Use of the Enzyme-Linked Immunosorbent Assay (ELISA)

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mr. 1977, p Copyright C 1977 Americn Society for Microbiology Vol. 5, No. 3 Printed in U.S.A. Use of the Enzyme-Linked Immunosorbent Assy (ELISA) nd Its Microdpttion for the Serodignosis of Toxoplsmosis KENNETH W. WALLS,* SANDRA L. BULLOCK, AND DONNA K. ENGLISH Prsitic Serology Brnch, Center for Disese Control, Atlnt, Georgi Received for publiction 30 September 1976 The enzyme-linked immunosorbent ssy (ELISA) hs proved to be sensitive nd specific quntittive procedure for the serodignosis of toxoplsmosis. Using the toxoplsm model, severl prmeters of the test were investigted. Dy-to-dy reproducibility ws 90% within one twofold dilution nd 98% specific when tested ginst btteries of ser from other diseses. Both the tube method nd the microtitrtion method were used successfully. ELISA results re equivlent to those found in the indirect immunofluorescence test, yet the ELISA procedure is simpler nd more rpid to perform. A number of serologicl tests hve been used for the detection of ntibodies to Toxoplsm gondii. The enzyme-linked immunosorbent ssy (ELISA) ppers to offer combintion of the best qulities of ll. Described first by Engvll nd Perlmnn (4), ELISA is modifiction of the rdioimmunosorbent technique (RIST), in which n enzyme is substituted for the rdiolbel of the ntiserum. In the short time since its inception, the test hs been pplied to the detection of number of metbolites (7, 12) nd bcteril (3, 5) nd prsitic diseses (2, 9-11, 15). Ruitenberg et l. (9-11) hve published numerous rticles on the use of ELISA in trichinosis, nd Voller nd others hve described its use in mebisis (1), schistosomisis (6), mlri (7), nd Chgs' disese (4). Recently, Voller et l. (13) described qulittive ELISA for toxoplsmosis. Although originlly described s single tube, photometriclly quntitted procedure, ELISA ws redily dpted to microtitrtion nd utomtion. In semiutomted system, Ruitenberg et l. (11) demonstrted tht on routine bsis s mny s 4,000 ser could be tested dily. Within the limits of the dsorbtive qulities of the polystyrene sorbent, ny soluble ntigen pprently cn be incorported into the test. Consequently, it seemed logicl tht soluble ntigen prepred from disrupted whole T. gondii orgnisms would contin both the cell wll ntigen (the ctive ntigen in the indirect immunofluorescence test [IIF] nd the methylene blue dye test) nd the cytoplsmic ntigen (which is ctive in the pssive hemgglutintion test). ELISA with soluble ntigen should 273 give rections chrcteristic of ll the former procedures. We describe here n ELISA procedure for toxoplsmosis in which n ntigen derived from solubilized whole orgnisms is used. Both the tube nd microtitrtion procedures hve been successfully used. MATERIALS AND METHODS Antigen. Tchyzoites of the RH strin of T. gondii were hrvested from mice infected 3 dys previously. The peritonel fluid ws withdrwn nd mixed with t lest 10 volumes of 0.5% formlinized phosphte-buffered sline (PBS), ph 7.2. After remining for 1 h t room temperture, the cells were collected by centrifugtion nd wshed three times with PBS. After the finl wsh, the pcked cells were resuspended to 1% concentrtion (vol/vol) in distilled wter nd disrupted in Ribi cell frctiontor t 20,000 lb/in2 t 7 C. After the mixture hd settled overnight, gross prticles were removed by centrifugtion t 2,000 x g for 30 min. The superntnt ws collected nd extrcted with n equl volume of trifluorotrichloroethne (Genitron 113, Allied Chemicl Corp., Morristown, N.J.). The resulting cler ntigen ws stored s 1-ml liqunts in the vpor phse of liquid nitrogen storge box. Conjugte. Horserdish peroxidse conjugted to got nti-humn immunoglobulin G-Fb ws prepred by the method of Kwoi nd Nkne (Fed. Proc. 32:840, 1973) modified by Ruitenberg et l. (9). The enzyme preprtion used ws horserdish peroxidse type VI (Sigm Chemicl Co., St. Louis, Mo.), with n RZ ctivity of A 10-mg mount of horserdish peroxidse ws dissolved in 2 ml of 0.3 M sodium bicrbonte, ph 8.1. Two-tenths milliliter of 1% (vol/vol) 1-fluoro-2,4-dinitrobenzene (Estmn Kodk Co., Rochester, N.Y.) in bsolute ethnol ws dded, nd the preprtion ws mixed for 1 h t room temperture. A 2-ml mount of queous 0.08 M

2 274 WALLS ET AL. sodium periodte ws dded nd mixed for 30 min t room temperture. Then, 2 ml of 0.16 M ethylene glycol ws dded nd mixed for 1 h. The resulting solution ws centrifuged t 900 x g for 10 min to remove the smill mount of precipitted impurities nd ws subsequently dilyzed t 4 C ginst four 1- liter chnges of 0.01 M crbonte buffer, ph 9.5. The dilyste ws mixed with 10 mg of nti-immunoglobulin for 3 h t room temperture nd then ws dilyzed ginst 0.15 M PBS, ph 7.2, t 4 C. The conjugted mteril ws seprted from the unconjugted protein nd enzyme by filtrtion on Sephdex G-200 column equilibrted with 0.15 M PBS contining 0.02% NN3. The peks tht showed opticl density ctivity t both 280 nm (for the protein) nd 403 nm (for the peroxidse) were collected nd concentrted to t lest 1 mg of protein per ml. The conjugte ws stored in the cold (4 C). Test ser. Test ser consisted of five pools of humn ser of known levels of rectivity in the IIF procedure for toxoplsmosis. Additionl ser used for testing sensitivity nd specificity were selected from ser submitted to the Center for Disese Control tht hd titers in the conventionl serologicl tests for the diseses suspected. Immunosorbent. Disposble polystyrene tubes (11 by 55 mm, Phrmci Lbortories, Inc., Pisctwy, N.J.) were filled with 1 ml of ntigen djusted to 5,ug of protein per ml in 0.1 M crbonte buffer, ph 9.6, contining 0.02% NN3. The tubes were incubted in 37 C wter bth for 3 h nd stored in the cold (4WC) until used. Substrte. The substrte ws prepred by dissolving 80 mg of 5-minoslicylic cid (Aldrich Chemicl Co., Inc., Milwukee, Wis.) in 100 ml of hot distilled wter (80 C). Immeditely before the solution ws used, the ph ws brought to 6.0 with 1 N NOH. To 9 prts of 5-minoslicylic cid solution, 1 prt of 0.05% (vol/vol) H2O2 ws dded. ELISA tube test. The method of Ruitenberg et l. (11) ws followed for the tube test, except for few minor modifictions. The dy before ssy, the immunosorbent tubes were emptied by suction, refilled with 2 ml of 1% (wt/vol) bovine serum lbumin in 0.1 M sodium crbonte buffer (ph 9.6) contining 0.02% NN3, nd incubted t 4 C overnight. To begin the test, the tubes were wshed with distilled wter three times for 5 min ech. Then, 1 ml of ech dilution of test serum in PBS ws dded to ech tube, nd the tubes were incubted t 37 C for 30 min. The tubes were wshed three times s before nd then incubted with 1 ml of 1% (wt/vol) queous bovine serum lbumin solution t 37 C for 30 min. The tubes were wshed gin nd then refilled with 1 ml of conjugte diluted to the proper dilution in PBS with 1% bovine serum lbumin. The tubes were incubted t 370C for 30 min nd then wshed s before. One milliliter of substrte ws dded nd llowed to rect t room temperture for 1 h. The rection ws terminted by dding 0.1 ml of 1 N NOH. The brown-colored product ws mesured in Colemn Jr. spectrophotometer t 450 nm. ELISA microtitrtion procedure. The procedure followed for the microtitrtion test ws tht of Ruitenberg et l. (10). All regents used were the sme J. CLIN. MICRODBIOL. s those for the tube tests. Disposble polystyrene microtitrtion trys with 96 flt-bottom wells (Flow Lbortories, Inc., Rockville, Md.) were coted with ntigen by dding 100,l of the diluted ntigen solution to ech well nd incubting the trys in wter bth t 37 C for 3 h. Trys contining the ntigen solution were then stored t 4 C until used. Before the ssy, the trys were wshed three times by flooding them with distilled wter nd then drining, inverting, nd vigorously shking them. Test ser were diluted with PBS, nd 100,ul of ech dilution ws trnsferred to well of try. The trys were incubted in 37 C wter bth for 30 min. They were then wshed s before, nd 100,ul of conjugte, diluted in PBS with 1% bovine serum lbumin, ws dded. The trys were gin incubted t 37 C for 30 min nd then were wshed. Finlly, 100,ul of substrte ws dded, nd the trys were llowed to remin t room temperture for 1 h while the rection occurred. The rection ws then terminted by dding 25,I of 1 N NOH. The brown rection product ws evluted visully. The lst serum dilution showing drker color thn the lowest dilution of the negtive serum ws regrded s the end point. RESULTS To increse objectivity nd reproducibility, we set 50% trnsmittnce (T) on the colorimeter s the mximum reding to be considered positive. We titered ll regents to the 50% end point to determine optiml dilution or serum end points. Our conjugte, prepred s n nti-humn immunoglobulin G-Fb, is highly specific nd rective. Tble 1 illustrtes typicl conjugte titrtion. In the lower dilutions, excessive bckground redings cused the negtive serum to pper to be positive. At the optimum dilution in this titrtion there ws essentilly no rectivity in the negtive serum (61% T t 1:10), but the positive serum ws rective to its known IIF titer of 1:2,560 nd gve shrp end point. Consistency in results of serologicl procedures for the dignosis of toxoplsmosis is extremely importnt. Since the most widely used procedure is the IIF test, which gives the sme results s the methylene blue dye test, we considered it importnt to compre the IIF nd ELISA procedures. Tble 2 illustrtes the comprtive results of these two tests. Becuse the ELISA test is reltively esy to perform, we tested it in twofold dilutions; the IIF test ws tested in fourfold dilutions. This resulted in n pprent skewing of the dt, but the dt still exhibit close stright-line reltionship. The only obvious discrepncy is the one serum tht is 1:16 by IIF nd 1:1,024 by ELISA. To determine reproducibility, we tested one positive serum (IIF 1:1,024) nd one negtive

3 VOL. 5, 1977 USE OF ELISA IN SERODIAGNOSIS OF TOXOPLASMOSIS 275 TABLE 1. Conjugte titrtion for ELISA in the toxoplsmosis system 6% T (t 450 nm) t serum dilution of: Conjugtedilu- Test serum tion ,560 5,120 10, Pos Neg Xb X X 600 Pos Neg X X X 800 Pos Neg X X X 1,000 Pos Neg X X X Pos, Positive control (IIF 1:1,024); Neg, negtive control (IIF < 1:4). b X, Not done. TABLE 2. Comprison of IIF nd ELISA tests for toxoplsmosis ELISA IIF < ,024-2,048 < , ,096 2 Number of ser. serum (IIF < 1:16) on 10 different dys. Tble 3 shows tht in 8 of the 10 tests, titers of 1:512 or 1:1,024 were obtined for the positive serum. In one instnce the titer ws 1:256, nd in only one instnce ws there mjor vrition, in which the titer ws 1:4,096. The high titer ws obtined in test tht normlly would hve been considered invlid becuse the negtive control ws strongly positive. Perhps better mesure of reproducibility is seen in the results of the negtive control serum. Since ll dilutions of the serum re negtive, only the colorimeter redings of the initil dilution were compred. In 8 of 10 tests, T vried only from 42 to 68%. In the one instnce tht ws low, 28% T, the positive serum ws similrly low. When the negtive serum gve 81% T, there ppered to be no dverse effect on the positive serum. (Although the dt do not show it, the titer of the positive serum ws 1:1,024 on tht dy.) Determining the specificity of tests for toxoplsmosis is complicted by the high prevlence of ntibody in the norml popultion. Tble 4 shows the results of btteries of ser submitted for the serodignosis of vrious diseses. With the single exception of the 1:256 result in n mebisis serum, ll ser positive in ELISA either were confirmed by being lso positive by IIF or hd titers too low to be of clinicl importnce. Of some concern is the one rheumtoid specimen tht ws positive by both IIF nd TABLE 3. Reproducibility of ELISA titers for toxoplsmosis on 10 successive dys Titer Serum tested ,024 2,048 4,096 Positive (times tested) l Negtive (% T t 1:16 64, 54, 68, 64, 81, dilution) 28, 50, 42, 48, 45 Tested on sme dy. TABLE 4. specificity ofelisa test for toxoplsmosis No. of ser Ser submitted for: + IIF +ELISA + IIF - IIF +ELISA - ELISA Amebisis Echinococcosis 32, Histoplsmosis Cytomeglovirus 16, Rheumtoid 1 12 Reciprocl of ELISA titer for ech serum negtive by IIF but positive by ELISA. ELISA. Although only 1 of 13 ser ws positive, rheumtoid serum hs been recognized s complicting fctor in the IIF procedure, so this finding justifies more thorough study of these ser by ELISA. DISCUSSION These dt clerly delinete the sensitivity,

4 276 WALLS ET AL. specificity, nd reproducibility of ELISA. As evluted here with toxoplsmosis s the test system, this procedure clerly shows gret promise s system for serodignosis of gret vriety of diseses nd conditions. We hve found it to be s sensitive nd specific s the IIF procedure nd highly reproducible. Although Voller et l. (13) found discrepncies between results from ELISA nd the methylene blue dye test nd between ELISA nd the indirect hemgglutintion test, we found excellent greement between ELISA nd IIF. With the microtitrtion techniques in prticulr, regents re inexpensive nd stble. The conjugte, s prepred, ws diluted 1:1,000 for use, nd it hs been used for 6 months without detectble deteriortion. The conjugte used in these studies ws prepred ccording to published techniques for ELISA, except tht NN3 ws included in the buffer for the Sephdex column. Since these dt were obtined, new conjugtes hve been prepred ccording to the method of Nkne nd Kwoi (8), nd the NN:, ws omitted since it is n enzyme inhibitor. This chnge provided superior conjugtes tht give more dependble rectivity, but the sensitivity nd specificity must still be evluted. The high working dilution nd long shelf life of the conjugtes mke them very economicl regents for lrge-scle testing. The stbility of horserdish peroxidse permits prepred conjugtes to be esily shipped nd stored. The high degree of reproducibility is very encourging. Plus or minus one twofold dilution is demnding criterion for serologicl procedure nd is not ttined by most with ny confidence; yet only one test fell mrkedly outside this rnge. The negtive serum only vried few percent T nd ws never considered positive in ny of the tests. This level of reproducibility ssures procedure tht is relible nd tht lends itself to modifiction nd refinement. Specificity ws well within expected limits. Clinicl informtion with which to evlute the toxoplsm rectivity of the one negtive-positive serum submitted for mebisis serology ws not vilble. Whether the IIF result is flse negtive or the ELISA result is flse positive is undecided. Of more importnce, however, is the positive rheumtoid serum. One of the mjor problems with the IIF test for toxoplsmosis is the rection with ser from utoimmune diseses. We hoped tht the prtil purifiction of the ntigen might eliminte this cross-rection. One of the 13 rheumtoid ser rected in both IIF nd ELISA. One questions whether the rection is specific toxo- J. CLIN. MICROBIOL. plsm rection in both tests or simply nonspecific rection in ech. Although ELISA hs been used for vriety of diseses nd conditions, it still needs to be stndrdized. We hve ttempted to delinete some of the prmeters of the technique itself, using toxoplsmosis s the test model. Nevertheless, optimum conditions for virtully every step of the procedure still need to be defined. All investigtors who hve reported so fr hve used the originl method of Engvll nd Perlmnn (4) or Ruitenberg et l. (10), or minor modifictions of them. Little hs been done to evlute nd stndrdize the methodology. Now tht we hve estblished the limits of specificity nd reproducibility with somewht purified toxoplsm ntigen, we cn use this quntittive procedure to define more clerly the optimum conditions for ELISA. LITERATURE CITED 1. Bos, H. J., A. A. vn den Eijk, nd P. A. Steerenberg Appliction of ELISA -enzyme linked immunosorbent ssy-in the serodignosis of moebisis. Trns. R. Soc. Trop. Med. Hyg. 69: Bout, D., J. C. Dugimont, H. Frg, nd A. Cpron Le dignostic immunoenzymologique des ffections prsitires. II. Immunoenzymologie quntittive (E.L.I.S.A.). Lille Med. 20: Crlsson, H. E., A. A. Lindberg, S. Hmmrstrom, nd A. Ljunggren Quntittion of Slmonell 0- ntibodies in humn ser by enzyme-linked immunosorbent ssy (ELISA). Int. Arch. Allergy Appl. Immunol. 48: Engvll, E., nd P. Perlmnn Enzyme-linked immunosorbent ssy (ELISA). Quntittive ssy of immunoglobulin G. Immunochemistry 8: Engvll, E., nd P. Perlmnn The enzymelinked immunosorbent ssy (ELISA), p In C. G. Heden nd T. Illeni (ed), Automtion in microbiology nd immunology. John Wiley & Sons, Inc. New York. 6. Huldt, G., B. Lgerquist, T. Phillips, C. C. Drper, nd A. Voller Detection of ntibodies in schistosomisis by enzyme-linked immunosorbent ssy (ELISA). Ann. Trop. Med. Prsitol. 69: Miedem, K., J. Boelhouwer, nd J. W. Otten Determintions of proteins nd hormones in serum by n immunossy using ntigen-enzyme conjugtes. Clin. Chim. Act 40: Nkne, P. K., nd A. Kwoi Peroxidse-lbeled ntibody: new method of conjugtion. J. Histochem. Cytochem. 22: Ruitenberg, E. J., I. Ljungstrom, P. A. Steerenberg, nd J. Buys Appliction of immunofluorescence nd immunoenzyme methods in the serodignosis of Trichinell spirlis infection. Ann. N.Y. Acd. Sci. 254: Ruitenberg, E. J., P. A. Steerenberg, nd B. J. Brosi Microsystem for the ppliction of ELISA (enzyme-linked immunosorbent ssy) in the serodignosis of Trichinell spirlis infections. Medikon Ned. 4: Ruitenberg, E. J., P. A. Steerenberg, B. J. Brosi, nd J. Buys Serodignosis of Trichinell spirlis infections in pigs by enzyme-linked immunosorbent ssys. Bull. W.H.O. 51:

5 VOL. 5, Vn Weemen, B. K., nd A. H. W. M. Schuurs Immunossy using hpten-enzyme conjugtes. FEBS Lett. 24: Voller, A., D. E. Bidwell, A. Brtlett, D. G. Fleck, M. Perkins, nd B. Oldehin A microplte enzyme-immunossy for toxoplsm ntibody. J. Clin. Pthol. 29: USE OF ELISA IN SERODIAGNOSIS OF TOXOPLASMOSIS Voller, A., C. Drper, D. E. Bidwell, nd A. Brtlett Microplte enzyme-linked immunosorbent ssy for Chgs' disese. Lncet i: Voller, A., G. Huldt, C. Thors, nd E. Engvll A new serologicl test for the detection nd mesurement of mlril ntibodies. Br. Med. J. 965: