Acta Tropica, 47(1990)1-10 Elsevier ACTROP 00043

Evaluation of an ELISA kit for epidemiological detection of antibodies to Plasmodium falciparum sporozoites in human sera and bloodspot eluates Fulvio Esposito 1, Paolo Fabrizi 2, Alessandro Provvedi 2, Paolo Tarli 2, Annette Habluetzel 1 and Stefania Lombardi 3 1Department of Cell Biology, University of Camerino, Camerino, Italy, 2Research and Development Reagents, Diagnostic and Instrument Division, Sclavo S.p.A., Siena, Italy, and 31nstitute of Parasitology, "La Sapienza" University, Rome, Italy (Received 2 December 1988; accepted 9 May 1989) Antibodies to Plasmodiumfalciparum sporozoites represent a serological transmission indicator, which can be applied in epidemiological studies to estimate the intensity of malaria transmission. An ELISA method has been developed as an industrial kit to detect these antibodies, using a chemically synthesized (NANP)40 peptide as antigen. The results obtained with this kit are compared in the present paper with those obtained by an ELISA test already applied in epidemiological studies. In testing sera from individuals living in endemic areas, a high diagnostic concordance (92.1%) was obtained between the two assays. The absorbances of these sera correlated well, as shown by a correlation coefficient r = 0.877. Sera from individuals never exposed to malaria gave very low absorbances with the kit. This minimum nonspecific binding increases the probability of comparable results in different studies. When the two ELISAs were evaluated for analytical sensitivity and precision, similar satisfactory results were achieved. The test can be performed not only with sera but also with eluates from filterpaper bloodspots. Modifications of the kit to reduce its cost and suggestions regarding distribution and funding are also proposed. Key words: Plasmodium falciparum; Synthetic circumsporozoite peptides; Antibodies; ELISA; Malaria epidemiology

Introduction A n t i b o d i e s to s p o r o z o i t e s r e p r e s e n t a serological t r a n s m i s s i o n i n d i c a t o r , w h i c h c a n be a p p l i e d in e p i d e m i o l o g i c a l studies to e s t i m a t e the i n t e n s i t y o f m a l a r i a t r a n s m i s s i o n ( D r u i l h e et al., 1986; E s p o s i t o et al., 1986; H o f f m a n n et al., 1986, 1987; T a n n e r et al., 1986; D e l G i u d i c e et al., 1987a). P r e v a l e n c e a n d levels o f a n t i b o d i e s to Plasmodium falciparum s p o r o z o i t e s , m e a s u r e d in i n d i v i d u a l s l i v i n g in e n d e m i c areas, h a v e b e e n s h o w n to c o r r r e l a t e with the e n t o m o l o g i c a l i n o c u l a t i o n rate assessed at the s a m e t i m e in the s a m e place ( E s p o s i t o et al., 1988). I n s i t u a t i o n s in w h i c h it is difficult to a p p r a i s e e n t o m o l o g i c a l d a t a q u a n t i t a t i v e l y , the serological t r a n s m i s s i o n i n d i c a t o r m a y p o s s i b l y e v e n serve as a s u b s t i t u t e for the e n t o m o l o g i c a l o n e in v e c t o r c o n t r o l p r o g r a m s ( L o m b a r d i et al., 1988).

Correspondence address." Dr. Paolo Fabrizi, Research and Development Reagents, Diagnostic and Instrument Division, Sclavo S.p.A., Via Fiorentina 1, 53100 Siena, Italy. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

Antibodies that bind to the surface coat of P.falciparum sporozoites have been demonstrated to bind to tandem repeats of the aminoacid sequence NANP (Asparagine-Alanine-Asparagine-Proline), which form the immunodominant part of the circumsporozoite protein (Zavala et al., 1985). Several laboratories have synthesized NANP repeats of variable length, either chemically (Zavala et al., 1985; Ballou et al., 1985) or by recombinant DNA technology (Young et al., 1985), and have set up immunoassays to detect humoral immunity to P.falciparum sporozoites (Zavala et al., 1986; Campbell et al., 1987; Del Giudice et al., 1987b; Webster et al., 1987; Habluetzel et al., 1989). As different peptides and assay protocols were applied, the possibility of comparing the results obtained becomes difficult. In addition, the use of the assay has been confined to institutions which have access, either directly or through collaboration to synthetic peptide or recombinant DNA technology. The commercial availability of standardized reagents could help to obtain more comparable results and facilitate the use of this epidemiological tool by all interested institutions, especially in endemic countries. An ELISA kit to measure antibodies to P. falciparum sporozoites using as antigen a chemically synthesized (NANP)4 o peptide (Verdini & Pinori, Italian Patent Application No. 1718, 1985) has been developed by Sclavo S.p.A., Diagnostic Division, Siena, Italy. The sensitivity, specificity and precision of the kit are discussed in the present paper in comparison with an ELISA protocol already applied in epidemiological studies.

Materials and Methods

Antigen, conjugate and substrate The peptide used to coat the polystyrene microtiter plates was (NANP)4 o, synthesized by Eniricerche/Sclavo, Monterotondo, Roma, Italy. The alkaline phosphataseconjugated goat antibodies to human immunoglobulins, the p-nitrophenylphosphate (p-NPP) substrate tablets and the diethanolamine substrate buffer were prepared by Sclavo S.p.A., Siena, Italy. These reagents were used in both ELISAs.

Sera Two panels of sera were assayed: panel 1, consisting of sera from 50 individuals never exposed to malaria infection, and panel 2, consisting of sera from 139 individuals living in endemic areas. To evaluate analytical sensitivity and precision, two pools of positive sera selected from panel 2 (sera b and c) and/or a positive serum from a single donor (serum a) were used.

Bloodspots Blood samples were prepared by mixing equal volumes of pelletted human erythrocytes from a non-immune European donor with individual sera from panels 1 and 2. Spots of 10 ~tl were blotted on Whatman No. 1 filter paper, dried and stored at room temperature.

Industrial kit (ELISA S2-kit) The kit includes a couple of microtiter plates (Nunc, Denmark, medium binding capacity, cat. No. 469078), one coated with (NANP)4 o (antigen plate) and the other with bovine serum albumin (BSA, control plate). The washing buffer provided is powdered phosphate-buffered saline (PBS=NaC1, 0.145M; Na2HPO4.2HzO, 0.0077 M; NaH2PO4-H20, 0.0027 M) containing Tween 20. The elution/dilution buffer for bloodspots, sera and conjugate is provided as a concentrated solution of 0.6 M PBS pH 7.2, containing 0.4% BSA, 4% Tween 20 and 0.02% sodium azide as preservative. This solution has to be diluted 4-fold before use. Lyophilized control sera at four different levels of antibodies to N A N P (negative, low, medium and high positive) are also included. To perform the assay, serum samples were diluted 1:200 and bloodspots were eluted for 60 min in 0.8 ml of buffer to get a final serum concentration of 1:160. Samples and control sera were transferred in duplicate to the antigen and control plate in volumes of 100 ~tl/well. After 1 h incubation, the contents of the wells were aspirated and the plates washed five times for 1 min with washing buffer. The conjugate was added to the wells (100 Ixl/well) at a final dilution of 1:600 and incubated for 1 h. The washing procedure was repeated and the substrate was dispensed into the wells (100 txl) as a 1-mg/ml solution of p-NPP in 1.1 M diethanolamine buffer pH 9.8, containing 0.6 mM MgC1 a. After 1 h the reaction was stopped with 25 ~tl/well of 3 M NaOH. All the steps were performed at room temperature (20-25°C). The absorbance against substrate blank was read at 405 nm. The net absorbance of each serum was calculated as the difference between the absorbances of the antigen- and control plates. The performance of the kit is not modified after 1 month of storage at 37°C or 12 months at 4°C.

The compared protocol (ELISA CAM) The following buffers, sterilized by filtration, were used: 0.15 M PBS, pH 7.2, containing 0.02% sodium azide, as coating buffer; PBS containing 0.05% Tween 20, as washing buffer; PBS containing 1% Tween 20 and 0.1% BSA (Serva, F.R.G., cat. No. 11930), as eluent/diluent for bloodspots and sera; PBS containing 0.05% Tween 20, 0.1% BSA and 5% pig serum, as conjugate diluent. The substrate buffer was the same as that used in the ELISA S2-kit. A general purpose microtiter plate (Greiner, Switzerland, cat. No. 655101) was chosen because of its low cost. Coating was done overnight with NANP4o and BSA at a concentration of 1 and 10 ~tg/ml respectively, adding volumes of 100 ~tl to the wells of the antigen plate and 150 ixl on the control plate. The antigen plate was washed four times with washing buffer and postcoated overnight with 150 Ixl/well of 10 ~tg/ml BSA. Antigen and control plates, after postcoating and coating respectively, were washed twice with washing buffer and three times with distilled water, dried under vacuum and stored in a desiccator. The same assay procedure was applied as described for the ELISA S2-kit, except that sera and bloodspot eluates were used at lower dilutions (1:100 and 1:80, respectively) and the conjugate at a lower concentration (1:800).

4 Results

Positivity threshold and binding to BSA The sera from the 50 individuals never exposed to malaria (panel 1) gave mean net absorbances of 0.010 (0.011 SD) and 0.029 (0.044 SD) with the ELISA S2-kit and ELISA CAM, respectively (Fig. 1). A positivity threshold was taken to be at least 0.050 absorbance units above the highest sample value. This criterion was fulfilled for both tests by taking ten times the mean value of the net absorbance. The positivity thresholds were therefore set at 0.100 (corresponding to the mean plus 8 × SD) for the ELISA S2-kit and 0.290 (mean plus 6 × SD) for ELISA CAM. Binding to BSA-coated plates was measured with sera from panel 1 and 2 (2 = exposed to malaria). The mean values obtained were 0.006 (0.005 SD) for panel 1, and 0.004 (0.005 SD) for panel 2 with the ELISA S2-kit. With ELISA C A M the values were 0.027 (0.021 SD) for panel 1 and 0.023 (0.029 SD) for panel 2.

Sensitivity The analytical sensitivity of the two ELISAs was compared by testing 3 positive sera (a, b and c) serially diluted 2-fold with negative sera as diluent. With the ELISA S2kit, sera a, b and c reached net absorbances (values on antigen-coated plate, minus values on BSA plate) lower than or equal to that of the positivity threshold at the dilutions 1:3200, 1:6400 and 1:6400 respectively (Fig. 2a).

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With E L I S A CAM, the corresponding dilutions for the same sera were 1:1600, 1:3200 and 1:6400 (Fig. 2b). Comparing the number of 2-fold dilutions starting from the working dilution of each E L I S A protocol, the positivity thresholds were reached with sera a and b at the 5th and 6th dilution with both assays. Serum c

became negative with the ELISA S2-kit at the 6th and with ELISA CAM at the 7th dilution.

Precision Three sera, with different levels of antibodies, were tested in 38 replicates to check the precision within one assay, and in duplicates over 4 successive days to evaluate the assay-to-assay precision. The mean coefficient of variation (CV) for the within-assay precision was 14.4 (range 8.5-19.1) with ELISA CAM and 11.9 (10.8-14.2) with the ELISA S2-kit. The same parameter assessed for the assay-to-assay precision was: 19.3 (10.5-33.5) for ELISA CAM and 17.2 (13.6-19.9) for the ELISA S2-kit. The lower assay-to-assay precision with both ELISAs, compared to the within-assay value, reflects the sensitivity of the assays to small day-to-day variation in room temperature and reagent preparation.

Correlation and diagnostic concordance The net absorbances obtained with the 139 sera from panel 2 correlated satisfactorily between the two assays, as shown by a correlation coefficient r = 0.877 (p < 0.001) and a slope of the regression line b = 0.651 (Fig. 3). The apparent upward trend for the

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