Sample collection and reference samples
Clinical samples (n=516) were collected between 2008 and 2012 from patients (6–50 years old) with fever and suspectable for malaria from Henan (n=90) and Yunnan (n=202) provinces of China (total n=292) and from Myanmar (Burma) region (n=224), where malaria is endemic and is caused mainly by Plasmodium falciparum as shown in Figure 1. The study was approved by the ethics committee of Yunnan Institute of Parasitic Diseases. All participants or legal guardians signed informed consent before participant enrolment and sample collection. Complete anonymity was maintained at each stage of the study. The research has been also approved by the national engineering laboratory of point-of-care testing (Wondfo Biotech company, Guangzhou, China).
The diagnosis of malaria follows Diagnostic Criteria for Malaria issued by the Ministry of Health of the People's Republic of China. Patients were included if they ①came from relatively serious malaria epidemic area and ②suffered from fever of unknown origin. Patients were excluded if they ①suffered from vivax malaria or ②had mixed infections. The samples were confirmed as single malignant malaria infection by microscopic examination of thick blood smear.
An expert medical technologist collected approximately 5 mL of blood from adult subjects and 3 mL from minor subjects by venipuncture. Thick and thin blood films were prepared in duplicate using two drops of blood for each sample. The remaining blood was preserved at −20°C in EDTA tube. Thick blood films were stained with 10% Giemsa and microscopic examined by experienced technicians.
Positive references provided by WHO were divided into 5 geographical areas, comprising Africa (Benin I and Nigeria XII), the Americas (Santa Lucia), Australia (Papua New Guinea-FC27/A3) and Southeast Asia (Philippines- PH1).
Expression and purification of HRP 2 protein
Plasmodium falciparum genomic DNA was extracted from clinical blood samples provided by Prof. Hui Liu (Yunnan Provincial Institute of Parasitic Diseases, Simao, Yunnan, China), using QIAamp DNA Mini Kit (QIAGEN, Germany). Pair of oligonucleotide primers was designed, complementary to forward and reverse strands of the HRP 2 exon II based on the sequence obtained from GenBank (U69551.1). The forward primer was HRP 2-F: 5׳-TATACATATGAATAATTCCGCATTTAATAATAACT-3׳, and the reverse primer HRP 2-R: 5׳-TACAGTCGACATAGACGTACTTCTTTTCGTAAG-3׳. The forward and reverse primers were designed to contain NdeI and XhoI restriction sites, respectively. Restriction endonucleases (NdeI and XhoI), DNA markers and PCR mix were purchased from Dongsheng Biotech Company (Guangzhou, China), while the T4 DNA Ligation mix was obtained from TaKaRa Biotech Co. Ltd (Dalian, China). The HRP 2 gene was cloned into a pET30a vector and transformed into CaCl2 competent E. coli BL21 (DE3) cells [17]. pET-30a plasmid and BL21 (DE3) cells were provided by Prof. Jufang Wang of South China University of Technology. Starter culture was grown overnight at 37°C in 5 ml LB medium, supplemented with kanamycin (50 mg/L). This stationary phase culture was used to inoculate 1 L LB medium to an OD600 of 0.6. The latter culture was inducted with 1 mM IPTG for target protein expression at 30°C and 225 g, overnight. Cells were harvested by centrifugation at 8000 g for 3 min and cell pellets resuspended in lysis buffer consisting of 100 mM Tris–HCl, pH 9.6. Cell suspensions were ultrasonicated on ice and centrifuged at 4°C at 12000 g for 15 min. The resulting supernatants were purified on a Hi-Trap Ni2+ column (GE, USA) using AKTA Purifier and the purified recombinant HRP 2 protein (rHRP 2) fractions visualized on 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [18]. The protein samples (>90% purity) were purified by dialyzing against 50 mM carbonate buffer, pH 9.6.
Animal immunization and mAb production, screening, purification and characterization
Six-to-eight-weeks-old Balb/c mice were purchased from the Animal Laboratory Center of Sun Yat-sen University (Guangzhou, China). All experimental procedures were in compliance with the Guide for Care and Use of Laboratory Animals (NIH version, revised 1996). All animals were allowed free access to food and water.
MAbs were produced as described by Kohler and Milstein [19] and screened by indirect ELISA described below. The IgG fractions were prepared by ammonium sulfate precipitation and purified on Protein A column. The titers were determined by indirect ELISA and mAb isotyping was carried out using mouse mAb isotyping kit purchased from Sigma-Aldrich (St. Louis, MO, USA) according to the manufacturer’s instructions. The specificity of monoclonal antibodies was determined by their binding affinity to both rHRP 2 and P. falciparum infected blood samples. SDS-PAGE was carried out on 12% separating and 5% stacking gels, followed by protein transfer to polyvinylidene fluoride (PVDF) membranes. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., USA) was applied for visualization. Determination of antibody affinity was carried out by a noncompetitive ELISA as described by David Beatty [20] with little modifications.
Indirect ELISA
Microtiter plates were coated with 1 μg/ml of purified rHRP 2 in coating buffer (0.05 M carbonate buffer, pH 9.6). After overnight incubation at 4°C, plates were rinsed with washing solution (0.15 M PBS 0.1% Tween 20, pH 7.4) and blocked with 3% bovine serum albumen (BSA) for 2 h. 100 μL hybridoma cell supernatants were added to wells and incubated for 1 h at 37°C. After washing, 100 μL goat anti-mouse IgG-HRP (ZSGB-BIO, China, 1:20000) antibodies were dispensed into each well followed by 1 h incubation at 37°C. The enzymatic reaction was visualized using TMB substrate with hydrogen peroxide and stopped with 2 M Sulphuric acid. Optical density (OD) was measured spectrophotometrically at 450 nm. Samples were considered positive with OD of cell supernatant (P) to negative control (N) ratios greater than 2.1.
Sandwich ELISA
Purified mAbs were paired, labeled with periodate oxidation and sandwich ELISA was performed as described before [21]. The lower the OD, the two strains of antibody antigen epitope closer; the higher the OD value, illustrate both the antigen epitope is more far.
Establishment of POCT lateral immunochromatographic assay
HRP 2 specific mAb 1A5 (1.5 mg/ml) and goat-anti-mouse IgG (2.0 mg/ml) were diluted in 0.05 M carbonate buffer and sprayed on a nitrocellulose membrane at 1.5 and 1 μl/cm2 to form test and control lines, respectively. The prepared nitrocellulose membranes were kept in a dry pack at 30°C and 30% humidity. 1C10 antibody were conjugated to colloidal gold as described by Oliver [22] and sprayed on glass fiber at 12 μL/cm2. The strip was assembled by placing 0.7 cm of antibody-gold conjugate glass fiber a few centimeters from the test line and 4 mm wide test strips were cut. Test strip preparation was done in a good manufacturing practice (GMP) workshop.
Sample evaluation and sensitivity calculation
Positive control samples provided by WHO were evaluated alongside 516 clinical samples and test results were compared with microscopic examinations. About 5 μL of each sample was applied to the sample site and three drops of dilution buffer added. Test strips were observed for 15 min and results were interpreted as positive when both the control and test lines turned red and negative if only the control line turned red. In cases where the control line did not appear, the results were considered invalid and the assay repeated.
Sensitivity of the test equals the ratio of total positives to total number of samples, specificity equals the ratio of total negatives to total number of samples, positive predictive value equals the proportion of the number of positive cases detected among actual positives cases and negative predictive value equals the proportion of the number of negative cases detected in relation to actual negatives.
Statistics
Statistical analysis was performed with SPSS 18.0 software (SPSS, Chicago, IL). Data obtained from both microscopic examination and immunochromatographic test were analyzed. Sensitivity and specificity of the assay and how it compares with the standard microscopic examination was analyzed. Agreements between both data were computed by Kappa statistics. Differences were considered statistically significant at p < 0.05.