Characterization of Latex Antigen and Demonstration of LatexSpecific Antibodies by EnzymeLinked Immunosorbent Assay in Patients with Latex Hypersensitivity Viswanath P. Kurup, Ph.D., Kevin J. Kelly, M.D., Abe Resnick, M.S., Naveen K. Bansal, Ph.D.,* and Jordan N. Fink, M.D.
ABSTRACT Two latex antigens, one extractedfrom surgical gloves (GE) and the other/rom the sap of Hevea brasiliensis plant (RPcj were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. These antigens were used to detect latex specijic IgE and IgG antibodies by enzyme-linked immunosorbent assay (ELISA) in the sera of patients with meningomyelocele (spina b(fida) and of normal controls. Of the 36 patients studied, 26 had wheal-and~flare skin-prick test reactivity to latex antigen with 11/26 having had a history of anaphylaxis to latex products. Twenty three of the 26 sera from skin-test positive patients and 10/11 patients with history of anaphylaxis demonstrated significant levels of latex specific IgE and 19G in the sera, whereas only 1/14 normals showed signijicant antibodies to latex. The remaining 10 patients, all skin-test negative with From the Allergy Immunology Division, Department of Medicine, The Medical College of Wisconsin, Milwaukee, and Research Service, V.A. Medical Center, Milwaukee, and * The Department q{ Mathematics and Statistics, Marquelle University, Milwaukee, Wisconsin Address correspondence to Viswanath P. Kurup, Ph.D., Research Service, 151-1, V. A. Medical Center, 5000 West National Ave., Milwaukee, WI 53295-1000
Allergy Proc.
latex antigens, showed only loll' lel'els of antibodies. The findings indicate that the ELISA used in the present study employing partially characleri:::edantigens has sensilil'ilJ' and specijicily to delecl latex specijic antibodies in the sera (i{ suspected patients and can be used for presl/lnplive diagnosis of latex allergy. (Allergy Proc 13, 6: 329-334, 1992)
there have been increasing numbers of reRecently ports of allergic reactions resulting from exposure to latex products.'-3 The responses to latcx products include rhinitis, bronchospasm, angioedema, contact urticaria, ul1icaria, anaphylaxis, and sudden death; the majority of these reactions have been associatcd with immediate skin-test reactivity to latex proteins. 1.4-7 However, skin tests may be hazardous in certain patients inasmuch as latex antigen may induce an anaphylactic reaction. The antigens/allergens used in diagnosis of latex allergy originate from diversc latcx products or from crude latex obtained from }[(!I'(!(/ brasiliensis plant.1.4.5 Using glove extract (GE) and rubber plant sap extract (RPE) as antigen sources, several methods including radioallergosorbent tcst RAST, basophil degranulation, and enzyme-linked immunosorbent assay (ELISA), have been developed to detect latex allergy. 1.8.9 However, none of the previous reports detailed the preparations of the antigens nor
329
provided any characterization of the extracts used in these assays. 1.4.7.10 The present investigation was undertaken to develop a reliable technique to demonstrate latex specific IgE in the sera of such patients suspected of having latex allergy. We isolated and partially characterized allergens from the latex sap of the rubber plant and from surgical gloves, used them in an ELISA, and compared the results with the clinical features.
top of the clear sap and a thick precipitate formed at the bottom of the centrifuge tubes. The clear fluid was removed with a syringe and needle from the middle layer and was extracted with equal volume of PBS, recentrifuged, and the supernatant collected, dialyzed, and lyophilized as above or preserved at -70°C as frozen aliquots. Characterization
MATERIALS Patients
AND METHODS
and Controls
total of 50 sera were studied for IgG and IgE antibodies to latex by ELISA. Of these 50 subjects, 36 were meningomyelocele (spina bifida) patients and 14 were normal controls. Some of the normal subjects included in the study were exposed to gloves and latex products while working in the laboratory.
A
Latex Antigens wo latex preparations were used in the present study, one preparation was obtained from the sap of II hrasiliensis plant and the other was an extract of unpowdered surgical gloves (Perry X-AM, Smith and Nephew, Massillon, OH). We have compared several extracts prepared from different latex products frequently used in health care procedures. Comparison of the protein profiles of these antigens in SDS-polyacrylamide gels and reactivity with patient sera by ELISA detected no significant differences. The differences among some of the preparations were quantitative, and hence only one glove preparation was selected for the present study.
T
Glo\'c Extract
(GE)
terile gloves were cut into I cm2 pieces and 10 g of the material was ground in a mortar with glass beads (1 mm diameter size) for approximately 5 to 10 minutes with phosphate buffered saline (PBS). The ground mixture was transferred to a flask and extracted by stirring with 150 m L PBS at 4°C on a magnetic stirrer. After 24 hours the supernate was collected, and the glove pieces extracted once again as described. The two extracts were mixed, dialyzed against deionized water at 4°C and freeze dried after sterile filtration. The freeze-dried product was dissolved in sterile distilled water and evaluated for quality by studying the immunochcmical characteristics as described below.
S
Rubber Plant Sap Extract
(RPE)
he raw sap from the rubber plant obtained from India and was transported with 0.1 % (vol/vol) formalin as preservative. On arrival, more than 75% of the sap remained uncoagulated. The sap was diluted I: I with PBS and centrifuged at 15,000 rpm for 30 minutes. A white fluffy surface film appeared on the
T
330
of Antigens
Protein estimation. The protein content
in the GE and RPE was estimated by the Bicinchoninic acid (BCA) method (Pierce Chemicals, Rockford, IL) according to the protocol provided by the manufacturer.
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). A 8 to 20% gradient polyacrylamide containing 0.5% SOS was used to study the protein components present in the extract. The method followed was the same as described previously. II The GE and RPE were treated with sample preparation buffer, and 10 to 25 J.Lg of proteins were applied to the gel. After electrophoresis the gels were stained with Coomassie brilliant blue and the protein profile compared. Molecular weight markers were run with all gels. Two-dimensional gel electrophoresis (2-DE). The method used was the same as reported previously except that a Bio-Rad (Burlingame, CA) mini 2-0 gel system was used.11•12 The proteins were subjected to isoelectric focusing in a pH gradient of 3.5 to 9.5 in capillary tubes followed by electrophoresis in a 8 to 20% linear SOS gradient gel in the second dimension. The first dimension gels were stained with Coomassie brilliant blue R250. Gels showing well-separated proteins were subjected to second dimension electrophoresis. After electrophoresis the gels were stained with silver stain 10 and protein bands compared. Crossed immunoelectrophoresis (C/E). Both GE and RPE antigens were analyzed by crossed immunoelectrophoresis to characterize them for their quality and reactivity. The method followed was exactly the same as described previously. 12 Ten J.LLof antigen containing 100 J.Lgprotein were electrophoresed in agarose gels at 10 V/cm for an hour. The separated proteins were then subjected to electrophoresis at 90° to the first dimension run, into a gel containing 7.5% antibody raised against RPE and GE in rabbits. The second direction electrophoresis was carried out at 0.5 V/cm for 18 hours at a constant temperature of 1a°e. The gels were then washed and stained with Coomassie brilliant blue R250, and the precipitin arcs developed were compared. The lectin-binding characteristics of the antigens by incorporating concanavalin A (Con A) in an intermediate gel between the antigen and antibody gel during the second dimension electrophoresis. The Con A binding antigens were retained in the intermediate gels, while the proteins will move into the antibody gel and react with the latex antibody.
November-December
1992, Vol. 13, No.6
Enzyme-Linked Immunosorbent Assay (ELISA) n the present study, a sensitive biotin-avidin linked sandwich ELISA was used to detect latex protein specific IgG and IgE as described previously.13,14 Polystyrene microtiter plates (Immulon II) were coated with antigens dissolved or diluted in PBS. One J.lg/mL protein of GE or RPE antigens in PBS was used to coat the wells of the ELISA plates. One hundred J.lL of the antigens were added to the wells and incubated for 3 hours at 37°C. This was followed by overnight incubation at 4°C and blocking with I % bovine serum albumin in PBS (BSA-PBS) for an hour at room temperature, The plates were washed thrice for 5 minutes each with PBS containing 0.05% Tween-20 (PBS-T) and treated with the dilution of the sera. The plates were then incubated at room temperature for 3 hours. The wells were washed again for three cycles of 5 minutes. followed by the addition of affinity purified goat antihuman IgG or IgE (I: 1000 dilution) and incubated at room temperature for an hour. The wells were washed as before and treated with 100 J.lL of 1:5000 dilution of rabbit anti-goat IgG coupled to biotin. After reincubation and washing, the wells were treated with streptavidin peroxidase (1 :20,000). The plates were then incubated for 30 minutes at room temperature and washed before adding the substrate (ortho phenyldiamine in citrate buffer [pH 5.5] and 0.15% H202). The optical density (00) of the color developed was read at 450 nm using an automated ELISA reader (Oynatech Labs, Chantilly, VA). All the reagents were obtained from Sigma Chemicals (St. Louis, MO) and reagents were prepared in BSA-PBS. The concentrations of the coating antigens and reagents were determined from results obtained from checkerboard titration studies using known positive and negative sera. Positive and negative control sera were run with each ELISA plate.
I
Specificity of Antibody pecificity of IgE antibody was studied by inhibition ELISA using positive sera as reported previously. 13 Serum dilutions were incubated with 50 ng to 50 J.lg/ mL of latex antigens for 3 hours at room temperature. After incubation, antigen-antibody complexes were removed by centrifugation at 15,000 rpm for 15 minutes, and the absorbed serum was used in ELISA as described above. The 00 values were compared with the uninhibited sera, and the dose-dependent inhibition was calculated. A dose-dependent inhibition of most of the antibody is indicative of the presence of specific antibody.
S
Skin Test 4.11 patients were skin tested by the prick technique with serial 10-fold dilutions of the GE extract
ft
Allergy Proc.
diluted in PBS ranging from I: 1000, to I: 1,000,000 according to the standard methods. IS The starting dilution was determined after reviewing the clinical history of the patient. The patients with history of anaphylaxis were initially tested with the I: 100,000 or I: I ,000,000 concentrations. All sites were read at 20 minutes for wheal-and-flare reactions in comparison with the reactivity with the diluent of the antigen (negative reaction) and histamine (positive skin-test reaction). Reactions were graded negative to 4+ depending on the size of the wheal and intensity of the flare.ls
Statistical Analysis hree dilutions of each sera for IgG (I :250, 1:500, and I: 1000) and two dilutions for IgE (I :50 and 1:100) were used to compare the 00. The means and standard deviations were calculated and the groups were compared by multivariate analysis of variance (MANGY A) using Hottelings T2 analysis. We have tested the 00 values of the dilutions individually against the three groups and between the two antigens. The p of :
,=;:"
Igf against GF in sera of patients •
at)!;ortl4!d
"'0
RPE. 5E 1: 1000
01
OF
u!l;ed
to
3l)!l;Orn
ant
19""
100
~
80
C
~
60
:.
40
o -l-,-..........,~,-.--.----,-. Amount
nPI
~
C
70 .', .
0.3
with
'..... ',' ,'-
~..•. ,'-:
,',".,
antibody
Q
Q
0.2
Igf against l,v
RPF in sera
.absorb@d with
Gf
of patlenls antigen
0.1
IgF
3g31nst
GF in
Sf!r3
of
Ilallf!nl"
oo:lorbed wllh RPf anllgen
1>0 , 00
0'.0
SPT
.•
~ ~
60
ABSTRACT Two latex antigens, one extractedfrom surgical gloves (GE) and the other/rom the sap of Hevea brasiliensis plant (RPcj were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. These antigens were used to detect latex specijic IgE and IgG antibodies by enzyme-linked immunosorbent assay (ELISA) in the sera of patients with meningomyelocele (spina b(fida) and of normal controls. Of the 36 patients studied, 26 had wheal-and~flare skin-prick test reactivity to latex antigen with 11/26 having had a history of anaphylaxis to latex products. Twenty three of the 26 sera from skin-test positive patients and 10/11 patients with history of anaphylaxis demonstrated significant levels of latex specific IgE and 19G in the sera, whereas only 1/14 normals showed signijicant antibodies to latex. The remaining 10 patients, all skin-test negative with From the Allergy Immunology Division, Department of Medicine, The Medical College of Wisconsin, Milwaukee, and Research Service, V.A. Medical Center, Milwaukee, and * The Department q{ Mathematics and Statistics, Marquelle University, Milwaukee, Wisconsin Address correspondence to Viswanath P. Kurup, Ph.D., Research Service, 151-1, V. A. Medical Center, 5000 West National Ave., Milwaukee, WI 53295-1000
Allergy Proc.
latex antigens, showed only loll' lel'els of antibodies. The findings indicate that the ELISA used in the present study employing partially characleri:::edantigens has sensilil'ilJ' and specijicily to delecl latex specijic antibodies in the sera (i{ suspected patients and can be used for presl/lnplive diagnosis of latex allergy. (Allergy Proc 13, 6: 329-334, 1992)
there have been increasing numbers of reRecently ports of allergic reactions resulting from exposure to latex products.'-3 The responses to latcx products include rhinitis, bronchospasm, angioedema, contact urticaria, ul1icaria, anaphylaxis, and sudden death; the majority of these reactions have been associatcd with immediate skin-test reactivity to latex proteins. 1.4-7 However, skin tests may be hazardous in certain patients inasmuch as latex antigen may induce an anaphylactic reaction. The antigens/allergens used in diagnosis of latex allergy originate from diversc latcx products or from crude latex obtained from }[(!I'(!(/ brasiliensis plant.1.4.5 Using glove extract (GE) and rubber plant sap extract (RPE) as antigen sources, several methods including radioallergosorbent tcst RAST, basophil degranulation, and enzyme-linked immunosorbent assay (ELISA), have been developed to detect latex allergy. 1.8.9 However, none of the previous reports detailed the preparations of the antigens nor
329
provided any characterization of the extracts used in these assays. 1.4.7.10 The present investigation was undertaken to develop a reliable technique to demonstrate latex specific IgE in the sera of such patients suspected of having latex allergy. We isolated and partially characterized allergens from the latex sap of the rubber plant and from surgical gloves, used them in an ELISA, and compared the results with the clinical features.
top of the clear sap and a thick precipitate formed at the bottom of the centrifuge tubes. The clear fluid was removed with a syringe and needle from the middle layer and was extracted with equal volume of PBS, recentrifuged, and the supernatant collected, dialyzed, and lyophilized as above or preserved at -70°C as frozen aliquots. Characterization
MATERIALS Patients
AND METHODS
and Controls
total of 50 sera were studied for IgG and IgE antibodies to latex by ELISA. Of these 50 subjects, 36 were meningomyelocele (spina bifida) patients and 14 were normal controls. Some of the normal subjects included in the study were exposed to gloves and latex products while working in the laboratory.
A
Latex Antigens wo latex preparations were used in the present study, one preparation was obtained from the sap of II hrasiliensis plant and the other was an extract of unpowdered surgical gloves (Perry X-AM, Smith and Nephew, Massillon, OH). We have compared several extracts prepared from different latex products frequently used in health care procedures. Comparison of the protein profiles of these antigens in SDS-polyacrylamide gels and reactivity with patient sera by ELISA detected no significant differences. The differences among some of the preparations were quantitative, and hence only one glove preparation was selected for the present study.
T
Glo\'c Extract
(GE)
terile gloves were cut into I cm2 pieces and 10 g of the material was ground in a mortar with glass beads (1 mm diameter size) for approximately 5 to 10 minutes with phosphate buffered saline (PBS). The ground mixture was transferred to a flask and extracted by stirring with 150 m L PBS at 4°C on a magnetic stirrer. After 24 hours the supernate was collected, and the glove pieces extracted once again as described. The two extracts were mixed, dialyzed against deionized water at 4°C and freeze dried after sterile filtration. The freeze-dried product was dissolved in sterile distilled water and evaluated for quality by studying the immunochcmical characteristics as described below.
S
Rubber Plant Sap Extract
(RPE)
he raw sap from the rubber plant obtained from India and was transported with 0.1 % (vol/vol) formalin as preservative. On arrival, more than 75% of the sap remained uncoagulated. The sap was diluted I: I with PBS and centrifuged at 15,000 rpm for 30 minutes. A white fluffy surface film appeared on the
T
330
of Antigens
Protein estimation. The protein content
in the GE and RPE was estimated by the Bicinchoninic acid (BCA) method (Pierce Chemicals, Rockford, IL) according to the protocol provided by the manufacturer.
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). A 8 to 20% gradient polyacrylamide containing 0.5% SOS was used to study the protein components present in the extract. The method followed was the same as described previously. II The GE and RPE were treated with sample preparation buffer, and 10 to 25 J.Lg of proteins were applied to the gel. After electrophoresis the gels were stained with Coomassie brilliant blue and the protein profile compared. Molecular weight markers were run with all gels. Two-dimensional gel electrophoresis (2-DE). The method used was the same as reported previously except that a Bio-Rad (Burlingame, CA) mini 2-0 gel system was used.11•12 The proteins were subjected to isoelectric focusing in a pH gradient of 3.5 to 9.5 in capillary tubes followed by electrophoresis in a 8 to 20% linear SOS gradient gel in the second dimension. The first dimension gels were stained with Coomassie brilliant blue R250. Gels showing well-separated proteins were subjected to second dimension electrophoresis. After electrophoresis the gels were stained with silver stain 10 and protein bands compared. Crossed immunoelectrophoresis (C/E). Both GE and RPE antigens were analyzed by crossed immunoelectrophoresis to characterize them for their quality and reactivity. The method followed was exactly the same as described previously. 12 Ten J.LLof antigen containing 100 J.Lgprotein were electrophoresed in agarose gels at 10 V/cm for an hour. The separated proteins were then subjected to electrophoresis at 90° to the first dimension run, into a gel containing 7.5% antibody raised against RPE and GE in rabbits. The second direction electrophoresis was carried out at 0.5 V/cm for 18 hours at a constant temperature of 1a°e. The gels were then washed and stained with Coomassie brilliant blue R250, and the precipitin arcs developed were compared. The lectin-binding characteristics of the antigens by incorporating concanavalin A (Con A) in an intermediate gel between the antigen and antibody gel during the second dimension electrophoresis. The Con A binding antigens were retained in the intermediate gels, while the proteins will move into the antibody gel and react with the latex antibody.
November-December
1992, Vol. 13, No.6
Enzyme-Linked Immunosorbent Assay (ELISA) n the present study, a sensitive biotin-avidin linked sandwich ELISA was used to detect latex protein specific IgG and IgE as described previously.13,14 Polystyrene microtiter plates (Immulon II) were coated with antigens dissolved or diluted in PBS. One J.lg/mL protein of GE or RPE antigens in PBS was used to coat the wells of the ELISA plates. One hundred J.lL of the antigens were added to the wells and incubated for 3 hours at 37°C. This was followed by overnight incubation at 4°C and blocking with I % bovine serum albumin in PBS (BSA-PBS) for an hour at room temperature, The plates were washed thrice for 5 minutes each with PBS containing 0.05% Tween-20 (PBS-T) and treated with the dilution of the sera. The plates were then incubated at room temperature for 3 hours. The wells were washed again for three cycles of 5 minutes. followed by the addition of affinity purified goat antihuman IgG or IgE (I: 1000 dilution) and incubated at room temperature for an hour. The wells were washed as before and treated with 100 J.lL of 1:5000 dilution of rabbit anti-goat IgG coupled to biotin. After reincubation and washing, the wells were treated with streptavidin peroxidase (1 :20,000). The plates were then incubated for 30 minutes at room temperature and washed before adding the substrate (ortho phenyldiamine in citrate buffer [pH 5.5] and 0.15% H202). The optical density (00) of the color developed was read at 450 nm using an automated ELISA reader (Oynatech Labs, Chantilly, VA). All the reagents were obtained from Sigma Chemicals (St. Louis, MO) and reagents were prepared in BSA-PBS. The concentrations of the coating antigens and reagents were determined from results obtained from checkerboard titration studies using known positive and negative sera. Positive and negative control sera were run with each ELISA plate.
I
Specificity of Antibody pecificity of IgE antibody was studied by inhibition ELISA using positive sera as reported previously. 13 Serum dilutions were incubated with 50 ng to 50 J.lg/ mL of latex antigens for 3 hours at room temperature. After incubation, antigen-antibody complexes were removed by centrifugation at 15,000 rpm for 15 minutes, and the absorbed serum was used in ELISA as described above. The 00 values were compared with the uninhibited sera, and the dose-dependent inhibition was calculated. A dose-dependent inhibition of most of the antibody is indicative of the presence of specific antibody.
S
Skin Test 4.11 patients were skin tested by the prick technique with serial 10-fold dilutions of the GE extract
ft
Allergy Proc.
diluted in PBS ranging from I: 1000, to I: 1,000,000 according to the standard methods. IS The starting dilution was determined after reviewing the clinical history of the patient. The patients with history of anaphylaxis were initially tested with the I: 100,000 or I: I ,000,000 concentrations. All sites were read at 20 minutes for wheal-and-flare reactions in comparison with the reactivity with the diluent of the antigen (negative reaction) and histamine (positive skin-test reaction). Reactions were graded negative to 4+ depending on the size of the wheal and intensity of the flare.ls
Statistical Analysis hree dilutions of each sera for IgG (I :250, 1:500, and I: 1000) and two dilutions for IgE (I :50 and 1:100) were used to compare the 00. The means and standard deviations were calculated and the groups were compared by multivariate analysis of variance (MANGY A) using Hottelings T2 analysis. We have tested the 00 values of the dilutions individually against the three groups and between the two antigens. The p of :
,=;:"
Igf against GF in sera of patients •
at)!;ortl4!d
"'0
RPE. 5E 1: 1000
01
OF
u!l;ed
to
3l)!l;Orn
ant
19""
100
~
80
C
~
60
:.
40
o -l-,-..........,~,-.--.----,-. Amount
nPI
~
C
70 .', .
0.3
with
'..... ',' ,'-
~..•. ,'-:
,',".,
antibody
Q
Q
0.2
Igf against l,v
RPF in sera
.absorb@d with
Gf
of patlenls antigen
0.1
IgF
3g31nst
GF in
Sf!r3
of
Ilallf!nl"
oo:lorbed wllh RPf anllgen
1>0 , 00
0'.0
SPT
.•
~ ~
60
