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Measurement of antigen-specific antibodies in human serum and saliva by multiple antigen simultaneous test
Allergology International (1996) 45: 177-179
Short Communication
Measurement serum
of antigen-specific
and saliva
by multiple
antibodies antigen
in human
simultaneous
test
Mikako Takasugi,1 Koji Yamada,1 Yuji Itoh,1 Tatsuya Igawa,2 Hideki Itaya2 and Michihiro Sugano1 Laboratory of1 FoodScience,Departmentof FoodScienceand Technology, FacultyofAgriculture, KyushuUniversity, Fukuokaand 2Pharmaceutical ResearchLaboratory, HitachiChemicalCo. Ltd,Hitachi,Japan
ABSTRACT Expression of antigen-specific serum IgE and IgA, and saliva IgA were measured in four volunteers using a Multiple Antigen Simultaneous Test (MAST).Two donors, expressing allergen-specific IgE in serum, expressed antigen-specific serum IgA strongly, but saliva IgA weakly. The other two donors, not expressing serum IgE, expressed serum IgA weakly and saliva IgA strongly. These results suggest a possibility that secretory IgA plays an important role in the prevention of food allergy. To clarify the role of IgA, further investigation on allergic patients is necessary. Key words; allergy, IgA, IgE, MAST,saliva INTRODUCTION Recently,food allergy has become a serious health problem not only for children but also for adults.1,2 Some types of food allergy are mediated by allergen-specific IgE bound to mast cells or basophils.1 The binding of allergen to the membranebound IgE induces degranulation of these cells and releases inflammatory mediators such as histamine, platelet activating factors and leukotrienes. Although the serum IgE levels of healthy individuals are very low, they often elevate markedly in allergic patients.3 Thus, the measurement of total and allergenspecific serum IgE is used for the diagnosis of food allergy. Though serum IgE plays an important role in the development of food allergy, other classes of antibodies such as IgA also affect the process. Secretory IgA inhibits the absorption of allergens from the small intestine.4 Therefore, secretory IgA may inhibit the incidence of food allergy. However, information on the expression of allergen-specific IgA is very limited. It is known
Correspondence: Mikako Takasugi, Laboratory of Food Science, Department of Food Science and Technology, Faculty of Agriculture 46-09, Kyushu University, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-81 Japan. Received 28 February 1996. Accepted for publication
13 June 1996.
that IgAis secretednot onlyin intestinalsecretions,but insaliva, tears and colostrum.4-5 We therefore tried to apply a multiple antigen simultaneoustest (MAST) for IgE determinationto the measurementof allergen-specificIgA. METHODS Serum and saliva samples were collectedfrom four volunteer students(2 malesand 2 females, 23-26 yearsold). One volunteer had been diagnosed as being allergicto soybean (MS)and one had been diagnosed as havingatopic dermatitis(MT).The others (MIand KY)have never been diagnosed as having had an allergicdisease and had no subjectivesymptoms.Informed consent was obtained from all volunteerstudents. Forthe measurementof antigen-specificserum IgE,reactions were done accordingto the standard methodrecommendedby the manufacturers(MastImmunosystems,MountainView,CA, USA).In brief, MAST pette TestChamberswere filledwithserum and allowedto stand at room temperaturefor 16 h. Peroxidase (POD)-conjugatedanti-IgEsuppliedwiththe kitwas dilutedwith dilutionbufferand introducedto the testchambers before being allowed to react at room temperaturefor 4 h. Afterincubation withthe substrate solutionsuppliedwiththe kit,the test chambers were set in a supplied Photocassetteand exposed to a Polaroidfilm.Chemiluminescentintensityof bands correspondingto each antigen was read usinga supplieddensitometer. Then,the MASTmethodwas applied to the measurementof antigen-specificIgAusing a POD-conjugatedanti-human IgA (Dakopatts,Glostrup, Denmark)in place of POD-conjugated anti-humanIgE.SinceIgAexpressedat much higherconcentrations thin IgE,sample dilutionwas necessaryfor its measurement. In assays using antibodiessuch as ELISA,bovine serum albumin or milk protein preparation are used as blocking agents to decrease non-specificabsorption of antibodies.b6,7 As these bovineproteinscould themselvesbe allergensto be studied, an alternate agent for blockingand sample dilutionwitha lowallergenicitywas needed. In ELISA determinations,we used 0.1% fish gelatin (SigmaChemical Co., St Louis,MO, USA)
178
M TAKASUGI ET AL .
according to the method reported by Fritsche and Bonzon.8 When serum or saliva samples were diluted with 0 .1% fish gelatin, negative control level decreased more quickly compared with the samples diluted with the buffer appended for POD-conjugate dilution. Thus, 0.1% fish gelatin for sample dilution was used in this study. The optimum dilution ratio to decrease the negative control level and to obtain the highest positive signals was 100 times for serum IgA and 25 times for saliva IgA.
Fig. 1 Expression of antigen-specific
antibodies
DISCUSSION Although we used fish gelatin for sample dilution because of its low antigenicity,it has recently been reported that some individuals are sensitive to bovine gelatin injected with vaccines and most of them have bovine gelatin-specific IgE.9However, only two of 11 patients showed allergic symptoms after eating gelatincontaining candies. This suggests that the allergenicity of orally administered gelatin is much weaker than that of other allergens.
in human sera and saliva.
MEASUREMENT
Figure 1 shows the expression of allergen-specific antibodies measured by the MASTmethod. One volunteer (MS)expressed serum IgE strongly to Japanese cedar and cat dander and weakly to house dust, egg white, milk and soybean. Volunteer MT expressed IgEto mite and cat dander, but the others, MIand KY,did not. The two donors expressing allergen-specific IgE showed strong and widespread expression of serum IgA and weak expression of saliva IgA. On the other hand, the two IgEnegative donors showed limited expression of antigen-specific serum IgA, except for cat dander, milk and beef, and stronger expression of antigen-specific saliva IgA. Most saliva IgA is dimeric and derived from the mucosal immune system, while serum IgA is largely monomeric and derived from plasma cells in the bone marrow.10The difference between expression of serum. and saliva IgA suggests that their production or secretion is regulated independently. Secretory IgA of mucosal tissues such as the small intestine and respiratory tract is known to be the first barrier against various pathogens such as bacteria, viruses and food antigens.4 In the case of IgAproduction, IgAplasma cells primed at intestine migrate via the systemic circulation to secrete IgA in saliva and tears.11,12To understand the activity of the gut-associated lymphoid tissue, we measured the expression of antigen-specific IgA in saliva and observed a negative relationship between the expression of serum IgE and saliva IgA. Deficiency or poor expression of IgA in infants is related to the development of food allergy.12,14These results suggest that secretory IgA plays an important role in the prevention of food allergy. However, because of the small number of cases in our study, further study with a large number of patients is necessary to clarify the role of secretory IgA. We showed here that the MASTsystem was useful for determination of allergen-specific IgA as well as IgE. Studies of antigen-specific IgA expression in allergic patients using this system will contribute to the elucidation of the mechanism of allergy development.
OF ANTIBODIES
BY MAST
179
REFERENCES 1 Metcalfe DD. Food allergy. Curr. Opin. Immunol. 1991; 3: 881-6. 2 Plaut M, Zimmerman EM. Allergy and Mechanisms of Hypersensitivity. In: PaulWE, ed. FundamentalImmunology,3rd edn. NewYork:RavenPressLtd,1993; 1399-425. 3 BlorkstenB,AhlstedtS, BlorkstenF et al. ImmunoglobulinE and immunoglobulinG4 antibodies to cow's milk in children with cow's milkallergy.Allergy1983; 38: 119-24. 4 ShorterRG,TomasiTBJr.Gut immunemechanisms.Adv.Intern. Med. 1982; 27: 247-80. 5 WatanabeT,Nagura H, Watanabe K,BrownWR.The bindingof human milk lactoferrinto immunoglobulinA. FEBSLett. 1984; 168: 203-7. 6 YamadaK,AkiyoshiK,MurakamiH et al. Partialpurificationand characterizationof immunoglobulinproductionstimulatingfactor derivedfrom Namalwacells. In VitroCell. Dev.Biol.1989; 25: 243-7. 7 Yamada K,MatsumuraK, SuzakiM, ShirahataS, MurakamiH. Stimulationand inhibitionof interferon-Bproductionof human diploid fibroblastsby foodstuffs.Agric.Biol.Chem. 1991; 55: 829-32. 8 FritscheR, BonzonM. Determinationof cow milkformula allergenicityin the rat modelby in vitromast celltriggeringand in viva IgEinduction.Int.Arch.AllergyAppl.Immunol.1990; 93: 289-93. 9 SakaguchiM, Ogura H, InouyeS. IgEantibodyto gelatin in children withimmediate-typereactionsto measles and mumpsvaccines.J.AllergyClin.Immunol.1995; 96: 563-5. 10 WolfHM,FischerMB,PuhringerH,SamstagA,VogelE, EeiblMM. Human serum IgA downregulatesthe release of inflammatory cytokines(tumornecrosisfactor-a, interleukin-6)in human monocytes.Blood1994; 83: 1278-88. 11 Jackson DE,LallyET,Nakamura MC,MontgomeryPC. Migration of IgA-bearinglymphocytesinto salivaryglands. Cell. Immunol. 1981; 63: 203-9. 12 VisakorpiJK. The immune response of the intestinalmucosa to foreignproteins.ActaPaediatr.1982; Suppl.296: 56-9. 13 Sloper KS, Brook OGD, KingstonD, Pearson JR, Shiner M. Eczemaand atopyin earlychildhood:LowIgAplasma cellcounts inthe lelunalmucosa.Arch.Dis.Child.1981; 56: 939-42. 14 KaufmanHS,HobbsJR.Immunogobulindeficienciesin an atopic population.Lancet1970; 21: 1061-3.
Short Communication
Measurement serum
of antigen-specific
and saliva
by multiple
antibodies antigen
in human
simultaneous
test
Mikako Takasugi,1 Koji Yamada,1 Yuji Itoh,1 Tatsuya Igawa,2 Hideki Itaya2 and Michihiro Sugano1 Laboratory of1 FoodScience,Departmentof FoodScienceand Technology, FacultyofAgriculture, KyushuUniversity, Fukuokaand 2Pharmaceutical ResearchLaboratory, HitachiChemicalCo. Ltd,Hitachi,Japan
ABSTRACT Expression of antigen-specific serum IgE and IgA, and saliva IgA were measured in four volunteers using a Multiple Antigen Simultaneous Test (MAST).Two donors, expressing allergen-specific IgE in serum, expressed antigen-specific serum IgA strongly, but saliva IgA weakly. The other two donors, not expressing serum IgE, expressed serum IgA weakly and saliva IgA strongly. These results suggest a possibility that secretory IgA plays an important role in the prevention of food allergy. To clarify the role of IgA, further investigation on allergic patients is necessary. Key words; allergy, IgA, IgE, MAST,saliva INTRODUCTION Recently,food allergy has become a serious health problem not only for children but also for adults.1,2 Some types of food allergy are mediated by allergen-specific IgE bound to mast cells or basophils.1 The binding of allergen to the membranebound IgE induces degranulation of these cells and releases inflammatory mediators such as histamine, platelet activating factors and leukotrienes. Although the serum IgE levels of healthy individuals are very low, they often elevate markedly in allergic patients.3 Thus, the measurement of total and allergenspecific serum IgE is used for the diagnosis of food allergy. Though serum IgE plays an important role in the development of food allergy, other classes of antibodies such as IgA also affect the process. Secretory IgA inhibits the absorption of allergens from the small intestine.4 Therefore, secretory IgA may inhibit the incidence of food allergy. However, information on the expression of allergen-specific IgA is very limited. It is known
Correspondence: Mikako Takasugi, Laboratory of Food Science, Department of Food Science and Technology, Faculty of Agriculture 46-09, Kyushu University, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-81 Japan. Received 28 February 1996. Accepted for publication
13 June 1996.
that IgAis secretednot onlyin intestinalsecretions,but insaliva, tears and colostrum.4-5 We therefore tried to apply a multiple antigen simultaneoustest (MAST) for IgE determinationto the measurementof allergen-specificIgA. METHODS Serum and saliva samples were collectedfrom four volunteer students(2 malesand 2 females, 23-26 yearsold). One volunteer had been diagnosed as being allergicto soybean (MS)and one had been diagnosed as havingatopic dermatitis(MT).The others (MIand KY)have never been diagnosed as having had an allergicdisease and had no subjectivesymptoms.Informed consent was obtained from all volunteerstudents. Forthe measurementof antigen-specificserum IgE,reactions were done accordingto the standard methodrecommendedby the manufacturers(MastImmunosystems,MountainView,CA, USA).In brief, MAST pette TestChamberswere filledwithserum and allowedto stand at room temperaturefor 16 h. Peroxidase (POD)-conjugatedanti-IgEsuppliedwiththe kitwas dilutedwith dilutionbufferand introducedto the testchambers before being allowed to react at room temperaturefor 4 h. Afterincubation withthe substrate solutionsuppliedwiththe kit,the test chambers were set in a supplied Photocassetteand exposed to a Polaroidfilm.Chemiluminescentintensityof bands correspondingto each antigen was read usinga supplieddensitometer. Then,the MASTmethodwas applied to the measurementof antigen-specificIgAusing a POD-conjugatedanti-human IgA (Dakopatts,Glostrup, Denmark)in place of POD-conjugated anti-humanIgE.SinceIgAexpressedat much higherconcentrations thin IgE,sample dilutionwas necessaryfor its measurement. In assays using antibodiessuch as ELISA,bovine serum albumin or milk protein preparation are used as blocking agents to decrease non-specificabsorption of antibodies.b6,7 As these bovineproteinscould themselvesbe allergensto be studied, an alternate agent for blockingand sample dilutionwitha lowallergenicitywas needed. In ELISA determinations,we used 0.1% fish gelatin (SigmaChemical Co., St Louis,MO, USA)
178
M TAKASUGI ET AL .
according to the method reported by Fritsche and Bonzon.8 When serum or saliva samples were diluted with 0 .1% fish gelatin, negative control level decreased more quickly compared with the samples diluted with the buffer appended for POD-conjugate dilution. Thus, 0.1% fish gelatin for sample dilution was used in this study. The optimum dilution ratio to decrease the negative control level and to obtain the highest positive signals was 100 times for serum IgA and 25 times for saliva IgA.
Fig. 1 Expression of antigen-specific
antibodies
DISCUSSION Although we used fish gelatin for sample dilution because of its low antigenicity,it has recently been reported that some individuals are sensitive to bovine gelatin injected with vaccines and most of them have bovine gelatin-specific IgE.9However, only two of 11 patients showed allergic symptoms after eating gelatincontaining candies. This suggests that the allergenicity of orally administered gelatin is much weaker than that of other allergens.
in human sera and saliva.
MEASUREMENT
Figure 1 shows the expression of allergen-specific antibodies measured by the MASTmethod. One volunteer (MS)expressed serum IgE strongly to Japanese cedar and cat dander and weakly to house dust, egg white, milk and soybean. Volunteer MT expressed IgEto mite and cat dander, but the others, MIand KY,did not. The two donors expressing allergen-specific IgE showed strong and widespread expression of serum IgA and weak expression of saliva IgA. On the other hand, the two IgEnegative donors showed limited expression of antigen-specific serum IgA, except for cat dander, milk and beef, and stronger expression of antigen-specific saliva IgA. Most saliva IgA is dimeric and derived from the mucosal immune system, while serum IgA is largely monomeric and derived from plasma cells in the bone marrow.10The difference between expression of serum. and saliva IgA suggests that their production or secretion is regulated independently. Secretory IgA of mucosal tissues such as the small intestine and respiratory tract is known to be the first barrier against various pathogens such as bacteria, viruses and food antigens.4 In the case of IgAproduction, IgAplasma cells primed at intestine migrate via the systemic circulation to secrete IgA in saliva and tears.11,12To understand the activity of the gut-associated lymphoid tissue, we measured the expression of antigen-specific IgA in saliva and observed a negative relationship between the expression of serum IgE and saliva IgA. Deficiency or poor expression of IgA in infants is related to the development of food allergy.12,14These results suggest that secretory IgA plays an important role in the prevention of food allergy. However, because of the small number of cases in our study, further study with a large number of patients is necessary to clarify the role of secretory IgA. We showed here that the MASTsystem was useful for determination of allergen-specific IgA as well as IgE. Studies of antigen-specific IgA expression in allergic patients using this system will contribute to the elucidation of the mechanism of allergy development.
OF ANTIBODIES
BY MAST
179
REFERENCES 1 Metcalfe DD. Food allergy. Curr. Opin. Immunol. 1991; 3: 881-6. 2 Plaut M, Zimmerman EM. Allergy and Mechanisms of Hypersensitivity. In: PaulWE, ed. FundamentalImmunology,3rd edn. NewYork:RavenPressLtd,1993; 1399-425. 3 BlorkstenB,AhlstedtS, BlorkstenF et al. ImmunoglobulinE and immunoglobulinG4 antibodies to cow's milk in children with cow's milkallergy.Allergy1983; 38: 119-24. 4 ShorterRG,TomasiTBJr.Gut immunemechanisms.Adv.Intern. Med. 1982; 27: 247-80. 5 WatanabeT,Nagura H, Watanabe K,BrownWR.The bindingof human milk lactoferrinto immunoglobulinA. FEBSLett. 1984; 168: 203-7. 6 YamadaK,AkiyoshiK,MurakamiH et al. Partialpurificationand characterizationof immunoglobulinproductionstimulatingfactor derivedfrom Namalwacells. In VitroCell. Dev.Biol.1989; 25: 243-7. 7 Yamada K,MatsumuraK, SuzakiM, ShirahataS, MurakamiH. Stimulationand inhibitionof interferon-Bproductionof human diploid fibroblastsby foodstuffs.Agric.Biol.Chem. 1991; 55: 829-32. 8 FritscheR, BonzonM. Determinationof cow milkformula allergenicityin the rat modelby in vitromast celltriggeringand in viva IgEinduction.Int.Arch.AllergyAppl.Immunol.1990; 93: 289-93. 9 SakaguchiM, Ogura H, InouyeS. IgEantibodyto gelatin in children withimmediate-typereactionsto measles and mumpsvaccines.J.AllergyClin.Immunol.1995; 96: 563-5. 10 WolfHM,FischerMB,PuhringerH,SamstagA,VogelE, EeiblMM. Human serum IgA downregulatesthe release of inflammatory cytokines(tumornecrosisfactor-a, interleukin-6)in human monocytes.Blood1994; 83: 1278-88. 11 Jackson DE,LallyET,Nakamura MC,MontgomeryPC. Migration of IgA-bearinglymphocytesinto salivaryglands. Cell. Immunol. 1981; 63: 203-9. 12 VisakorpiJK. The immune response of the intestinalmucosa to foreignproteins.ActaPaediatr.1982; Suppl.296: 56-9. 13 Sloper KS, Brook OGD, KingstonD, Pearson JR, Shiner M. Eczemaand atopyin earlychildhood:LowIgAplasma cellcounts inthe lelunalmucosa.Arch.Dis.Child.1981; 56: 939-42. 14 KaufmanHS,HobbsJR.Immunogobulindeficienciesin an atopic population.Lancet1970; 21: 1061-3.