ORIGINAL PAPER

Improving the detection limit of quantitative diagnosisof anti-S. haematobium antibodies using Falcon AssayScreening Test (FAST) ELISA by developing a newstandard curve

Mohamed Abdel-Fattah & Maged Al-Sherbiny &

Ahmed Osman & Ragia Charmy & Victor Tsang

Received: 4 October 2010 /Accepted: 26 November 2010 /Published online: 16 December 2010# Springer-Verlag 2010

Abstract Immunodiagnosis of schistosomiasis are currentlybased on parasitological examinations of stool and urine foregg detection, which is laborious and lacks sensitivity. Thereare many assays that detect the anti-schistosomal antibodies inpatient sera. One of these assays is the Falcon assay screeningtest (FAST) ELISA that uses adult worm microsomal antigenfor Schistosoma haematobium and Schistosoma mansoni,HAMA, MAMA antigen, respectively. This assay dependson quantitative detection of anti-schistosome antibodies,using a standard curve, but the detection limit of FAST-HAMA assay is low due to the small range of the standardcurve. In our study, a new wide range (0–80 μl/μl) of standardcurve for FAST-HAMA assay was constructed, and the cut-off value of the assay, using the new curve, was determinedto be 1.2 units/μl. Screening of 41 S. haematobium-infectedsera with FAST-HAMA, using the new constructed curve,showed a sensitivity of 95%. The purity of HAMA antigenand highly specific Abs for S. haematobium lends the FAST-HAMA with the new constructed wide range standardcurve as a diagnostic assay with high detection limit forS. haematobium infection.

Introduction

Endemic to many countries in the developing world,schistosomiasis continues to be a serious public healthproblem (Hayunga et al. 1987). Schistosomiasis is causedby digenetic trematodes known as schistosomes and affectsabout 200 million persons in the world. There are threespecies of major importance in humans, Schistosomamansoni, Schistosoma haematobium and Schistosomajaponicum. In Egypt, schistosomiasis, caused by S.mansoniand S.haematobium, is the most important endemic disease(Abdel-Wahab 1982; Abdel-Wahab and Mahmoud 1987)

Estimation of the intensity of schistosomal infection iscurrently based on quantitative egg counts, which can behighly variable (Deelder et al. 1989) and may depend onimmune-response status of the host (Doenhoff et al. 1978).Therefore, patient management, based solely on thepresence of ova, is overly conservative and may result inpatients with low egg count being undiagnosed and able tocontinue to transmit the disease (Maddison 1987).

Antibody-detecting assays with the proper antigens arehighly specific and sensitive (Maddison et al. 1985;Maddison 1987; Tsang et al. 1983c, 1984).

For the serodiagnosis of schistosomiasis, a number oftechniques and antigen preparations are available (Mott andDixon 1982; Mott et al. 1987). The detection of serumantibodies in many of these tests is specific and sensitive,but there are many variations between these tests in termsof sensitivity, specificity, complexity, and sophistication.

Tests utilizing crude or partially purified antigens, derivedfrom life cycle stages, can detect antibodies, but thespecificity and sensitivity of these assays were decreaseddue to the cross reactivity with the heterologous parasites(Nash et al. 1982). However, the purification of microsomal

M. Abdel-Fattah (*)R&D Department, Holding Company for Biological Productsand Vaccines (VACSERA),51 Wezaret El-Zeraa St., Agouza,Giza, Egypte-mail: mafmohamed@yahoo.com

M. Al-Sherbiny :A. Osman : R. CharmyDepartment of Zoology, Faculty of Science, Cairo University,Giza, Egypt

V. TsangCenter for Disease Control and Prevention,Atlanta, GA, USA

Parasitol Res (2011) 108:1457–1463DOI 10.1007/s00436-010-2198-y

antigen, systematically from adult worms (Tsang et al.1983b), has allowed the development of rapid, highlyspecific, and sensitive assays, such as Falcon assayscreening test-enzyme-linked immunosorbent assay(FAST-ELISA) (Hancock and Tsang 1986), for detectingantibodies in schistosomiasis infection sera. The assaysensitivity and specificity were 98% and 99%, respectively.

The results of a study performed by (Maddison et al. 1985)showed that a combination of MAMA and a standardizedmicrotest plate ELISA provides a sensitive and specific assayfor detection of homologous S.mansoni infection. The assaysensitivity and specificity were 96% and 99%, respectively.

The main target of this study is to increase the detectionlimit of FAST-ELISA that uses HAMA antigen (FAST-HAMA) for the immunodiagnosis of S. haematobiuminfection by developing a new standard curve for that assay.

Material and methods

S. haematobium adult worm microsomal antigen (HAMA)fractionation

The preparation of HAMA was performed according toTsang et al. (1983b) as follows:

A lot of 20 gm of liquid N2 frozen adult S.haematobiumworms were quickly thawn at 37°C. Thawed worms weretransferred into five volumes (w/v) of cold sucrose/HEPESbuffer, pH 7.02. The protease inhibitors (pepstatin A,leupeptin, phenylmethyl-sulfonyl fluoride) were added toa final concentration of 1 μg/ml sucrose/HEPES buffer.

Worms were homogenized, and the homogenate wasfiltered through four layers of prewashed, sucrose/HEPES-soaked cheesecloth. The filtrate was then centrifuged at 480×G for 20 min. Lipoic material on top was removed by suction,and the supernatant was decanted and spun at 7,650×G for20 min. The 7,650×G supernatant was then centrifuged at360,000×G for 1 h in a Beckman 60-Ti rotor (Beckman,Fullerton, CA, USA). The resulting pellet was then resus-pended with 50 ml, 0.05M Tris–HCl, 8.0 M urea (urea buffer,pH 8.00) and sonicated at 100% power, 20% pulse duty cycle(1 s pulse and 1 s pause) for 1 min. using a heat systemsonicator model 375 (Heat system, Plain View, NY, USA).The sonicate was then centrifuged at 48,000×G for 30 min.

Fractionation of microsomal antigens by gel filtrationand ionic-exchange chromatography

Before subsequent fractionation by column chromatogra-phy, the 48,000×G supernatant was treated with n-butanolto remove lipids and lipoproteins.

Fractionation was performed by using the fast perfor-mance liquid chromatography (FPLC). The sizing column,

Superose 6 (Pharmacia Biotech Inc., Piscataway, NJ, USA)was equilibrated with 0.05 M Tris–HCl, pH 8.00 andpacked into K26/100 column (Pharmacia). The microsomalfraction was loaded onto this column by ascending flowrate of 50 ml/hr, and fractions were eluted with 0.05 MTris–HCl, pH 8.0, at the same flow rate, whereas proteinelution peaks were monitored by absorbance at 280 nm.

The second eluted peak (first inclusion peak after the voidvolume peak) and portions of the leading edge of the thirdpeak (representing the most reactive fractions) were collected,pooled, and further fractionated by ion-exchange FPLC. Thesample was applied to a MonoQ column (Pharmacia) byascending flow rate of 80 ml/hr. The column was eluted with0.05 M Tris–buffer pH 8.0 until the A280 returned to baselinereadings. Subsequent elution of bound materials wasachieved with a linear continuous salt gradient (0.0 M to0.5 M NaCl in 0.05 M Tris-buffer pH 8.00).

Individual peaks were pooled and assayed for reactivityin FAST-ELISA, and the most reactive fractions werecombined to form the final microsomal antigen (HAMA).

Enzyme-linked immunoelectrotransfer blot (EITB)

Electrophoresis and electrotransfer were done according toTsang et al. (1983a). The HAMA was resolved by sodiumdodecyl sulfate gradient (5–22%)–polyacrylamide gel elec-trophoresis. The resolved bands were then electrophoreti-cally blotted onto a nitrocellulose sheet. After transfer, thenitrocellulose was cut into 2-mm wide strips, and the stripswere exposed to individual sera diluted 1:50 with PBS/Tween/5% nonfat milk for 1 h at room temperature. Stripswere washed four times with PBS/Tween. After the lastwash, goat anti-human IgG-peroxidase (Tsang et al. 1995)was added and incubated for 1 h at room temperature.Strips were washed four times with PBS/Tween and oncewith PBS. Antigen bands with bound antibodies werevisualized with 3, 3′-diaminobenzidine/H2O2 substrate.Sera, recognizing HAMA GP23, were recorded.

Standard preparation

A standard S. haematobium serum pool was prepared fromthe sera of patients reacting only with species-specific S.haematobium antigen (Gp 23) in immunoblotting assay andwith high titer in FAST-ELISA using a reference primary S.haematobium standard curve, kindly provided by Prof Dr.Victor C. W. Tsang, CDC, Atlanta, GA, USA.

For delipidization, half volume of Trichlorotrifluoro-ethane was added to the serum pool, and the mixture wasstirred for 2 h at maximum speed at +4°C and centrifugedat 3,000 rpm, at +4°C for 30 min. The supernatant(delipidized serum) was removed and filtered through a 5-μm syringe filter, and protease inhibitors were added.

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Serial dilutions for S.haematobium delipidized serumusing a delipidized normal human serum as diluent weredone and different concentrations of the SH-standards wereprepared.

FAST-ELISA assay

FAST-ELISAwas developed byHancock and Tsang (1986) todetect antibodies against MAMA antigen. The beads, whichare molded into sticks and attached to 96-well lid, weresensitized with HAMA prepared from adult worms (2 μg/ml0.05 M Tris/KOH, 0.3 M KCl, 2 mM EDTA, pH 8.0) for 2 hat room temperature with gentle agitation on an orbitalshaker (Bellco). The sticks were washed by spraying for 20 swith 0.15 M NaCl/ 0.01 M Na2 HPO4 /Na H2 PO4, pH 7.2(Phosphate-buffered saline, PBS), containing 0.3% Tween-20 (Sigma) (PBS-TW), using a compressed air sprayer(Flomaster, Michigan, USA) and then rinsed with deionizedwater. Three microliter of calibrated reference standard, andserum samples were diluted 1:33.3 (vol/vol) with PBS-TW intriplicate into individual wells of a 96-well microtitrationplate (Becton Dickinson, San Jose, CA, USA), using theDigiflex automatic diluter (ICN Biomedicals, Irvine, CA,USA). The beads were incubated with serum for 5 min atroom temperature with mixing on a mini orbital shaker atspeed 5 (MOS-5) (Bellco, Vineland, NJ).

After washing the beads, they were incubated for5 min in another 96-well plate, containing 100 μl/well ofdiluted (1:1000 PBS-TW) peroxidase-labeled, immunoaf-finity, purified goat antihuman IgG, M or A (H+L)(Kirkegaard & Perry Laboratories (KpL), Gaithersburg,MD, USA), with mixing on MOS-5 (Bellco). The beadswere then washed and placed into another 96-well platecontaining 150 μl of 3, 3′, 5, 5′-tetramethyl benzidine(TMB) and H2O2 mixed at a ratio of 1:1 (vol/vol) (KpL).The plate was incubated for 3 min at room temp on MOS-5. The absorbance at 650 nm was measured using a UVmax ELISA plate reader (Molecular Devices Corp.). TheELISA reader-controlling software (Softmax) readilyprocesses the digital data of raw absorbance value into astandard curve from which FAST-ELISA antibody units ofactivity of unknown samples can be derived directly froma four parameters line equation.

Sera with activity greater than 1.2 U/ml were consideredpositive.

Results

FAST-ELISA

FAST-ELISA beads were sensitized with HAMA antigen(2 μg/ml) and sequentially incubated with diluted sera(1:33.3 PBS-0.3%TW), diluted conjugate (1:1000 PBS-0.3%TW), and finally, with substrate solution (TMB:H2O2,vol:vol) to determine serum units of reactivity using aprimary standard curve for S.haematobium (0–9 units/μl).On screening 41 S.haematobium-infected sera collected byour laboratory in a previous study (Al-Sherbiny et al. 1999)with FAST-HAMA, the results were over the upper limit ofthe standard curve (9 units/μl). Therefore, another new

Fig. 1 Reactivity of four S.haematobium-infected sera with HAMA inEITB. HAMA (0.1 μg/μl/mm) resolved on 5–22.5% SDS-PAGE,transferred onto NC sheet, cut into 0.2 cm identical stripes and reactedwith normal human serum (NHS), S.haematobium positive referencecontrol and four sera of S.haematobium-infected patients, diluted 1:50in PBS-0.3%TW/5% milk. Left arrows indicate the positions of the M.Wt protein markers, while the right arrow points to the species-specific 23.0 KDa band

Fig. 2 S.haematobium infection sera titration in FAST-ELISA.Antigen: HAMA (2 μg/ml); sera: patient sera 1,2,3,4 and normalhuman serum NHS, diluted (1:5–1:105) in PBS-TW (5 min);conjugate: peroxidase-labeled goat anti-human IgG diluted 1:1000 inPBS-TW (GAHG-POD) (5 min); substrate: TMB/H2O2 (2 min). (Thefigure was done by Sigmaplot 10 software)

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secondary standard curve for S.haematobium (SH-STD)was constructed.

Secondary S.haematobium standard curve

Four patient sera were selected for the construction of thesecondary SH-STD curve. The selected sera had highreactivity in FAST-ELISA, using MAMA antigen(≥75units/μl) and strongly reacted with S.haematobiumspecies-specific glycoprotein Gp23 in immunoblottingassay using HAMA antigen (Fig. 1).

In comparison to normal human serum (NHS), selectedsera were diluted with PBS-TW (1:5–1:105) and titratedagainst HAMA antigen in FAST-ELISA to determine thechanges in Ag–Ab affinity with dilution. Figure 2 depicts

the titration patterns of each selected serum. Serum ofpatient #4 was excluded as it showed an unstable affinity toHAMA antigen with titration.

Meanwhile, the other three patients sera were pooled(SH positive pool) in equal amounts, delipidized andtitrated in FAST-HAMA assay (Fig. 3) against the primarystandard curve. SH positive pool was diluted 1:10, 1:20 and1:40 with PBS-TW and assayed in FAST-HAMA. Thesedilutions had 8.07, 4.1, and 2.0 units/μl, respectively.Therefore, each microliter of the delipidized SH positivepool was assigned 80.3 units.

From the SH positive pool, a standard curve wasconstructed by serial dilutions of the pool using thedelipidized normal human serum as diluent, thus giving 0,1.25, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80 units/μl. A typicalsecondary S.haematobium standard curve, using four-parameter line equation Y ¼½ ½ A� Dð Þ= 1þ X=Cð ÞB

� iþ

DÞ with a correlation coefficient of 0.999, is shown inFig. 4, where Y is Δ A650 nm, X is the antibody activity inunits (U/μl of serum), A is zero antibody concentrationresponse, D is the infinite antibody concentration response, Band C are derived constants describing the slope of thecurve.

Sensitivity and specificity of FAST-HAMA assayusing the secondary constructed standard curve

To determine the COV of FAST-HAMA using the second-ary SH-STD curve, a total of 34 different normal humansera were assayed. Figure 5 depicts normal human seravalues, which ranged from 0.07–1.2 units/μl (Mean±SD=0.357±0.290). The COV (1.2 units/μl) was then chosen forFAST-ELISA assay using HAMA antigen (FAST-HAMA).

Fig. 3 S.haematobium pooled sera titration in FAST-ELISA. Antigen:HAMA (2 μg/ml); sera: pooled sera and normal human serum NHS,diluted (1:5–1:105) in PBS-TW (5 min); conjugate: peroxidase-labeledgoat anti-human IgG diluted 1:1000 in PBS-TW (GAHG-POD)(5 min); substrate: TMB/H2O2 (2 min). (The figure was done bySigmaplot 10 software)

Fig. 4 A typical secondary S.haematobium standard curve,using four-parameter line equa-tion, using ELISA reader controlsoftware (Softmax)

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The positive control sera of S.mansoni, S.japonicum, andS.haematobium were reactive in FAST-HAMA, and allgave positive reaction more than the COV (>1.2 units/μl)(Fig. 6).

To determine the specificity of FAST-HAMA assay,different heterologous sera were assayed (Fig. 7).

The results showed that only one infection serum (1 outof 11) of Taenia-infected patients was slightly over theCOV (1.3 units/μl).

FAST-ELISA beads were sensitized with HAMA anti-gen, and the new secondary SH-STD curve was used toassay 41 parasitologically positive S.haematobium patientsera. The results showed that the assay sensitivity was 95%(39 out of 41 had >1.2 units/μl).

Discussion

Currently, demonstration of schistosome eggs in urine isused as the definitive diagnosis for schistosomiasis.However, the sensitivity of corporal examination ishighly limited by the sporadic fecundity of the adultworms and the low rate of recovery of parasitic ova frompatient samples. Therefore, patient management, basedsolely on the presence of ova, is overly conservative andmay result in patient with low egg count beingundiagnosed and able to continue to transmit the disease(Maddison 1987). Diagnosis of schistosomiasis by detec-tion of specific antibodies is likely to be more sensitivethan diagnosis by traditional parasitological techniques(Hamilton et al. 1998).

The systematic purification of microsomal antigens fromS. mansoni (MAMA) and S. haematobium (HAMA) adultworms (Tsang et al. 1983a) has allowed the development ofrapid, highly specific, and sensitive assays, such as theFAST-ELISA (Hancock and Tsang 1986; Tsang andWilkins 1991) and enzyme-linked immunoelectrotransferblot (EITB) (Brand and Tsang 1989) for detecting anti-bodies in schistosomiasis. When MAMA was used in theFAST-ELISA, the sensitivity and specificity were 98% and99%, respectively in detecting sera infected with S.mansoni. (Brand and Tsang 1989).

The EITB is capable of differentiating infections causedby the three species of the genus Schistosoma of majorimportance in humans (Tsang et al. 1984) Antibodyreactivity with GP30 of MAMA identifies a S. mansoniinfection, GP18, GP23, or GP29 of S. japonicum adult

Fig. 6 Reactivity of positive control sera in FAST-ELISA. Antigen:HAMA (2 μg/ml); sera: S.mansoni (SMPR1,2-SMB5,52,54,55), S.japonicum (AHJ-14,15), and S.haematobium (SH-3,4,39,92) infectedsera diluted 1:33.3 PBS-TW (5 min); conjugate: GAHG-POD diluted1:1000 PBS-TW (5 min); substrate: TMB/H2O2 (2 min). (The figurewas done by Sigmaplot 10 software)

Fig. 5 Reactivity of 34 negative control sera in FAST-ELISA.Antigen: HAMA (2 μg/ml); sera: control sera (C1–C34) diluted1:33.3 PBS-TW (5 min); conjugate: GAHG-POD diluted 1:1000 PBS-TW (5 min); substrate: TMB/H2O2 (2 min). Broken line indicates theCOV. (The figure was done by Sigmaplot 10 software) Fig. 7 Reactivity of heterologous sera in FAST-ELISA. Antigen:

HAMA (2 μg/ml); sera: heterologous sera (designated by letters)diluted 1:33.3 PBS-TW (5 min); conjugate: GAHG-POD diluted1:1000 PBS-TW (5 min); substrate: TMB/H2O2 (2 min). Broken lineindicates the COV. (The figure was done by Sigmaplot 10 software)

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worm microsomal antigens (JAMA) identifies S. japoni-cum, and GP23 of HAMA identifies S. haematobium(Andrews et al. 1989) Thus, the FAST-ELISA and EITBwere used as screening and confirmatory assays, respec-tively for prior or present schistosomiasis (Rossi et al.1991).

The concept of screening schistosomiasis patients withFAST-MAMA, irrespective of the infective species andconfirming with EITB was found to be not as valid inscreening of infected sera from S.haematobium endemicsite with FAST-MAMA, where the sensitivity of the assaywas only 80.5% (Al-Sherbiny et al. 1999). But when FAST-HAMA was used in screening the S.haematobium-infectedpatients, the assay sensitivity reached to 97%; meanwhile,most of these patient sera also recognized Gp23 in EITB.

Herein, we used FAST-HAMA to screen 41 S. haema-tobium-infected sera, but the low detection limit of theassay leads to most of them were over the maximum limitof FAST-HAMA standard curve (9 U/μl). It is stated thatthe microsomal antigen isolated from adult worm of S.haematobium contain large amount of specific component(Gp23) and minimal quantities of non specific or irrelevantmaterials. Therefore, the specific activity of these reagentsis high, lending them to be especially critical for assays,such as FAST-ELISA (Hancock and Tsang 1986). So byincreasing the range of the FAST-HAMA detection to80 U/μl, it is easy to detect all infected sera without dilutionand time consuming. Also, the sensitivity of the assaywas 95% which is comparable with that obtained byAl-Sherbiny et al. (1999).

Tsang et al. (1984) used homologous adult microsomalantigens which allowed the detection of 23 KDa bandhighly specific for infection of S. haematobium in EITB.We herein selected three S. haematobium-infected sera thatcontain specific anti-S .haematobium antibodies as theyhighly reacted with Gp23 in EITB and used them inconstructing the new standard curve. Increasing thedetection range of FAST-HAMA (80 U/μl) leads to easydetection of all infected S. haematobium sera withoutdilution and time consuming.

The purity of HAMA antigen and highly specific Absfor S. haematobium lends the FAST-HAMA with the newconstructed wide range standard curve as a diagnostic assaywith high detection limit for S. haematobium infection.

Acknowledgments The authors would like to acknowledge thecontributions and efforts exerted by the Division of Parasitic Diseases(DPD/CDC, Atlanta, GA, USA).

Financial support The research described in this article wasperformed under a research grant agreement with the SchistosomiasisResearch Projects, 263-0140.2, grant # 09-02-82, funded by theMinistry of Health and Population of Egypt and the United StatesAgency for International Development, Egypt.

References

Abdel-Wahab MF (1982) Schistosomiasis in Egypt. CRC, Boca RatonAbdel-Wahab MF, Mahmoud SS (1987) Schistosomiasis mansoni in

Egypt. Clin Trop Med Comm Dis 2:371–395Al-Sherbiny M, Osman AM, Hancock K, Deelder AM, Tsang VCW

(1999) Application of immunodiagnostic assays: detection ofantibodies and circulating antigens in human schistosomiasis andcorrelation with clinical findings. Am J Trop Med Hyg 60(6):960–966

Andrews J, Tsang VC, Maddison SE (1989) Evidence of tripleinfections with S.japonicum, S. haematobium, and S. mansoni intwo siblings. J Infect Dis 160:171–173

Brand JA, Tsang VCW (1989) A rapid immunoblot assay (Westernblot) to detect specific antibodies for Human ImmunodeficiencyVirus, S.mansoni and Taenia solium (Cysticercosis). Immunoas-say 10:237–255

Deelder AM, De Jonge N, Boerman OC, Fillies YE, Milberath GW(1989) Sensitive determination of circulating anodic antigen inSchistosoma mansoni infected individuals by an enzyme-linkedimmunosorbent assay using monoclonal antibodies. Am J TropMed Hyg 40:268–272

Doenhoff M, Musallam R, Bain J, Mcgregor A (1978) Studies on thehost–parasite relationship in Schistosoma mansoni—infectedmice: the immunological dependence of parasite egg excretion.Immunology 35:771–778

Hamilton JV, Klinkert M, Doenhoff JM (1998) Diagnosis ofschistosomiasis: antibody detection, with notes on parasitologicaland antigen detection methods. Parasitology 117:S41–S57

Hancock K, Tsang VCW (1986) Development and optimization of theFAST-ELISA for detecting antibodies to Schistosoma mansoni. JImmunol Methods 92:167–176

Hayunga EG, Möllgârd I, Duncan JF Jr, Sumner MP, Stek M Jr,Hunter KW Jr (1987) Early diagnosis of S.mansoni in mice usingassays directed against cercarial antigens isolated by hydrophobicchromatography. J Parasit 73(2):351–362

Maddison SE (1987) The present status of serodiagnosis andseroepidemiology of schistosomiasis. Diagn Microbiol InfectDis 7:93–105

Maddison SE, Slemenda SB, Tsang VCW, Pollard RA (1985)Serodiagnosis of Schistosoma mansoni with microsomal adultworm antigen in an enzyme-linked immunosorbent assay using astandard curve developed with a reference serum pool. Am JTrop Med Hyg 34:484–494

Mott KE, Dixon M (1982) Collaborative study on antigens forimmunodiagnosis of schistosomiasis. Bull World Health Organ60:729–753

Mott KE, Dixon H, Carter CE, Garcia E, Ishii A, Matsuda H, MitchellG, Owhashi M, Tanaka H, Tsang VCW (1987) Collaborative studyon diagnosis of antigens for immunodiagnosis of Schistosomajaponicum infection. Bull World Health Organ 65:233–244

Nash TE, Cheever AW, Ottesen EA, Cook JA (1982) Schistosomeinfections in humans: perspective and recent findings. NIHconference. Ann Intern Med 97:740–754

Rossi CL, Tsang VCW, Pilcher JB (1991) Rapid, low technology,field- and laboratory applicable enzyme-linked immunosorbentassays for immunodiagnosois of S.mansoni. J Clin Microbiol29:1836–1841

Tsang VCW, Wilkins PP (1991) Immunodiagnosis of schistosomiasis.Screen with FAST-ELISA and confirm with immunoblot. ClinLab Med 11:1029–1039

Tsang VCW, Peralta JM, Simons AR (1983a) Enzyme-linkedimmunoelectrotransfer blot techniques (EITB) for studying thespecificities of antigens and antibodies separated by gel electro-phoresis. Meth Enzymol 92:377–391

1462 Parasitol Res (2011) 108:1457–1463

Tsang VCW, Tsang KR, Hancock K, Kelly MA, Wilson BC,Maddison SE (1983b) Schistosoma mansoni adult microsomalantigens, a serologic reagent. I. Systematic fractionation, quan-titation, and characterization of antigenic components. J Immunol130:1359–1365

Tsang VCW, Hancock K, Kelly MA, Wilson BC, Maddison SE (1983c)Schistosoma mansoni adult microsomal antigens, a serologicalreagent. II. Specificity of antibody responses to the S.mansonimicrosomal antigen (MAMA). J Immunol 130:1366–1370

Tsang VCW, Hancock K, Maddison SE, Beatty AL, Moss DM(1984) Demonstration of species-specific and cross-reactivecomponents of the adult microsomal antigens from Schistosomamansoni and S.japonicum (MAMA and JAMA). J Immunol132:2607–2613

Tsang VCW, Greene RM, Pilcher JB (1995) Optimization of thecovalent conjugating procedure (NaIO4) of horseradish peroxi-dase to antibodies for use in enzyme-linked immunosorbentassay. J Immunoassay 16:395–418

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