Human-Isotype-Specific Enzyme Immunoassay for Antibodies to Pneumococcal Polysaccharides

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JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1988, p. 1575-1579 Vl. 26, N. 8 0095-1137/88/081575-05$02.

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1988, p Vl. 26, N /88/ $02.00/0 Cpyright D 1988, American Sciety fr Micrbilgy Human-Istype-Specific Enzyme Immunassay fr Antibdies t Pneumcccal Plysaccharides G. N. S. SHYAMALA, DONAL M. ROBERTON, AND CLIFFORD S. HOSKING* Immunlgy Department, Ryal Children's Hspital, Flemingtn Rad, Parkville, Victria 3052, Australia Received 1 June 1987/Accepted 28 April 1988 A simple enzyme immunassay has been develped t allw the quantitatin f the human respnse t immunizatin with pneumcccal plysaccharide. The assay uses the 14-valent vaccine (Pneumvax) as a cnvenient antigen t adsrb t the slid-phase micrdilutin plate wells and cmmercially available istype-specific antibdy cnjugates. The results have been expressed as arbitrary pneumcccal plysaccharide antibdy units by reading ff a standard curve cnstructed by using hetergeneus pled serum. All nnimmunized subjects tested had immunglbulin G (IgG) antibdies present in serum. All six cntrl subjects wh were immunized with Pneumvax demnstrated an IgG respnse, and the majrity respnded with a rise in IgA- and IgM-specific antibdy cncentratins at a mean f 6 weeks pstimmunizatin. Five ut f six crd sera tested cntained IgG antibdies nly, which were present in cncentratins similar t thse seen in adults, whereas in 6- t 12-mnth-ld infants nly lw levels f IgG and IgM and n IgA antibdies were detected. Serum taken frm 10 hypgammaglbulinemic patients immediately prir t infusin f immunglbulin shwed lw t negative IgG antibdy cncentratins, and n IgA r IgM antibdy was present. The majrity f studies f the antibdy respnse t pneumcccal plysaccharide (PNPS) vaccinatin have been perfrmed by radiimmunassay (12), which is designed t measure the quantity f precipitable antibdy, irrespective f the immunglbulin class. Estimates f the prtective cncentratin f anti-pnps antibdy in humans have been frmed n the basis f results btained by the abve methd (16). Enzyme-linked immunassay (EIA) has several advantages ver radiimmunassay, including greater reagent stability, greater ease f use with avidance f the need fr gamma cunting, and greater adaptability fr measuring istype-specific antibdy respnses (8). EIAs are nw being used mre extensively fr detectin f PNPS antibdies (1, 4, 5, 17r 19, 20). Different immunglbulin classes have different psnic capacities (3). Giebink et al. (9) have shwn that splenectmized, as well as nnsplenectmized, children may shw an increase in ttal serum antibdy, as measured by radiimmunassay, withut shwing enhanced psnic activity after PNPS vaccinatin. T mre cmpletely understand the bilgy f the immune respnse t PNPS, an assay which detects immunglbulin class-specific antibdy is needed. In develping this EIA, we have used a 14-valent pneumcccal vaccine (Pneumvax; Merck Sharp & Dhme Pty. Ltd.) as the slid-phase antigen. This prvided a cnvenient means f determining the istype-specific respnse t immunizatin. MATERIALS AND METHODS Serum samples. Serum samples were cllected frm the fllwing grups and stred at -70 C until assayed: (i) 14 labratry persnnel (these sera representing nnimmunized cntrl adults [aged 22.7 t 46.9 years]); (ii) 6 healthy adult vlunteers, aged 24.5 t 44.9 years (mean, 28.5 years), wh received Pneumvax, a plyvalent pneumcccal vaccine cntaining 50,ug f capsular plysaccharide frm each f 14 pneumcccal sertypes (serum samples were cllected * Crrespnding authr befre immunizatin and at 4 t 8 weeks pstimmunizatin, with an average pstimmunizatin perid f 6 weeks); (iii) 10 hypgammaglbulinemic patients receiving fur weekly infusins f intravenus gamma glbulin (sera were cllected immediately prir t infusin); (iv) 6 nrmal babies, aged 6 t 12 mnths; (v) 6 crd bld samples. As well as the abve, the Australian reference serum ASPS 78/1 (22) and a 1:10 dilutin f nrmal human gamma glbulin (Cmmnwealth Serum Labratries) were assayed fr PNPS antibdy. EIA. The sera were assayed by EIA using a mdificatin f the methds f Melville-Smith and Sheffield (17). The tests were carried ut in dispsable 96-well flat-bttm (Inter Med) Nunc Immunl plates. Pneumvax was dialyzed against 0.05 M carbnate buffer (ph 9.8). The ptimal binding cncentratin was btained by checkerbard titratin. A final cncentratin f 3.5,ug/ml was used in 100 pul f 0.05 M carbnate buffer (ph 9.8) t cat the apprpriate wells nt n the edge f the plate. After an initial incubatin at 37 C fr 3 h, the cated plates were left at 4 C vernight. All incubatins were carried ut in sealed mist chambers. At the cmpletin f incubatin, the wells were washed fr 15 s with 0.05 M phsphate-buffered saline (PBS) (ph 7.2) cntaining 0.05% Tween 20 (PBS-Tween) by using an autmatic micrdilutin plate washer (Immunwash-12 machine; Meds Cmpany Pty. Ltd.). Apprpriate dilutins f serum samples were added in 50-pul amunts and incubated fr 1 h at 37 C. The diluent used was 0.05 M PBS (ph 7.2) with 1% bvine serum albumin (BSA, fractin V; Cmmnwealth Serum Labratries) (PBS-BSA) t reduce nnspecific binding Ȧfter incubatin, the wells were washed as abve and drained. The apprpriate wells were then incubated with 50 pul f ptimal dilutins (in PBS-BSA) f affinity-purified gat anti-human immunglbulin G (IgG) (1:2,000), IgA (1:1,000), r IgM (1:1,000) cnjugated t alkaline phsphatase (immundiagnstic reagents frm Tag, Inc.). Incubatins were perfrmed fr 1 h at 37 C, and the wells were washed as befre. The substrate used was p-nitrphenyl phsphate (alkaline phsphatase test kit; Behringer Mannheim), which was diluted t 1 mg/ml in 10% diethanlamine buffer

2 1576 SHYAMALA ET AL. with MgCl2 (ph 9.8). Substrate slutin (50,ud) was added t each test well and incubated fr 30 min at 37 C in light-sealed cntainers. The reactin was quenched by the additin f 25,ul f 5 N NaOH, and the ptical density was read at 405 nm by using an autmated Micrelisa reader (Flw Titertek, Multiscan; Flw Labratries). The ptimal times and temperatures fr all the abve steps were determined in separate experiments by using paired pre- and pstvaccinatin sera. Standard sera and cntrls. The first rw f wells in every micrdilutin plate received buffer in each step f the assay. The same wells als received substrate and reactinquenching slutin. These wells were used as blanks t zer the Micrelisa reader. Backgrund absrbances were calculated frm cntrl wells n each plate with n cnjugate and n sera. These absrbance readings were generally less than The backgrund absrbance reading was subtracted frm standard, cntrl, and test sample absrbance values fr the calculatin f PNPS antibdy unit values. The fllwing sera were included in each Micrelisa plate. (i) Fr cnstructin f the standard curve and assigning f PNPS antibdy unit values, three different sera were used fr each f the IgG, IgA, and IgM pneumcccal antibdydetecting EIAs. The standards were made frm a pl f sera frm immunized and nnimmunized adult vlunteers. The starting dilutin f the sera used fr standard curve cnstructin was assigned an arbitrary value f 1,000 U/ml. (ii) A crd bld sample with n detectable (<2 U/ml) IgG, IgA, r IgM antibdies t pneumcccal plysaccharide was used as a negative cntrl serum. (iii) Three different sera, A, B, and C, cvering three pints n the standard curve, were used in duplicate in each f the assays fr IgG, IgA, and IgM antibdies. The antibdy units f the abve sera had been previusly determined frm the mean absrbance value f 20 assays each, read against the standard curve. The alltted values f psitive internal cntrl sera (in PNPS antibdy units per ml) were as fllws: 500 fr serum A, 62.5 fr serum B, and 2 fr serum C. Reprducibility f assays. The psitive internal cntrl sera A, B, and C were tested 15 times each during ne assay t assess intrarun variatin. This was repeated n tw different days. T determine the interrun variatin f the assay, the internal cntrl sera A, B, and C were tested repeatedly in assays ver many mnths. T minimize variatin, all f the serum samples, cntrls, and standards were assayed in duplicate and in a pattern described previusly (21). The absrbance values f duplicates were averaged, and the unit values were read frm the standard curve. If the absrbance values fr a sample did nt fall within the linear part f the curve, the test was repeated using a different dilutin f the serum. The assay results were accepted nly if (i) the PNPS antibdy unit values f the internal cntrls fell within the interrun variatin, (ii) the unit value f the negative cntrl serum was less than 2 U/ml, and (iii) the backgrund absrbance was less than Liquid-phase absrptin studies. Six paired pre- and pstimmunizatin sera with a furfld r greater rise in PNPS antibdy after immunizatin, as determined by EIA, were used. The abve serum samples at a dilutin f 1:80 were added t equal vlumes f a 1:10 dilutin f PNPS r buffer (PBS-BSA). The mixtures were incubated at 37 C fr 30 min and were left sealed vernight at 4 C. Bth unabsrbed and absrbed sera were then assayed simultaneusly in duplicate by EIA fr IgG PNPS antibdies. Serum IgG quantitatin. A Technicn Aut Analyzer II J. CLIN. MICROBIOL. Flurnephelmeter was used t quantitate IgG levels (7) in the pre- and pstimmunizatin samples used in the absrptin studies. RESULTS Titratin curves fr determinatin f the ptimal cncentratin f PNPS fr precating are given in Fig. 1. The cncentratin f PNPS chsen fr further assays was 3.5,ug/ mi ṖBS-BSA was chsen as the diluent fr sample and cnjugate dilutin steps after cmparisn f PBS alne, PBS-BSA, and PBS with 0.1% gelatin. Each diluent gave similar absrbance readings with a psitive reference serum, but unacceptably high readings were seen with serum frm a hypgammaglbulinemic patient in the presence f PBS and PBS-gelatin. The effect f liquid-phase absrptin f serum at a 1:80 dilutin with an equal vlume f a 1:10 dilutin f PNPS r PBS-BSA n the absrbance readings btained in the IgG EIA are shwn in Fig. 2. A decrease in absrbance values is seen in the presence f PNPS in prevaccinatin serum and in immune serum, demnstrating assay specificity fr PNPS. The lwer limit f detectin was represented by tw-time backgrund readings fr IgG, IgA, and IgM antibdy t PNPS and was apprximately 2 U/ml fr each istype. The intrarun cefficient f variatin fr the assay, as determined by the three internal cntrls (A, B, and C) fr IgG, IgA, and IgM PNPS antibdy, was 14.1, 17.8, and 19.5%, respectively. The interrun cefficient f variatin was 20.5% fr IgG, 21.0% fr IgA, and 20.9% fr IgM. The 14 nnimmunized adult cntrls had medium titers f PNPS f 50.9 x 103 U/ml fr IgG (range, 16.6 x 103 t 98.3 x 103 U/ml),<10 U/ml fr IgA, and <10 U/mI fr IgM. Nne f the sera frm the 10 hypgammaglbulinemic patients had measurable IgA and IgM antibdies, and IgG antibdy cncentratins in all 10 were less than 10 U/ml. All but ne f the six crd bld sera tested had IgG antibdy levels in the range f the adult cntrls. The sera frm the six infants aged 6 t 12 mnths had IgG and IgM antibdies at less than 10 U/ml, and n detectable IgA antibdies. Istype-specific antibdy cncentratins in arbitrary units per ml fr pre- and pstimmunizatin sera fr the six immunized adult vlunteers are given in Table 1. All vlunteers had a threefld r greater rise in IgG antibdy after immunizatin, fur had a twfld r greater rise in IgA antibdy, and three had a twfld r greater rise in IgM antibdy. DISCUSSION The virulence f encapsulated Streptcccus pneumniae is attributed t the antiphagcytic capacities f the PNPS capsule. The develpment f specific anticapsular antibdies is the aim f immunizatin with purified PNPS (13, 23). The EIA described in this reprt has been used t detect the human istype respnse t PNPS. The assay is simple, reprducible, and specific and ffers an pprtunity t btain additinal infrmatin with respect t the immune respnse t pneumcccal vaccine. The respnse t Pneumvax nt nly represents the respnse t type-specific plysaccharides but als the respnse t the pneumcccal C plysaccharide which the vaccine cntains (14). Similarly the "resting" r preimmunizatin specimens almst certainly cntain antibdies t the C plysaccharides, as well as t a variable number f the type-specific plysaccharides.

3 VOL. 26, 1988 HUMAN ANTIBODIES TO PNEUMOCOCCUS _ - -&- - backgrund *..O... pre - vac serum O pst - vac serum *0. '4&( * O* {). _ PNPS (ug/ml) FIG. 1. T determine the ptimal PNPS cncentratin, a preimmunizatin (pre-vac) serum and crrespnding pstimmunizatin (pst-vac) serum were assayed at varius cncentratins f PNPS precat slutin. A cncentratin f 3.5,ug/ml was chsen as the ptimal cncentratin f PNPS fr further assays. Backgrund is defined in text. T enhance sensitivity and minimize variability, a number f parameters affecting the assays were examined. In the experiments reprted here, the PNPS adsrbed t the plates cnsistently at ph 9.8. Sme wrkers have fund success in direct cating f PNPS nt micrdilutin plates (2, 12, 18), whereas thers have failed (1, 5, 18-20). Perhaps the dis- UJ «t 0.8 s w z 1.25 _ 1 cc,.75 *.5.25 j UNABSORBED ABSORBED UNABSORBED ABSORBED POST VACCINATION PRE VACCINATION FIG. 2. Liquid-phase absrptin fllwed by detectin f IgG antibdy f a representative pair f pre- and pstimmunizatin sera. Backgrund absrbance was crepancies in adsrptin reprted in the literature are a reflectin f the type f slid supprts used, the cating buffer and the cnditins f precating. In the present study, these questins were nt pursued further. The ptimal cncentratin f PNPS was 3.5,ug f Pneumvax per ml (Fig. 1). In studies reprted previusly, a range (1 t 200 pg/ml) f cncentratins f PNPS have been used fr detecting anti-pnps antibdies (1, 2, 5, 12, 17-19). Liquid-phase absrptin with PNPS f paired pre- and pstimmunizatin sera caused a decrease in IgG antibdy levels (Fig. 2). The reasn fr the inability t absrb ut all f the antibdy is a reflectin f the absrptin technique. Liquid-phase absrptin is inefficient in remving all f the antibdy in the serum samples, as cmpetitin between fluid- and slid-phase antigens fr the antibdy ccurs unless high cncentratins f fluid-phase antigen are used. Anther pssibility is that a cmpnent in the Pneumvax may be selectively binding t the wells in preference t the ther cmpnents. In this case, there wuld be verrepresentatin f antigen n the slid phase cmpared with the liquid phase and relative inability t absrb ut antibdy. The EIA was used t measure serum IgG, IgA, and IgM antibdy cncentratins fllwing immunizatin f nrmal adults with Pneumvax. Defining sercnversin as at least tw times the intrarun cefficient f variatin, all f these cntrls shwed IgG sercnversin when tested after an average pstimmunizatin perid f 6 weeks (Table 1). One f the cntrls (cntrl D in Table 1) failed t have any IgA respnse, and ne (cntrl E) did nt have an IgM respnse. Measurement f ttal serum IgG cncentratins in preand pstimmunizatin sera frm a number f immunized individuals did nt shw any significant change ver that perid (data nt shwn). Therefre, the increase in the PNPS antibdy cncentratins detected by EIA between the pre- and pstimmunizatin sera are nt a result f a nnspecific increase in the IgG cncentratin f the sera. AL

4 1578 SHYAMALA ET AL. J. CLIN. MICROBIOL. TABLE 1. Istype respnse f individual cntrl subjects t PNPS Respnse f the fllwing subject t PNPS: Cntrl A B C D E F Preb Pstc Pre Pst Pre Pst Pre Pst Pre Pst Pre Pst IgG , IgA IgM a 103 PNPS antibdy units (sec b text). Pre, Prevaccinatin. ' Pst, 4 t 8 weeks pstvaccinatin. Thus, if sercnversin as defined is used as an index f sensitivity, the assay culd be said t be 100% sensitive fr IgG and 92% sensitive fr IgA and IgM. Unfrtunately we have n similar data fr specificity, which wuld have required testing unimmunized cntrls at weekly intervals fr 6 weeks t determine the prprtin wh failed t sercnvert ver that interval. In initial experiments, a series f paired preimmunizatin sera and 4- t 8-week pstimmunizatin sera were titrated and the absrbance versus serum dilutin curves were cmpared (results nt shwn). The shapes f paired pre- and pst-sera dilutin curves were similar fr individual pairs f sera, cnfirming previus findings (5, 18). This suggests that the antibdy measured befre and after immunizatin has equal affinity fr the antigen phase in the ne individual. Kskela and Leinnen (15) als nticed n marked variatin f the slpes f titratin curves between serum samples frm different patients. Hwever, this was nt the bservatin f the present study, in which titratin curve slpes were fund t vary between individuals. Samples taken frm 10 hypgammaglbulinemic patients just prir t intravenus immunglbulin administratin were assayed. In these patients, preinfusin IgG cncentratinis represent small amunts f endgenus IgG prductin and residual IgG 1 mnth after the previus infusin f immunglbulin. The PNPS IgG antibdy levels were lw r undetectable. This was cnsistent with the lw preinfusin serum IgG cncentratins in these patients. They als did nt have IgA r IgM PNPS antibdies, cnsistent with the absence f these immunglbulins (less than 1 IU/ml) in their serum. Residual IgG PNPS antibdy fund in the sera f the abve patients was likely t have been frm the administered immunglbulin, especially since antibdies t cell wall plysaccharides are t be fund in mst nrmal human sera. In the present study, IgG antibdy t PNPS was shwn t be present in the standard human serum ASPS 78/1 (22) and in nrmal human gamma glbulin. Recently, Hetcheringtn and Giebink (11) demnstrated psnic activity t type 14 pneumcccal plysaccharide in intravenus immunglbulin preparatins. Crd bld sera have IgG antibdy t PNPS but n IgM antibdy, althugh the IgG antibdy and psnic activity levels are said t be significantly iwer than in maternal serum (6). In the present study, IgG antibdy cnsistent with adult IgG levels was fund in all except ne f the crd sera. There was n IgM r IgA antibdy in any crd serum samples tested. In the sera f infants aged 6 t 12 mnths, IgG antibdy levels were lw. IgA antibdy was nt detected, and IgM antibdy als was lw. Evidence has been presented f the greater ability t discriminate between patient grups using the results f antibdy levels t 12 plysaccharide types cmpared with using the gemetric mean f thse results (10). This wuld seem t imply that discriminatry infrmatin wuld be lst by using the verall respnses as we have dne. Hwever, n data were given fr the results f testing new samples using the calculated discriminant functin, which detracts cnsiderably frm what culd ptentially be an imprtant finding. On the ther hand, ur aim has been t devise a simple technlgy that culd be widely and cheaply applied. The istype-specific EIA described in this reprt uses cmmercially available pneumcccal antigen which adsrbs readily t the slid phase chsen, and it is capable f demnstrating sercnversin in healthy adult vlunteers. This assay may be useful in determining the antibdy status f at-risk patients, such as thse underging splenectmy, and ffers a simple nieans f determining the respnse t immunizatin. LITERATURE CITED 1. Barrett, D. J., A. J. Ammann, S. Stenmark, and D. W. Wara Immunglbulin G and M antibdies t pneumcccal plysaccharides detected by enzyme-linked immunsrbent assay. Infect. Immun. 27: Berntssn, E., K. Brhlm, and B. Kaiser Serlgicai diagnsis f pneumcccal disease with enzyme-linked immunsrbent assay (ELISA). Scand. J. Infect. Dis. 10: Brss, T., and H. J. Rapp Cmplement fixatin n cell surface by 19S and 17S antibdies. Science 150: Callahan, L. T., III, A. F. Wdhur, J. B. Meeker, and M. R. Hilleman Enzyme-linked immunsrbent assay fr measurement f antibdies against pneumcccal plysaccharide antigens: cmparisn with radiimmunassay. J. Clin. Micrbil. 10: Carlsn, B. A., G. S. Giebink, J. S. Spika, and E. D. Gray Measurement f immunglbulin G and M antibdies t type 3 pneumcccal capsular plysaccharide by enzyme-linked immunsrbent assay. J. Clin. Micrbil. 16: Chudwin, D. S., D. W. Wara, G. Schiffmann, S. G. Artrip, and A. J. Ammann Maternal-fetal transfer f pneumcccal capsular plysaccharide antibdies. Am. J. Dis. Child. 139: Eckman, I., J. B. Rbbins, C. J. A. Van Den Hamer, J. Lentz, and I. H. Scheînberg Autmatin f a quantitative immuncheniical micranalysis f human serum transferrin: a mdel system. Clin. Chem. 16: Engvall, E., and A. J. Pesce (ed.) Quantitative enzyme immunassay. Scand. J. Immunl. 8(Suppl. 7): Giebink, G. S., J. E. Fker, J. Kim, and G. Schiffman Serum antibdy and psnic respnse t vaccinatin with pneumcccal capsular plysaccharide in nrmal and splenectmized children. J. Infect. Dis. 141: Giebink, G. -S., C. T. Le, and J. S. Spika Summarised vs. type specific analysis f antibdy t pneumcccal plysaccharides. Rev. Infect. Dis. 3(Suppl.):S43-S Hetcheringtn, S. V., and G. S. Giebink Opsnic activity f immunglbulin prepared fr intravenus use. J. Lab. Clin. Med. 104: Hsea, S. W., E. J. Brwn, C. G. Burch, R. A. Berg, and M. M.

VOL. 26, 1988 HUMAN ANTIBODIES TO PNEUMOCOCCUS 1579 Frank. 1981. Impaired immune respnse f splenectmized patients t plyvalent pneumcccal vaccine. Lancet i:803-807. 13. Kass, E. H. (ed.). 1981. Assessment f the pneumcccal plysaccharide vaccine.

5 VOL. 26, 1988 HUMAN ANTIBODIES TO PNEUMOCOCCUS 1579 Frank Impaired immune respnse f splenectmized patients t plyvalent pneumcccal vaccine. Lancet i: Kass, E. H. (ed.) Assessment f the pneumcccal plysaccharide vaccine. Rev. Infect. Dis. 3(Suppl.):S1-S Kskela, M Serum antibdies t pneumcccal plysaccharide in children: respnse t acute pneumcccal titis media r t vaccinatin. Pediatr. Infect. Dis. J. 6: Kskela, M., and M. Leinnen Cmparisn f ELISA and RIA fr measurement f pneumcccal antibdies befre and after vaccinatin with 14-valent pneumcccal capsular plysaccharide vaccine. J. Clin. Pathl. 34: Uandesman, S. H., and G. Schiffmann Assessment f the antibdy respnse t pneumcccal vaccine. Rev. Infect. Dis. 3(Suppl.):S184-S Melville-Smith, M., and F. Sheffield Enzyme-linked immunsrbent assays fr the estimatin f immune respnse t pneumcccal plysaccharides. J. Bil. Stand. 8: Pedersen, F. K Antibdy respnse t pneumcccal vaccine in splenectmized children. Acta Pathl. Micrbil. Scand. Sect. C 91: Pedersen, F. K., and J. Henrichsen Detectin f antibdies t pneumcccal capsular plysaccharides by enzymelinked immunsrbent assay. J. Clin. Micrbil. 15: Russell, H., L. R. Edwards, E. W. Wrtham, and R. R. Facklam Enzyme-linked immunsrbent assay fr detectin f antibdies against Streptcccus pneumniae capsular plysaccharides. J. Clin. Micrbil. 11: Stemshrn, B. W., D. J. Buckley, G. St. Amur, C. S. Lim, and J. R. Duncan A cmputer-interfaced phtmeter and systematic spacing f duplicates t cntrl within-plate enzyme immunassay variatin. J. Immunl. Methds 61: Turner, K. T., P. M. Carter, C. S. Hsking, and W. K. Finger The Australian reference preparatin f human serum immunglbulins. Aust. J. Exp. Bil. Med. Sci. 58: Wikelstein, J. A Opsnins: their functin, identity and clinical significance. J. Pediatr. 82:

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PROTOCOL 1850 Millrace Drive, Suite 3A Eugene, Oregon PROTOCOL Cmplex II Enzyme Activity Micrplate Assay Kit 1850 Millrace Drive, Suite 3A Eugene, Oregn 97403 MS241 Rev.0 DESCRIPTION Cmplex II Enzyme Activity Micrplate Assay Kit Sufficient materials are prvided