Two site immunoradiometric assay for detection of Plasmodium falciparum antigen in blood using monoclonal and polyclonal antibodies

JOURNALOF CLINICAL MICROBIOLOGY, Aug. 1987, p. 1467-1471 0095-1137/87/081467-05$02.00/0

CopyrightC 1987, American Society for Microbiology

Vol. 25, No. 8

Two-Site

Immunoradiometric

Assay for

Detection

of Plasmodium

falciparum

Antigen in Blood Using Monoclonal and

Polyclonal Antibodies

SRISIN KHUSMITH,l* SAVANATTHARAVANIJ,' RATANAPORNKASEMSUTH,2 CHUTRUDEE VEJVONGVARN,1 AND DANAIBUNNAG3

Departmentsof Microbiology andImmunology,l TropicalRadioisotope,2 and Clinical Tropical Medicine and Hospitalfor

Tropical

Diseases,3

Faculty of

Tropical

Medicine,

Mahidol

University,

Bangkok

10400,

Thailand

Received5November 1986/Accepted 14 April 1987

Three systems ofimmunoradiometric assays (IRMAs), a two-site monoclonal antibody sandwich IRMA

(MAb-IRMA), two-site polyclonal antibody-monoclonal antibody sandwich IRMA (PAb-MAb-IRMA), and

two-site polyclonal antibodysandwich IRMA (PAb-IRMA), were developed to detect low-grade infections with

Plasmodium

falciparum.

The assays showed good correlationwith parasitemia when tested against parasites frominvitro cultures(r= 0.996,0.994, and 0.998 for MAb-, PAb-MAb-, and PAb-IRMA, respectively), with

theabilitytodetect asfewas0.24, 0.67, and 1.82 parasites per107erythrocytes,respectively. The assays were

specific forP.falciparum, since aserially diluted specimen from a patient with vivax malaria with an initial

parasitemia of 0.8% and almostal of the undiluted specimensfrom five other vivax malaria patients were

negative. The assays were performed on patients with falciparum malaria before and after treatment with

antimalarialdrugs. Before treatment, all 24 patients were positive by all three systemsof two-site sandwich

IRMAs. Twoweeksaftertreatment,81.8% (18 of 22) of the patients were positive bymicroscopicexamination,

but the IRMA positivity rates were 90.9% (20 of22), 86.4% (19 of 22), and 81.8% (18 of 22) for MAb-,

PAb-MAb-, and PAb-IRMA, respectively. Four weeks after treatment, all 19 patients were negative by

microscopicexamination, but 52.6% (10 of 19) ofthe patients werestill positive with MAb- and

PAb-MAb-IRMA and31.6% (6of 19) werepositive with PAb-IRMA. Comparisonbetween the three systems of IRMA

showed that the MAb-IRMA wassuperior tothe othertwosystems forthree reasons. First, it gave a lower countwhen testedwith blood fromhealthy individuals. Second,it gave ahighercountwhen tested withblood

from patients with falciparum malaria. Third, itgave bettercorrelation with parasitemia whenblood from falciparum malaria patientswastested. MAb-IRMA isrecommended forusefor thedetectionoflow-grade P.

falkiparum

infection.

Demonstration ofantigens isbecoming increasingly pop-ular in the diagnosis of parasitic diseases because they are more closely related to present illness than are antibodies which can be detected even when the etiologic agent has disappeared. Diagnostic tests based on antigen detection have beendeveloped forseveralparasitic diseases, including amebiasis (9, 15), giardiasis (8, 16), toxoplasmosis (1), and schistosomiasis(6). Detectionofsporozoites of Plasmodium falciparum and Plasmodium vivax in infected mosquitoes has been done successfully by immunoradiometric assay (IRMA) or enzyme-linked immunosorbent assay (4, 5, 20, 21). The presence ofblood-stage antigen in P.

falciparum-infectederythrocyteshas beendemonstrated witha compet-itive binding radioimmunoassay (RIA) using

polyclonal

an-tibodies (PAb) with a sensitivity of detecting one to

eight

parasitesper

106

erythrocytes (2, 11, 12).

We have recently

developed

a

competitive

binding

RIA with anti-P. falciparum immunoglobulin G

(IgG) prepared

from people living in an area where malaria is endemic in ThailandandIgG fromamonoclonal

antibody

(MAb)

against

blood stages ofP.

falciparum prepared

in our

laboratory,

with sensitivities ofdetecting 13 and 2.2

parasites

per 106 erythrocytes, respectively (10). We also showed that our

MAb bound to theparasite

antigen

more

specifically

than did the polyclonal IgG (PIgG). The

competitive-binding

RIA appearstobe lesssensitivethan

microscopic

examination

by

* Correspondingauthor.

anexperiencedmicroscopist, who could detect as few as one parasiteper 5 x 106 erythrocytes (14). Theobjectives ofthe present study were todevelopa two-site sandwich IRMA, whichis considereda moresensitivemethodfor

detection

of antigen than the competitive-binding RIA, using MAb and PAb and to select the assay system for future use by comparingtheresults withMAb, PAb, andacombinationof the two.

MATERIALSANDMETHODS

Subjects. (i)Patients withfalciparummalaria.

Twenty-four

falciparum malaria patients admitted to the Hospital for Tropical Diseases, Faculty ofTropical Medicine, Bangkok, from June to October 1984 were studied. Blood samples were obtained from all patients during the pretreatment period, from22patients forasecond time

during

thesecond week after treatment, andfrom 19 patients fora third time duringthefourthweek.

(ii)

Patients with vivax malaria.Included inthe

study

were

apatient with vivax malaria(0.8% parasitemia) admittedto the Hospital for

Tropical

Diseases and five other vivax malariapatientsresidingatPobPhra, MaeSod

District,

Tak Province, 460km northwest of

Bangkok.

(iii) Healthy controls.

Thirty healthy

persons

residing

in Bangkok,wheremalaria isnot

endemic,

werestudied.

They

denied

travelling

toany endemicareain thepast 2 years and hence would be most

unlikely

to have been

exposed

to malaria

during

the timeof

study.

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Blood samples. Three milliliters of blood was collected in screw-cap tubes containing 5 mM EDTA, from which two thick and two thin blood films were made on clean

micro-scopic slides, stained with Giemsa, and examined by light microscopy. For thethick film, 10,ulof bloodwasused. The

number of infected erythrocytes per

107

erythrocytes was

determinedby counting the number of parasitized cells in the thin blood smear per 104 erythrocytes; for the thick blood

film, the entiresmear wasexamined (100oil immersion fields

in approximately 3 min), and the number of parasites was

counted and expressed per

107

erythrocytes, with the

as-sumption thataThai patient with falciparum malaria hadon

the average4 x

10'

erythrocytes per p1.

The blood collected in the tubeswascentrifuged at 500 x

gfor 10 minatroomtemperaturetoremovetheplasma, and the remaining packed cells were washed twice by

centrifu-gation with phosphate-buffered saline, pH 7.2 (PBS; 0.01 M

phosphate buffer, 0.14 M NaCl). The final cell suspension

wascentrifugedat9,980 x gfor 5 minatroomtemperature, and the packed cells were stored at -70°C until used. The packedcellswerethawedonceandtreated with 9 volumes of

borate-buffered saline (BBS; 0.025 M disodium tetraborate, 0.1 M boricacid, 0.075 M sodium chloride, pH8.6)

contain-ing 0.5% Nonidet P-40 (BDH Chemicals Ltd., Poole, En-gland) (BBS-NP40) for 10 min at room temperature by the techniqueof RenuDayal (World 1lealth Organization Immu-nology Research and Training Centre, Faculty of Medicine, University ofGeneva, Geneva, Switzerland; personal

com-munication).

Standardantigen. The SOstrainof P.falciparumwasused

(17). The parasites were grown in RPMI 1640 medium

(GIBCO Laboratories, Grand Island, N.Y.) with 10% heat-inactivated ABserumbythe technique of Trager and Jensen (18). The culture was harvested when parasitemia reached

16.5%, of which approximately 50% were ring forms. The

cells were washed twice in RPMI 1640 without serum by

centrifugation at500 x g for 10 min at roomtemperature.

The packed infected erythrocytes were then treated with

BBS-NP40 as described above. Before use, infected-erythrocyte extract was adjusted to give an initial

concen-tration equivalent to 105 infected cells in

107

erythrocytes and serially diluted 1:5 eight times to a final dilution of

1:390,625.

Control antigen. Normal erythrocytes from healthy indi-vidualswerewashed three times in PBS by centrifugation at 500 x gfor 10 minatroomtemperature, and the plasmaas

wellasthebuffycoatlayerwasremoved. The washed cells

werelysed in 9volumesofBBS-NP40atroomtemperature,

followedby centrifugationat9,980 x gfor 10 minatroom

temperatureto remove nuclei and cell debris. The

superna-tantwas then usedasthecontrolantigen andasthediluent

for the standard parasite antigen (11).

Preparation of anti-P. falciparum antibody. (i) PAb. A

rabbit was initially immunized subcutaneously at multiple sites with 1 ml ofa suspension of107 parasites of the SO

strain of P. falciparuft, of which most were in the late

trophozoite and schizont stages, in physiological saline

(0.85% NaCI solution) incorporated in an equal volume of

Freund complete adjuvant.Thiswasfollowed bythree other

monthly injections of1 x 107,4 x 107,and4 x 107parasites,

respectively, in Freund incomplete adjuvant. The animal

wasbled by cardiacpuncture3weeks afterthelast injection. Theserumwasinactivated byheatingat56°Cfor30min and

was absorbed by mixing 1 ml of the serum with 4 ml of

packed group AB erythrocytes from healthy individuals, incubatedat37°C for30minandat4°Covernight,followed

bycentrifugation at 500 x g for 10 min at room temperature, and the supernatant was kept at -70°C until used.

(il) MAb. The F2W22C1 MAbprepared from theculture supernatant was used.This MAb wasproduced byfusion of Sp2/0 myelomacells withspleencells fromaBALB/cmouse

immunized with the SO strain of P.falciparumasdescribed previously (10). It reacted in the indirect fluorescent-antibody test with thering as well as all other stages of P. falciparum in 27of31 isolates tested(10).

Radioiodination of anti-P. falkiparum IgG. IgG fractions from either PAb or MAb against P. falciparum were pre-pared byprotein A-Sepharose 4B(Pharmacia) chromatogra-phy by the technique provided by the manufacturer. Two hundredmicrograms ofIgG, determinedby a spectrophoto-metric method (19), was labeled with 2 mCi of carrier-free 125I (Amersham, England) by the iodogen tubemethod (13). The percentincorporation and specific activitywere97.69% and 4.2 x 106 cpm/,ug of protein, respectively, for labeled PIgG and 95.44% and 9.6 x 106 of cpm/,ug of protein, respectively, for monoclonal IgG (MIgG). The labeled IgG was kept in small portions inPBS (pH 7.2) containing 1% bovine serum albumin (BSA) fraction V (RIA grade; US Biochemical Corp.) at -70°C and thawed only once, just before use. The labeled IgGsolutionwascentrifugedat9,980

x g for 10 min at roomtemperature to remove anyformed aggregates, further diluted with BBS containing0.5%BSA, and used at aconcentration of0.1 and0.4 ,ugofproteinper 30 pI for MIgG and PIgG, respectively.

IRMA. Wells of96-well U-bottom assay plates (Falcon 3911 Microtest III; Becton Dickinson and Co., Paramus, N.J.) were each coated with 50 ,ulofMIgG orPIgGin 0.05 Msodiumbicarbonate buffer, pH 9.6, at aconcentration of 10 ,ug/ml, followed byincubation at 37°C for 3 h or at4°C overnight. The plates were washed three times with BBS-0.5%BSA, saturatedwith100 ,ulof BBScontaining 1% BSA(BBS-1%BSA), and keptfor 1 h at roomtemperature andat4°C thereafteruntilused. Theplates werethoroughly washed with BBS-0.5% BSA to which 30 pil ofeither the erythrocyte preparations to be tested or standardantigen in various dilutions was added to each well, followed by incubation for 3 h at room temperature. The plates were washed twice withBBS-0.5% BSA containing 0.05%Tween

80and oncewith BBS-0.5% BSA. After washing, 30 ,ul of

[1251]MIgG

or

[251I]PIgG

(approximately 5 x

105

to106 cpm) inPBS-1% BSAand10%normalhuman serum were added, and the plates were incubated for another 3 h at room temperature. After beingwashed three times with BBS, the wells were cut out and placeddirectly into counting tubes,

and

bound radioactivity was measured in aganimacounter (Mini-Assay type 6-20; Mini Instruments Ltd., London, England). Three systems of IRMA were developed: (i) a two-site MAb sandwich IRMA(MAb-IRMA), in which the plates were coated with unlabeled MIgG and detected with labeled MIgG; (ii) a two-site PAb-MAb sandwich IRMA (PAb-MAb-IRMA), in which the plates were coated with unlabeled PIgG anddetected with labeled MIgG; and

(iii)

a two-site PAb

sandwich

IRMA (PAb-IRMA), in which the plates were coated with unlabeled PIgG and detected with labeled PIgG. All assays were done in duplicate, with an intrarun difference of less than 5%, and the means (± standarddeviation[SDI)of thecoefficient of variationfor12 samples in fourdifferentruns were 10.2 + 3.1%(range, 5.2

to 14.9%), 9.1 + 4.6% (range, 2.96 to 14.6%), and 10.12 ±

3.3% (range, 5.45 to 16.5%) for MAb-, PAb-MAb-, and PAb-IRMA, respectively.

Statisticalanalysis.Regression analysis wasusedto study

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IRMA FOR DETECTION OF P. FALCIPARUM ANTIGEN

11

1~~~ ~

I

5

I

i

~-. a, > .

-4 -2 o0 2 6 8 10 12

LN

psraslte.la

FIG. 1. Sensitivity of MAb- (-) PAb-MAb- (---), and PAb-IRMA(- - - )indetecting P. falciparum-infectederythrocytes from in vitro culture and P. vivax-infected erythrocytes from one patient with vivax malaria. Based on the cutoff MLC of 6.9792, 7.1433, and 7.729 for MAb-, PAb-MAb-, andPAb-IRMA, respec-tively, the sensitivity was -1.4, -0.4, and 0.6 In parasites, which is

equivalentto0.24, 0.67, and 1.82 parasites per107erythrocytesfor MAb-, PAb-MAb-, and PAb-IRMA, respectively. Symbols: *, O,

El, P. falciparum-infected cells in MAb-, MAb-, and PAb-IRMA, respectively; A, A, *, P. vivax-infected cells in MAb-, PAb-MAb-, and PAb-IRMA, respectively.

the correlation between parasite counts by microscopic examination and IRMA binding activity with the NWA STATPAK version 2.1computer program (Northwest Ana-lytical, Inc., Portland, Oreg.).

RESULTS

Sensitivity and specificity of IRMA. The assay performed on blood samples from 30 healthy individuals showed that themeanIn cpm (MLC)

(±

SD)was6.2032 ±0.194, 6.6113 ± 0.133, and7.209 ± 0.130cpmforMAb-, PAb-MAb-, and PAb-IRMA, respectively. Asamplewasconsidered positive ifitsbinding wasabove the MLC + 4 SDof6.9792forthe MAb-IRMA, 7.1433 for the PAb-MAb-IRMA, and 7.729 for the PAb-IRMA(equivalent to 1,074, 1,265, and 2,273 cpm, respectively).

Thecutoff levels in the above threesystems were used to determine the

sensitivity

ofIRMA whenvariousdilutions of P.

falciparum-infected

erythrocytes from in vitro cultures were allowed to react with antiplasmodial antibodies. The sensitivity ofthe IRMA was 0.24, 0.67, and 1.82 parasites per

107

erythrocytes with MAb-, PAb-MAb-, and PAb-IRMA,

respectively (Fig.

1).

The

specificity

ofthe assay was determined in a

serially

diluted suspension ofP. vivax-infected erythrocytes from one

patient

with vivax malariawithaninitial

parasitemia

of 0.8%

(Fig.

1) and in undiluted blood

samples

from fiveother vivax malaria patients, one of whom was

asymptomatic

(Table 1).Bindingwasbelow thecutofflevelfor each system in all cases except one

(patient

3), in which the PAb-MAb-IRMAbinding activity (7.2269Incpm)was alittleabove the cutoff MLC levelof7.1433 In

cpm).

ApplicationofIRMA toclinicalspecimens. The blood from malaria patients collected before treatment and at various times thereafter was tested in

duplicate only

once with all three systemsofIRMA, andthe resultswere

compared

with

TABLE 1. Detection of P.falciparum-infected erythrocytes in patients with vivax malaria by two-siteMAb-, PAb-MAb-, and

PAb-IRMA

Initial IRMAbinding activity(Incpm)

Patient

Patient

Age

parasitemia

Age (no. of

no. tyr) parasites/107 MAb-IRMA PAb-MAb-IRMA PAb-IRMA

erythrocytes)

1 6 72 6.5610 6.9197 6.8221

2 8 77 6.7742 6.9402 7.5858

3 7 43 6.6080 7.2269 7.3460

4 il 71 6.7708 6.4425 7.5011

5 23 41 6.6657 6.9431 7.4702

those found by microscopic examination. All 24 patients before antimalarial treatment were positive, with MLC of 10.23 + 0.58 (27,723 cpm; range, 6,002 to 46,879), 9.95 ± 0.54 (20,952 cpm; range, 5,640 to 35,850), and 9.66 ± 0.64 (15,678 cpm; range,1,569 to 28,998) with MAb-, PAb-MAb-, and PAb-IRMA, respectively. In the second week after treatment, 18of 22 patients (81.8%) tested were positive by microscopic examinationbut 20(90.9%), 19 (86.4%), and 18 (81.8%) patients were positive by MAb-, PAb-MAb-, and PAb-IRMA, respectively.TheMLC declinedto 9.08 ± 1.21 (8,778cpm; range, 725 to 26,827), 8.92 ± 1.13 (7,480 cpm; range, 994 to 25,274), and 8.50 ± 0.69 (4,915 cpm; range, 1,503 to 13,248) with MAb-, PAb-MAb-, and PAb-IRMA, respectively. In the fourth week after treatment, all 19 patients tested were negative by microscopic examination, but 10 (52.6%) were still positive by MAb- and PAb-MAb-IRMAandonly 6(31.6%)were positive by PAb-IRMA. The MLC were 7.72 ± 1.12 (2,252 cpm; range, 715 to 36,240), 7.67 ± 0.95 (2,143 cpm; range, 843 to 23,568), and7.59 ± 0.47 (1,978 cpm; range, 1,089 to 6,272) for MAb-, PAb-MAb-, and PAb-IRMA, respectively. The relationship be-tween parasite count and MAb-IRMA binding activity be-fore and after treatment is presented in Fig. 2. When parasitological counts for all samples positive by micro-scopic examination were plotted against IRMA binding activities, a positive correlation was demonstrated (r =

i

_aE

à

Wb

13,

12

il

10

j9

S8

O .1t',g;0Io..*z

Pretreatent 2 wecsafter 4 weeks after controls treatment treatment

FIG. 2. Parasitemia(Inparasitesper107erythrocytes)and MAb-IRMA binding activity (In cpm) in blood from patients with falciparum malaria before and 2 and 4 weeks after antimalarial

treatmentcomparedwithhealthycontrols.

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C>

-J

en

3

il

10

9

8-0

b

6 7 8 9 10 il LN parasitemia

FIG. 3. Correlation between parasitemia (ln parasites per 107 erythrocytes)detectedbymicroscopic examination inpatientswith

falciparum malariaand MAb-IRMAbinding activity (In cpm) (r =

0.886).

0.886, Fig. 3). With the two other assay systems (data not shown), similar findings were observed, but with a lower

degreeofcorrelation(r=0.803 and 0.638 forPAb-MAb-and

PAb-IRMA, respectively).

DISCUSSION

Detection of low-grade parasitemia has recently been a

subject ofintense investigation by RIA,enzyme immunoas-say, andDNAprobes (2, 3, 7, 11, 12).Thehighest sensitivity

ofthe assays ranged from 1 to 10parasites per106 erythro-cytes.ThislevelofsensitivitywasconsideredbyMeuwissen (14)to belowerthan thelevelofparasitemiadetected byan

experienced microscopist, who in a3-min examination (100

oilimmersionfields)couldidentifyasfewasoneparasiteper

5 x 106erythrocytes. Inan attempt toimprovethe

sensitiv-ity of the antigen detection assay, we first developed a

competitive-binding RIA with either MAb or PAb in the assay to yield, at best, the sensitivity ofdetecting only 2.2

parasites per 106 erythrocytes (10). In the present study,

three systems oftwo-site sandwich IRMAs, MAb-,

PAb-MAb-, and PAb-IRMA, were developed with the ability to

detect as few as 0.24, 0.67, and 1.82 parasites per 107

erythrocytes, respectively. Our assays were specific, since

they were positive only with P.falciparum-infected eryth-rocytes and negative when erythrocytes from 30 healthy individuals and P. vivax-infected cells were tested.

Cross-reactivity against Plasmodium malariae or Plasmodium

ovale hasnot beendetermined, mainlybecause of therarity

ofinfectionswiththese twospeciesinThailand. Ourassays

showed good correlation with parasitemia when tested against P. falciparum-infected erythrocytes from in vitro

culture aswell asfrom clinical specimens, especially when

the MAb-IRMA was used (r = 0.998 and 0.886,

respec-tively). It was observed that in most patients with parasitemiaof more than44,000 parasites per 107

erythro-cytes (0.44%), the radioactive binding did not exceed

10.7579Incpm(47,000 cpm).Thisapparentlack of increased binding with a level of parasitemia beyond 0.44% was

probably due to exhaustion of the radiolabeled antibody,

makingitunable to reactwiththeexcessnumberof parasites overthemaximum limit of theassay.

Comparisonbetweenthe three assaysystemsused in this

studyshowedthattheMAb-IRMAwassuperiortothe other

twoassaysystems for threereasons.First, itgavethe lowest

MLC (6.20) when tested with erythrocytes from healthy

individuals,whereasthose assayed with thePAb-MAb- and

PAb-IRMA showed a higher MLC (6.61 and 7.20, respec-tively).

Second,

it correlated more closely with

parasitemia

in clinical

specimens

(r =

0.886)

than the PAb-MAb- and PAb-IRMA (r = 0.803 and 0.638, respectively).

Third,

it gave higher radioactive counts with P.

falciparum-positive

specimens than the other two systems. It is therefore

rec-ommendedthat theMAb-IRMA be used in future trials with clinical specimens and in the field.

The natureof theantigendetectedwith ourassay systems has not been determined. Neither do we know whether the antigen detected is associated withlivingordead

parasites

or solubleparasite products. The high

IRMA-positive

rates for patients 4 weeks after treatment, when parasites were no longerdetectedby microscopic examination, suggested that the parasite moleculesreacting with our MAb orPAb were present evenwhenthey were degeneratedandthuscouldnot

be recognized even by experienced microscopists. Our IRMA is therefore inferiorto parasitological examination in the assessment of therapeutic cure, but it is more

practical

than parasitological examination in a large-scale survey of specimens collected from the field, especially when parasitemia is low.

The MAb-IRMA developed in our laboratory could have wide applications, including field monitoring of the malaria control program, assessing the effectiveness offield trials of afuture malaria blood-stage vaccine, detecting early

recru-descence of malaria after chemotherapy, and

screening

blood donors. The radioisotopetechnique usedinthis

study

may, at present, preclude the assays from field use, but it couldbe substituted for the enzymatic techniques,whichwe

are planning to develop in the nearfuture.

ACKNOWLEDGMENTS

We acknowledge Dasayanee Chandanayingyong, Blood Bank,

Siriraj Hospital, forherhelp in theprocurement of AB serumused for the invitroculture ofP.falciparum.

This investigation was supported in part by the International Atomic Energy Agency.

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