Monoclonal antibodies to Lepidoglyphus destructor: delineation of crossreactivity between storage mites and house dust mites

Clinical and Experimental Allergy. 1992. Volume 22. pages 1032-1037

Monoclonal antibodies to Lepidoglyphus destructor.delineation of crossreactivity between storage mitesand house dust mites

B. HARFAST*, M. VAN HAGE-HAMSTEN*, I.J.ANSOTEGUI*t,E. JOHANSSON*, M. JEDDI-TEHRANIt and S.G.O. JOHANSSON*

Departments of*Clinical Immunology and f Immunology. Karolinska Institute, Karolinska Hospital. Stockholm,Sweden

Summary

We have developed monoclonal antibodies {MoAbs) to the storage mite Eepidoglyphusdestructor (Ld). Fmploying these anti-Ld MoAbs (Ld-MoAbs) in ELISA and HLISAinhibition techniques we have analysed the reaction pattern of Ld-MoAbs to both non-pyroglyphid and pyroglyphid mites. The storage mite Glycyphagus domesticus (Gd)exhibited most efficient inhibition, followed by Acarus siro (As). Tyrophagus putrescen-tiae (Tp), Dermatophagoules pteronyssinus (Dpt) and Euroglyphus maynei (Em). Of thetwo pyroglyphid species, Dpt showed at least 1000 times less inhibition than Gd. Two ofthe MoAbs immunoprecipitated a band of 39 kD whereas the third reacted weakly, witha high-molecular band of approximately 110 kD. The Ld extract was also subjected tovarious reagents and conditions and the antigen was heat stable, it was not aflected bylow pH, or sensitive to dimethyl sulphoxide (DMSO) or paraformaldehyde. Afterexposure ofthe extract to various reagents, such as protease trypsin and periodate. weconclude that the epitopes recognized by Ld-MoAbs were of carbohydrate rather thanof protein nature. It would thus seem that MoAbs recognize the carbohydrate part of aglycoprotein.

Clinical and Experimental Allergy, Vol. 22. pp. 1032-1037. Submitted 22 October 1991;revised 17 March 1992; accepted 24 April 1992.

In trod ucf ion

Mites are considered to bo one of the major causes ofallergies in many parts of the world. House dust mites,belonging to the genus Dermatophagoides. have beenextensively studied [I -4]. Two major allergens, a 25 kDagroup I and a 14 kDa group II, have been identified,cloned and sequenced [5 7]. For several years we haveconcentrated on the much less studied storage miteEepidoglyphus destructor iLd). These mites are predomin-antly found in storage barns in farming environmentswhere they feed on straw and grain dust. Farm workersexposed to barn dust are known to develop allergicreactions such as asthma, cough and rhinoconjunctivitis

Correspondence: Dr B. Hiirfiisl. Department of Clinical Immunology,Karolinska Hospilal, S-10401 Stockholm, Sweden.

1032

[8-13]. Among Swedish farmers, we found mites to be themost important allergen, by far exceeding the allergicsymptoms caused by pollen or anm-ial danders [14]. Wehave previously described the allergen of Ld using serafrom mite-allergic farmers and an immunobiotting tech-nique [15]. Further, MoAbs have recently been developedby us [16] and others [17] to identify allergens in Ld.

The relation between storage mites and house dustmites as to allergenic crossreactivity has been addressed inseveral studies, using RAST and RAST-inhibition tests[18-20]. Naturally, as each serum contains its specific setsof polyclonal IgE antibodies, the pattern of reactivity iscomplex. We thought that the use of MoAbs might addsome information as to the degree of crossreactivity todefined allergens between different mite species. In thepresent investigation we have used a set of MoAbs to Ldin an attempt to delineate crossreactivity between Ld andfive other mite species.

MoAbs to Lepidoglyphus destructor 1033

Material and methods

Mite extracts

Lepidoglyphus destructor (Ld). Acarus siro {A.s), Tyropha-gus putrescentiae (Tp), Glycyphagus domesticus (Gd).Dermatophagoides pteronyssinus (Dpt) and Euroglyphusmaynei (Em) were prepared from raw material obtainedfrom Allergon Ab (Angelholm, Sweden). Extracts wereprepared in 015 M NaCl as 1/20 (w/v) for 18 hr at roomtemperature.Theextracts were then centrifuged at f>600^for 10 mm and filtered through a 0'8 ^m Miliex-HA filter(Millipore, Bedbord, U.S.A.). The protein concentrationsof these extract were determined to be around 2 mg'ml.For some purposes, extracts were desalted on a PDIOSephadex column (Pharmacia, Uppsala. Sweden) andsubsequently lyophilized. All extracts were stored at— 20 C until used.

Monoclonal antibodies

BALB/c mice, aged 6-8 weeks, were immunized threetimes intraperitoneally with Ld extract. For the firstinjection, Ld was mixed with Freund's complete adjuvantwhereas in the following two. Freund's incomplete adju-vant was used. The injections were given fortnightly.Hybridomas were produced according to standard pro-cedures [2!]. Briefly, 3 days after the final injection themice were killed, spleen cells were mixed with SP2/0myeloma cells and fusion was performed in a suspensionof 50"'o polethylene glycoi 4000 (gas chromatographypure, Merc, Darmstadt, Germany). Cells were subse-quently resuspended in a selective medium containinghypoxanthine. aminopterinc, thymidme and seeded tomicrotitrc plates containing peritoneal macrophages asfeeder cells.

For assessment of antibody activity, culture super-natants were tested against Ld extracts using an enzyme-linked immunosorbent assay (ELISA] [22]. These assayswere performed in microtitre plates (Nunc, Roskilde,Denmark). Briefly, wells were coated with 200 fi\ of asolution oi Ld extract (1 /fg/m!) in 005 M carbonatebulVer, pH 9-6, overnight at room temperature. Phos-phate-bufTered saline (PBS) containing 0 2"o bovinescrum albumin andO-l'^Twecn 20 was used as a diluent.PBS containing and OO5'V,. Tween 20 was used forwashing throughout. First, the culture supernatants to betested were diluted, added to wells and incubated for 4 hr.Next, 100 fi\ alkaline-phosphatase-conjugated rabbitanti-mouse immunoglobulin (Dakopatts A/S, Copenha-gen, Denmark) diluted I/lOO was added and incubatedovernight. Finaily, the substrate/'-nitrophenyl phospha-tase disodium (Sigma, St Louis. Missouri, U.S.A.) wasdiluted in I M diethanolaminc hufier, pH 9 8, and added.

The absorbance at 405 nm was measured 30-60 min laterwith an ELISA plate reader. Clones displaying antibodyactivity were further cultivated, cloned and retested inELISA against several other mile extracts. MoAbs fromselected clones were subsequently isotyped and subclassdetermined using ELISA (and reagents from SouthernBiotechnology Associates, Birmingham. Alabama,U.S.A.). In thisstudy we selected three MoAbs for furtheranalyses. One was of IgG3 subclass (42B6) and two wereIgM (83F3 and 814C7). MoAbs of IgG isotype were insome instances purified from cell culture supernatantusing protein G-Sepharose according to the instructionsof the manufacturer (Pharmacia, Uppsala, Sweden).Unless otherwise stated, cell culture supernatants wereused as the source of MoAbs.

Treatment of Ed extraet

In an attempt to further characterize the nature of antigenrecognized by the MoAbs, we treated the Ld extract invarious ways. ELISA plates were coated with Ld asdescribed above and were subsequently subjected to oneof the treatments listed below. However, boiling of Ldextract was performed prior to coating oi the ELISAplates. The iV")liowmg treatments oi Ld extracts wereperformed. 90"n dimethyl sulphoxide {DMSO), 80%methanol, 4"/n paraformaldehyde, 1 "/o glutaraldehyde,3".. 2-mercaptoethanol (2MF), 2",, sodium dodecylsul-phate (SDSl,curic buffer. pH3 (these were all treated for 1hrat room temperature); boiling (5-30 min), 0' 1 M sodiummetaperiodate (periodate), for 3 days at 4 C in the dark;0-5% trypsin 37 C, protease 250 /ig ml 37 C. A^-GIycosi-dase F 18 units/ml (Boehringer, Mannheim. Germany),O- Glycosidase 20 units/ml (Boehringer). The enzymetreatments were all performed at 37 C for 18 hr. To testfor the effects of these treatments, the ELISA activity ofMoAb 42B6 lo treated and untreated antigen wascompared.

EAJSA inhihitioi] assay

To sUidy the specificity oi the Ld-MoAbs and to assessthe distribution of the antigen detected, we performedinhibition experiments. Ld-MoAbs diluted 1:200 werepreincubated with six different mile extracts. In eachexperiment 12 different concentrations of each extractwere tested. Starting with a 1:30 dilution, each extractwas successively diluted using three-fold steps. Followinga 2 hr incubation, the mixtures were transferred tomicrotitre plates coated with Ld and allowed to incubatefor an additional 3 hr. followed by over-night incubationwith rabbit anti-mouse immunoglobulin conjugate. Thisapproach allowed us to determine the dilution oi eachextract which gave a 50",. inhibition.

1034 B. Harfasieta\.

2-5

2-0

Q I -5

1-0

0 - 010 100 1000 10

Dilution of mile ex-iracis

Fig. 1. The ability of six different mite extracts to inhibit thereaetion oi MoAb 42B6 to Ld in ELISA. • . As. .A,Tp. v. Em.D. Dpt. O. Ld. • . Gd.

Radiolabelling o/Lepidoglyphus destructor

One milligram of Ld extract was iodinated with 1 mCi ofsodium '"iodine, using the commercial solid phaseradioiodination reagent l,3,4,6-tetrachloro-3a. 6a-diphe-nyl glycouril (Iodo-Gen) (Pierce Chemical Company,Rockford, U.S.A.) according to the manufacturer'sinstruction.

Immunoprecipitation and sodiutn dodecylsulphate poly-acrylaniide gel electrophoresis (SDS P.4GEI

The anti-Ld MoAbs and immunoprecipitation controlMoAb (1B3, a MoAb to p56'̂ M [-3] were incubated with100 /(I of protein-A Sepharose coated with goat anti-mouse immunoglobulins. After 1 hr, the antibody-boundprotein A-Sepharose beads were washed three times withPBS. Samples were then incubated for I hr undercontinuous mixing with 10 x 10'' c.p.m. of preabsorbedradiolabelled Ld extract. The immunocomplex-boundprotein A-Sepharosc beads were washed six times withPBS, and then 60 fi\ of sample buffer was added to thesebeads. After boiling for 3 min, the protein-containingsample buffers were analysed using 10% SDS-PAGE.Pre-stained BioRad (BioRad, Richmond. California.U.S.A.) molecular weight markers were used as SDS-PAGH standards. Gels were dried and autoradiographedon Amersham film with Agfa-60 intensifying screens(Agfa-Gevaert, Germany) at — 70'C.

Results

Specificity

We selected three MoAbs 42B6, 82F3. 814C7 for athorough investigation of their reactions with Ld and five

other mite extracts. Tn a set of inhibition experiments thepattern of crossreactivity was investigated (Figs 1 -3). Inthis assay, MoAbs 42B6, 82F3 and 814C7 were preincu-bated with serial dilutions of the panel of mite extracts.After preincubation, the antibodies were assessed againstLd in ELISA. The four storage mite extracts exhibitedvery strong inhibition. The most effective inhibitorystorage mite extracts were, in order of efficiency; Gd, Ld,As, Tp. The two house dust mite species were ineffectiveby comparison (Figs i-3). The six mite species may bedivided into three groups according to their inhibitorycapabilities (Table 1). The two Glycyphagidae specieswere more efficient than the tw o Acaridae species which inturn were more efficient than the two Pyroglyphidaespecies. For inhibition, 1 f.tgim\ ofthe extracts were used.The pattern of inhibition was similar whichever of theMoAbs was used.

Resistance oj Ed extract

To characterize further the antigen recognized by theMoAb. extracts were exposed to various treatments(Table 2). These antigen samples were subsequently usedas coating antigens in ELISA and compared withuntreated antigens. The epitope recognized by MoAb42B6 was very resistant to protease, trypsin. boiling (5min), SDS, glutaraldehyde. paraformaldehyde, DMSO.methanol, low pH (pH 3) and 2ME. However, periodatetreatment and boiling for 30 min reduced the ELISAresponse considerably. Also, treatment wth ,V-glycosi-dase F reduced the response by 29% whereas 0-Glycosi-dase was ineffective.

To check the effect of some of these treatments we alsoused another MoAb we recently raised against Ld. ThisMoAb is directed to the 15 kD band as analysed inimmunoblotting. We now used MoAb 12D8 as a controlto ensure that periodate oxidation would not generallydestroy protein structures oi Ld under the conditionsused. As can be seen in Table 3, MoAb 12D8 displayedreduced response when tested against protease-treatedextract but was not influenced by periodate oxidation oiLd. In contrast, 42B6 had the reversed pattern oisensitivity for these treatments of Ld.

[nwnunopreeipitation

Confirming our previous results [16]. we found that 42B6precipitated a band of 39 kD. Here MoAb 814C7 alsoreacted with the 39 kD component. In contrast, MoAb82F3 did not react with 39 kD component. Instead itprecipitated a faint high-molecular band of approxima-tely 110 kD. Although this faint band was repeatedlyfound its significance remains to be proved. Despite this,82F3 displayed high ELtSA activity.

MoAbs to Lepidogiyphus destructor 1035

2-5r-

2-0 -

0-5 -

0-0100 1000

Dilulion of mile exirocis

10" 10=

Fig. 2. The ability of six difTerent mite extracts to inhibit thereaction of MoAh X2F3 to Ld m ELISA. • , As. A.Tp. v, Em.D, Dpt. O. Ld. • , Gd.

0-5 -

0-010 100

Dilution of miie enirocis

Fig. 3. The ability of six difTerent mite extracts to inhibit thereaction of MoAb 8I4C7 to Ld in ELISA. • , As. A.Tp. v, Em.D. Dpi. O, Ld. • , G d .

Table I. Comparison oi Vhe percentage inhibition oblaineiJ bvdifl̂ erent mite .species on the ELISA response to L, destructor

MoAbs42B6814C782F3

% Inhibition

Glycyphagidae

Ld809096

Gd809596

by mite extracts

Acaridae

As386281

Tp20314i

(1 fig/TTll)

Pyroglyphidae

Dpt0

152^>

Em410

Table 2. Influence of treatments of Eepidoglyphus destructorextract on the ELISA reaction of MoAb 42B6

Exposure to Diminished ELISA response

Methano!ParaformaldehydeGlutaraldehydeSDSDMSOMercaploelhanolProteaseTrypsinPeriodateA'-Glyeosidase F0-GlycosidasepH 3Boiling 5 minBoiling 30 min

9000

i2704

8729

04

1084

Periodate

760

Treatments

Protease

6SI

Table 3. EPTecl of treatment of L. destructor extract withperiodate or protease on the ELiSA results using MoAb 42B6directed to 39 kD and MoAb I2D8 directed to 15 kD

% Diminished ELISA response

MoAbs

42B6I2D8

Discussion

In the present study we describe a set of MoAbs raisedagainst the storage mite Ld. These MoAbs wore used astools to investigate the relationship between Ld and fiveother mite species. In a number of previous investigationswe [10.19.24,25] as well as other investigators [4.18,20]studied the clinical significance of allergenic crossreacti-vity between mites of various species. These experimentswere performed using sera of mite-allergic patients. Thespecificity and pattern ofreaction oflgE in these sera wereassessed by means of RAST and RAST-inhibitionstudies. Although the results are not idenlical, a patternemerged of a close relationship, within the storage mitesand within house dust mites, respectively. Also, eachspecies studied seemed to possess unique allergens inaddition to allergens shared by closely related species. In

1036 B. Harfasteta\.

contrast, the crossreactivity belween the two groups waslimited, but not absent. For example, using an immuno-blot technique we have recently shown that Ld sharessome antigens with Dpt [26]. Such crossreactivity has alsobeen suggested by others [18,20,27]. The present experi-ments using Ld-MoAbs are in line with the main findingthat a closer taxonomical relationship infers more cross-reactivity. Thus the two house dust mite species displayedpoor inhibition of the anti-Ld MoAbs, whereas within'the storage-mite group", species belonging to the familyof Glycyphagidae (Gd and Ld) were more efficient thanspecies ofthe Acaridae family (As and Tp) in this respect.These results agree with our previous RAST inhibitiondata except for the reposition of Tp and As, i.e. Tpcrossreacts more than As with Ld in experiments withsera from mite-allergic patients [24].

In agreement with previous characterization [16], wefound that Ld-MoAbs recognized an antigen of 39 kD asdetermined by immunoprecipitation. In addition, wefound a MoAb (82F3) that recognized a weak band ofapproximately 110 kD. This antibody seems to crossreactsomewhat more strongly with Dpt than the other two Ld-MoAbs do. Whereas the 39 kD has been shown to be anallergen [16] this remains to be determined for thecomponent recognized by 82F3, A series of experimentson the antigen recognized by 42B6 indicated that this wasextremely resistant to a variety of treatments. Its resis-tance to trypsin and protease seems to suggest that theepitope recognized is not of protein nature but rather ofcarbohydrate. This is further supported by the sensitivityof Ld extract to peroxidate oxidation prior to ELTSAanalysed by 42B6. As control, in these experiments, weused a recently raised MoAb (12D8) directed to the 15 kDof Ld. In contrast to 42B6, the FLISA reaction was notinffuenced by periodate oxidation of Ld, whereas pro-tease treatment decreased ELTSA reactivity. This experi-ment implies that periodate oxidation does not generallyunder these conditions destroy the proteins of Ld.Moreover, treatment oi Ld by A'-Glyeosidase F, anenzyme which will cleave intact oligosaccharides fromglycoproteins. partly reduced the response of 42B6. Thereduction was far from complete. However, according tothe manufacturer's (Boehringer) information, completedigestion is not to be expected. It would thus seem that42B6 recognizes the carbohydrate part of glycoproteinsof 39 kD. Alternatively, the antigens could be purecarbohydrates of the same size. The latter possibilityseems, however, to be excluded as the radiolabellingtechnique we used labels proteins only. Consequently.only antigens of protein nature could be visualized byimmunoprecipitation. However, these antigens seem tobe exclusively or preferentially expressed in ihe storagemite species.

Interestingly, in RAST-inhibition studies with Derma-tophagoids farinae. TgE bmding to carbohydrate struc-tures has been demonstrated [28]. In preliminary experi-ments using MoAb 42B6 and patient sera in aradioimmunoassay, the results show that native 39 kD isrequired for detection by patients" IgE. This finding iscompatible with the observation that the 39 kD band isnot detected in SDS-PAGE and immunoblotting. as bothsuggest that this protein is sensitive to denaturatingconditions. In contrast. 42B6 reacts with a very stablestructure on the same 39 kD allergen.

Acknowledgments

We thank Susanna Olsson for help in the development ofMoAb 12D8. Financial support for this work wasreceived from the Swedish Work Environment Fund (83-1182, 82-0881), the Swedish Medical Research Council(grant 16X-105), the Tore Nilsson Foundation, the EllenWaltes and Lennart Hesselman Foundation, the Petrusand Augusta Hedlund Foundation, the Swedish Societyoi Medicine, the Margit and Folke Pehrzon Foundationand the National Association for Prevention of Asthmaand Allergy (RmA).

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