Scribd Logo
Importer

Bienvenue sur Scribd !

  • Immunochem 1976

    Transféré par

    petergw1
    18 vues7 pages
    Willadsen P and Williams P (1976). Isolation and Characterisation of an Antigen from the Cattle Tick, Boophilus microplus. Immunochem 13:591-597

    Copyright

    © © All Rights Reserved

    Formats disponibles

    PDF ou lisez en ligne sur Scribd

    Avez-vous trouvé ce document utile ?

    Ce contenu est-il inapproprié ?

    Signaler ce document
    Willadsen P and Williams P (1976). Isolation and Characterisation of an Antigen from the Cattle Tick, Boophilus microplus. Immunochem 13:591-597

    Droits d'auteur :

    © All Rights Reserved
    Téléchargez comme PDF ou lisez en ligne sur Scribd
    Signaler comme contenu inapproprié
    18 vues7 pages

    Immunochem 1976

    Transféré par

    petergw1
    Willadsen P and Williams P (1976). Isolation and Characterisation of an Antigen from the Cattle Tick, Boophilus microplus. Immunochem 13:591-597

    Droits d'auteur :

    © All Rights Reserved
    Téléchargez comme PDF ou lisez en ligne sur Scribd
    Signaler comme contenu inapproprié
    sur 7
    ISOLATION AND PARTIAL CHARACTERIZATION OF AN ANTIGEN FROM THE CATTLE TICK, BOOPHILUS MICROPLUS P, WILLADSEN and P. G, WILLIAMS CSIRO, Divison of Animal Health, Long Pocket Laboratories, Private Bag No. 3, P.O. TIndooroopily, Queensland 4068, Ausiraia (First reece 11 November 1975: Abstract—The isolation is reported of a component ‘revised form 16 January 1976) Of B. micropls larvae which causes an immediate ‘Npersensiity reaction in eae which have ben exposed to the tick, It has teen purified and shown fo be a protein with a mol wt of approx 6.000. Evidence i als given that the component is an thtcrase whowe inreversile inhibition by the orgasophosphate reagent, DFP, shows the presence of {serine reside essential for enzymatic activity INTRODUCTION ‘At the present time, the immunology of « number fof host_parasite relationships is being increasingly in ‘vestigated. The association between the cattle tick and its host has, however, despite its interesting features, received litle attention. Although cate develop an ‘mmunologically-mediated resistance to infestation by the cattle fick, BL microplus (Roberts, 19684) the nature ofthe antigens and the types of immunological esponse involved are unknown. The resistance ‘mechanism is likely to be complex. Complete im- ‘munity to infestation is rarely achieved and cattle dis- play a range of fairly stable resistance levels (Roberts, 1968a), The cellular responses in cattle skin to the attachment of larvae and their relationship to host resistance have been described (Sehleger et al, 1975) Further, it has been suggested that many of the events ‘observed histologically may be duc to the host's reac- tion to tick antigens (Tatebell, 1969). 1 seemed likely to us that skin reactions of cattle to tick components ‘would provide a suitable way of investigating those Antigens which the tick has injected into the host in the course of natural infestation. Riek (1956, 1962) has reported that animals which have been exposed to ticks show an immediate hypersensitivity t0 tick extracts which is small or absent in unexposed ani ‘mals and may increase in intensity with increasing exposure, Ill stages of the life-cycle of B. microplus could bbe sources of antigenic material but a major expres sion of resistance isthe rejection of larvae during the first 24 hr of the life cycle (Roberts, 1968) This indi- cites that the eritical antigens must be present at that ‘Stage. In this paper therefore, we report the isolation ‘of a component of H. microplus larvae which produces fan immediate hypersensitivity reaction in hosts whieh hhaye been naturally infected with ticks Tt is possible that in a parasitic infection a protein ‘which is isolated as an antigen could have additional ‘and possibly non-antigenic sites with some other phy- siolopical function. One ean only guess at possibilities for such a function, buta likely one is that of a hydro- lytic enzyme, We have therefore tried to see if the 91 antigen being purified had an identifable enzymatic activity, MATERIALS AND METHODS Souree of materials Fluorescsin dibutyrate was obtained from ICN Pharma- ceutical and trun lablled isopropyl phosphoroflur idate. DEP, 3.9G/mmole, from the Radiochemieal Contte, Amersham. Stach for gel electrophoresis, Lot 1é-. was from Connaught Medical Research Labora: tories and Cyanogum-4l from BDH. The DEAE-cellulose wed was a mediam mesh product Wom Sigma and the Sephadex G-200 was of normal grade. B, microplus larvae trove obtained as desribed by Roberts (1971) and frozen Mt =20°C, 7-14 days after hatching. Immediate hyperseniety est ‘The animals used were Bos taurus eatle which had received substantial natural exposure to ticks. Materials {o be tested were dilated in phosphate-buflered saline con taining | mgim bovine serum albumin and 0. ml was i= jected intradermally, The oedematous reaction reached its ‘maximum size between 20 and 30min after injetion and Gi not alter in size for another 30min. The diameter was measured in two dictions at right angles and the product ‘Of these two measurements taken as tbe result. Tt was found. that. within the seul range of response (a 10-30 mm dia swelling the iz of the reaction was pro- portional to the logarithm of the amount of material in- [Feted, During porifeation procedures, a standard line was ‘bianed from dilutions of the initial crude extract. Using this, the concentration of antigen in any fraction was felated to that in the erude extract, and expressed as the ‘umber of of crude extract which would give the same Sized reaction as one pl of the fraction. The response of Sifrent animals varied and the standardization with crude Shtigen was repeated with each animal and usually each Seri of determinations. Tests for non-apecfic activity were flone in the same way, using catle which had not been exposed to teks Esterase assay Enzyme activity was measured in 005 M phosphate bufier pH 75 containing LmM 2-mereaptoethanol and (05 mM EDTA (EDTA and meteaptocthanol were added 92 routinely to all burs at these concentrations and hence- forth in this report, buffers will be identified only by th bulleting component, Fluorescein dibutyrate was used substrate ta fnalconeentration of 81> 10° ML An ali- {quot of the solation to be assayed was added t0 3 mi of the substrate solution and incubated at 37°C. Incubation times were 6 12 oF O mia, depending on the activity of the sample, The reaction was terminated by addition of Tim 10%, TCA. the solution centrifuged if here was a sig- nificant precipitate and the fluorescence was measured in {1 Petkin-Elmer Model 203, Fluoresence Spectrophot- tometer using excilaion and emission wavelengths of 440 fand $15:nm respectively. Not more than 02%, ofthe sub SMrate was hydrolyzed in any assay. The ‘Muorescence change was proportional to the duration of the incubation fnd the amount of enzyme added "To convert the enzyme activity from the arbitrary Auor scence units info the normal units of moles of substrate Uydrolyzed per min st was necessary to prepare a fuores- ‘sence standard. For this 40989 4M solution of uorescein Sibutyrate in the assay bufer was hydrolyzed enzymically. tint absence of a change on addition of more exzyme br prolongation ofthe reaction time showed the hydrolysis to be complete, The fluorescence of this solution, when nessuredl ae described for the normal assay procedure, was taken asthe standard Gel electrophoresis Starch gel electrophorcses were run in a 122% gel, 22em Jong. set ds «thin layer in an LKB21I7 Multiphor appar- aus. Small wells were cut in the gel. $0 samples were pipetted in and, afer electrophoresis for She at 40D V. the fel vas cut into Smm sections These were then eluted ‘overnight into |S ml electrophoresis bulk prior to testing for antigenic and esterase activities ‘Polyacrylamide gels were prepared from 7% Cyanogum in 0PM Tris 004M glycine buffer. and set as Sem ts in narrow bore glass tubes. After pre-electrophoresis for 3hr at 400V in the same buller, to which had been ‘added 1 mMf mercaptoethanol, samples were loaded and fun for 2hr at 400. The ges were stained for protein P. WILLADSEN and P. G. WILLIAMS ‘with Coomassie blue (Chrambach etal, 1967). To localize Tadioactive components, the gels were cut into 2mm se tions which were solubilized in 05 ml 30% hydrogen pes bxide(Tisler & Epstein, 1968) and counted, afer addition of 6m) Packard Instigl, in a Packard TrisCarb liquid Scintillation counter Protein determinations ‘The mcthod of Warburg & Christan (1941) and Kalckar (1947) was used. RESULTS Purification of antigenic activity Tick larvae were homogenized, with cooling, in a Braun Cell Homogenizer. MSK for 2min, then ‘extracted for 30min in 0.05 M phosphate bulfer pH 75 using 25 ml buffer/s of larvae. This, and all sub- sequent steps of the purification, were carried out at °C. The extract was then centrifuged for 30min at 10.000 g followed by a further centrifugation for 30min at 25.000. The supernatant was filtered and the filtrate centeifuged once more at 25000 for 30 min, As a preliminary experiment intended to show ‘the number of antigens present, 2 ml of the superna tant was chromatographed in 0.10M- phosphate buller pH 7.5 on a 25 x 62cm column of Sephadex ‘G-200 and the fractions were tested for antigenie and esterase activity. The results are shown in Fig. 1. The Figure shows the results when 2.8 void volumes had been collecteg, representing molecules with apparent mol, wts of about 20,000 or larger. There were no additional peaks of antigenic activity in the lower ‘mol. svt range, The breadth ofthe peak of hypersensit- ivity activity strongly suggests that more than one antigen is involved, The work to be described con- ‘ems the purification of the material shown at the Fig. 1 Chromatography of crude extract on G-200, Experimental points for antigen concentration ‘are shown as dots Antigenic Material from B. microplus Larvae 93 os F = E Paes | ie ea ee Toe olen bl. 3 gCie ee See ee Ge CES Fig. 2. Chromatography of salt-factionaed extract on DEAE-cellulose, Experimental points for antigen Cuncentrauon as in Fig. 1 The preliminary washing of the column with starting buffer bas been ‘omitted from the diagram leading edge in this chromatography and the co-puri- fication of an esterase activity "A typical purification will be described. The super- natant from the extraction of 100g larvae, prepared fas before, was fractionated between 40% and $: saturation ammonium sulphate at pH 7.5. The ipitate was dissolved in. approx. 40ml of 005 M Phosphate buffer pH 75 and dialyzed overnight ‘against two changes of a X0old excess of 0415 M Tris buffer pH &7. Approximately half of the protein pre Cipitated during dialysis. After centrifugation, the Supernatant was loaded on a 2.5 x 27em column of DEAE-cellulose equilibrated in the same Tris bul. ‘After washing with 230 mi of buffer. the protein con- centration of the eluate fell to 0.1 mg/ml and a 11 Beetee se VOLUME (mi) 3+Logjg ANTIGEN CONCENTRATION linear gradient 0-03 M_ in. sodium chloride was applied. The results in Fig. 2 show that one of the two major antigenic components co-chromato- trraphed with the esterase activity. This material was pooled and brought to 60% ammonium. sulphate Saturation at pH 8.2, centrifuged and the precipitate Gissolved in, and dialyzed for 3hr against, 0.1 M phosphate buffer pH 75. The product, in 2.7 ml solu tion, was then chromatographed on a 2.6 x 62em ‘column of Sephadex G-200 in phosphate buffer. The result, Fig. shows that the antigen and esterase ac- tivities also’ co-chromatograph on the Sephadex column. The active material was finally concentrated fon an Amicon UM-10 membrane. The purification procedure is summarized in Table 1 gq a PROTEIN CONCENTRATION (mg/ml-—+—) 0 aio. Fig. 3. Chromatogeaphy of the prosuct of ion-exchange chromatography on G-200. Experimental points for antigen concentration asin Fig P. WILLADSEN and P. G. WILLIAMS ‘Table 1, Purification of larval antigen and esterase activities Parification Purification Spact Esterase factor of Antigen factor of Fraction Total protein esterase x 10% yield) esterase (Ye yield) antigen Exact 770mg 0036 100 1 100 7 40-55%, Satd (NH),804, before dialysis 1900 oon 2 21 8 2 40-55%, Satd (NH,S0., afer Aialgss 960 aus 2 42 sa 5 DEAE chromatography product 4 oss 2 239 4 ° Sephadex chromatography 41 ote ° 13 2 © Starch gel electrophoresis of the product of this preparation was carried out using two bullers-a 0.05 M Tris buller pH 860 and an 002M HEPES buffer pH 7.31, After electrophoresis and elution, the samples were tested for antigenic and estorase activity. The results are shown in Fig. 4 where, for case of comparison, the antigen concentrations are shown on, ‘linear scale and not a logarithmic one as in previous ‘diagrams. In this experiment, antigen concentrations ‘were standardized using dilutions of the material applied to the gel "The purified antigen produced a dermal reaction ‘only in animals which had been exposed to ticks, In- jection of 100 times as much as the maximum amount This butter pH 8.6 ~ t ‘Origin ‘normally used in antigen quantitation into an unex posed animal produced no significant swelling. Determination of apparent mol. wt of the esterase ‘The apparent mol. wt of the esterase was deter- mined from its elution volume in gel filtration on Sephadex G-200, under the conditions used in the final step of the purification. Ovalbumin, bovine scrum albumin, rabbit muscle lactate dehydrogenase and yeast alechol dehydrogenase were used a stat dards with the mol, wls assigned to them by Andrews (1970), The valve thus obtained for the esterase was 60,000, The chromatographic behaviour of the ‘enzyme was not significantly dependent on its concen Beck [ENZYME ACTIVITY PER FRACTION (munits) & MIGRATION DISTANCE (em) Fig. 4 Electrophoresis of purified antigen on starch gol The shaded azea shows the esterase activity, the thick Tine the antigen activity Antigenic Material from B. microplus Larwe COUNTS PER MINUTE x10~° o 3 a ° Oem1 5 6 395 8 woo 12:13 14 15 Fig. 5. Electrophoresis of tritunlablled esterase. The gel is stained for protein and the bars show dlstbution of ttism. tration, within the range of concentrations used. Dur- ‘ng the final step of the purification, where total ‘of 4mg esterase was present in the sample loaded, the elution volume was 190 void volumes. Rechroma- tography of 20 ug gave & ratio of 191 Inhibition of esterase activity Despite the fact that meveaptoethanol was necess- ary for the stability of the esterase, particularly of purified material, the esterase was not sensitive to the rormal sulphydtyl reagents. Incubation at 37°C in a Tris buffer pH 4,6 with 0.7mM iodoacetamide for hr, 25m iodoncetic acid for 40min or 09.mM p-mereuribensoate for 30 min failed fo cause any inhi bition. For these experiments, although the EDTA concentration was maintained at 0.5 mM, the concen- tration of mercaptoethanol was 0. mM or less. $0 aS not to interfere with possible enzyme inhibition, However, the esterase was completely and irrevers- bly inhibited by DEP in a very rapid reaction. The effect of DEP on esterase activity was measured by ‘adding an aliquot of DFP in ethanol to a solution fof enzyme in 0.05 M phosphate buffer at 37°C, then ‘withdrawing 200g samples at 10-15 sec intervals and. fadding these directly to an assay mixture, the assay reaction being stopped after 10min, For a second ‘order inhibition reaction, this dilution will result in ‘a decrease in the reaction rate of more than 2S0-fold and henee the duration of the inhibition was taken fas the time up to dilution of the 200 ul samples. The inhibition was difficult to follow accurately because ‘of the low levels of activity which had to be used. [At the lowest concentration of DFP, 1.8 x 10-°M ‘and with an enzyme concentration of 1.9 yg/m. the foss of activity still appeared to be first order or almost two half-lives. Further dilution of the DFP Jed to incomplete inhibition, presumably because the concentration of enzyme became greater than that of the inhibitor. Using DFP conesatrations in the range 18-52 x 10-* M, it was found that the reaction ‘was first order in DFP concentration, giving an ap- proximate sovond order rate constant for the reaction OF LT x 107A min? (range 1.5-22 x 10") Antigenic activity of DEP-inhibited esterase Inhibited esterase was prepared by reacting dupli- cate samples of the enzyme In phosphate buffer with O11 mM DFP at 37°C for 10min, After this time, there Was no residual enzymic activity. Overnight di alysis was carried out to remove DFP, without any ‘activity being recovered in the inhibited esterase. A fontrol simple which was treated identically except for the addition of DFP remained fully active. Tests for antigenic activity showed no measurable difference between inhibited and uninhibited esterase samples. Electrophoresis of tritiumlabelled esterase Purified antigen, 18 x 10-> units, in O8 ml 0.10 M phosphate buffer pH 7.5 was mixed with 101 of [H]DEP at 37°C. After 4 min, the enzyme was more than 99%, inhibited. ARer 7.5 min, the solution was dialysed against cold buffer. Overnight dialysis giving ‘total dilution factor of more than 10* was castied ‘out without recovery of enzyme activity. A 108 ul ali {quot of the product was run on polyacrylamide gel electrophoresis, stained for protein and then sectioned for counting of radioactivity. The correlation between protein bands and the radioactive label is shown in Fig. 5. Four bands of protein are visible The two bands botween 44 and 52cm were barely separated ‘and contain almost all of both the protein and tritium latch They ae presumably very similar forms of the ‘Sime enzyie. Two minor protein components are vis- ible. One, at 3.2m, is also tritium labelled and can teasonably be attributed to the small amount of slowly-migrating esterase activity observed on starch ‘gel electrophoresis (Fig. 4) This component was not fan antigen. It is uncertain ftom Fig. $ whether or ‘not the fourth component, at 5Sem, is radioactive $96 DIscUssioN The initial objective of this work was 10 puri, from tick larvac, components which were potentially relevent to the immunologically-based resistance of cattle to the tick. The isolation and characterization Of antigenic material which bas been described depends on the accuracy with which antigen eoncen- trations can be determined from the immediate hyper- sensitivity feaction. This accuracy is limited by two factors Firsly, any single estimate may be in error by a factor of two. That i, the deviation of points from the regression line obtained in standardizing an antigen-contaiing solution is commonly 03 log units on the concentration scale. Secondly, although itis necessary to estimate the antigen content of diferent fractions during a purification, this ean only be done rigorously if the dose response lines forall antigen fractions are parallel, This is not normally the case but the deviation from this situation varies from ani- mal to animal. The results which have been given for antigen purification were obtained with animals in which all actions dic give parallel lines, atleast Within experimental error. The results thos give a Semi-quantitative idea of the progress of purification, tht-do- not imply that identical figures would. be biained in all animals. Despite the limitations of the skin test, it has been show that one of the major antigens giving an imme- diate hypersensitivity reaction in exposed animals can be succesfully puried. At the maximum concen- tration used in skin testing, there was no detectable ‘clayed reaction. Moreover, tests for a reaction 10 the antigen with unexposed cattle were negative,

    Vous aimerez peut-être aussi