kd protein band of three

extracts, whereas AL, LG 1, and LG 2 were able to remove all the reactive IgE from the serum (Fig. 2, D, E, F) (data for L G 2 not shown). To examine the possibility that the incomplete inhibition by NAL was due to the lack of denatured proteins, heat denaturation was applied to the NAL. The heat-treated NAL completely inhibited the 29 kd protein band (Fig. 2, D, E, and F). DISCUSSION The two serum pools (adult health care workers and children with spina bifida) used to evaluate the proteins of different latex extracts showed similar

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Non-ammoniated Ammoniated

Non-ammoniated Ammoniated

Glove

Glove

Heated non a m m o n i a t e d

D

A --46

--30

o~ c

o* c --21.5 :~

.5~ --14.3 i.3 v --6,5

E

g

: --46

--30

o~ c

--21,5 m ~5

{

--14.3 --6.5

C

F --46

--46

--30

--30 c_

--21.5 --21.5 ::r

14.3 --14.3

6.5

--6.5

FIG. 2. Inhibition i m m u n o b l o t . A t o C, Serum pool f r o m adults inhibited with the extracts indicated at top. D t o F, Serum pool f r o m children inhibited with the extracts indicated at top. Blotted extracts were: A and D, NAL; B and E, AL; and C and F, LG. N is an i m m u n o b l o t f r o m n o n t r e a t e d serum, The concentration gradient at the t o p of each panel indicates the decreasing a m o u n t of c o m p e t i n g extract used. Undiluted extracts w e r e used in the far right lanes.

but not identical immunob|otting patterns. Data from our recent study indicated that these pools contain high levels of latex-specific IgE, and the immunoblot indicated that almost a|l |atex-reac-

tive lgE antibodies were included in these pools. J Alenius et al. 4 reported 10 IgE-reactive proteins (with molecular weights of 14, 18, 20, 30, 35, 39, 44, 54, 63, and 200 kd) in two brands of surgical gloves.

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A m o n g these proteins, the 14 and 30 kd proteins reacted strongly with sera. 4 The glove extracts used in our study were p r e p a r e d from two brands of latex gloves (Bantex and Triflex). L G 1 contains immunoreactive bands with molecular weights of 6.5, 14, 29, 39, 46 kd and a smear of around 50 kd, which were reactive to the adult serum pool; and L G 1 and L G 2 contain immunoreactive bands of 14, 16, 27, 29, 39, 46 kd and a smear of around 50 kd, which were reactive to the pediatric serum pool (Fig. 2, C, F, lane N). According to the results of inhibition ELISA, the general trends of inhibition were similar, regardless of which serum pool was used. The capacities to inhibit the IgE in allergic sera were different. The difference was m o r e obvious when the serum pool from children was used. Almost 100 times more N A L than LG was required to achieve 50% inhibition (Fig 1, E and F); whereas only approximately 10 times m o r e N A L was required to achieve 50% inhibition when the adult serum pool was used. The difference was also observed in the inhibition immunoblot study. A L and L G completely inhibited the reactive IgE in both serum pools, even though they have different immunoreactive patterns (Fig. 2, lanes labeled N). It is possible that the proteins in these preparations were denatured and/or degraded and thus migrated to different locations on the gel, but the epitopes remained intact to react with the specific IgEs in the serum pools. When two serum pools were first treated with NAL, the IgE-reactive species were greatly reduced but not completely absorbed (Fig. 2), suggesting exposure of new epitopes during sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. When the N A L was heat-treated, inhibition was increased. The 29 kd protein could not be inhibited with NAL, regard-

less of the concentration used, but the h e a t treated N A L completely inhibited this band. It is possible that this protein changes its c o n f o r m a tion w h e n it is in a m m o n i a buffer or treated with heat, thereby altering or occluding some of the epitopes. This study demonstrated that A L and L G contain a m o r e complete immunoreactive repertoire for detecting latex-specific IgE antibodies in both adult and pediatric serum pools. Our results also indicate that N A L contains many c o m m o n I g E reactive domains with denatured latex preparations. To increase the epitope repertoire of N A L in order to detect all of the latex-specific IgE, a mixture of N A L with h e a t - t r e a t e d N A L can be prepared. The relevance of these in vitro studies to optimizing diagnosis of latex allergy requires clinical confirmation. We thank Dr. Esah Yip, of the Rubber Research Institute of Malaysia, for providing us with the latex sap for our study. We also thank Dr. Robert Hamilton for the generous contribution of sera and extract for this study and Dr. Paul Turkeltaub and Dr. Arthur Karpas for their valuable suggestions and critical review of this manuscript. REFERENCES

1. Akasawa A, Hsieh L, Lin Y. Serum reactivities to latex proteins (Hevea brasiliensis) J Allergy Clin Immunol 1995; 95:1196-205. 2. Slater JE, Chhabra SK. Latex allergens. J Allergy Clin Immunol 1992;89:673-8. 3. Hamilton R, Charous L, Adkinson F, Yunginger J. Serologic methods in the laboratory diagnosis of latex rubber allergy: study of nonammoniated, ammoniated latex, and glove (end-product) extracts as allergen reagent sources. J Lab Clin Med 1994;123:594-604. 4. Alenius H, Makinen-Kiljunen S, Turjanmaa K, Patosuo T, Reunala T. Allergen and protein content of latex gloves. Ann Allergy 1994;73:315-20.