Establishment Of A Method For Rapid Malaria Diagnosis

Objective Malaria is a very important parasitic disease caused by the protozoan Plasmodium, it is endemic in tropical and subtropical regions and paid high attention to by these governments. The rapid, sensitive and reliable methods of malaria diagnosis not only can bring the treatment to patients in time, but also can avoid the abuse of the antimalarial drugs. New technologies aiming to achieve more rapid, simple and reliable diagnosis have been developed in order to overcome the limitations of traditional methods for malaria diagnosis. The microfluidics system is the technology of manipulating and controlling fluids and particles at micron and submicron dimensions, which could provide high surface area-to-volume ratio, fast mass-heat transfer and improved local control. The microfluidics technology also allows smaller, cheaper and smarter sensors and devices, promising to revolutionize the detection of pathogens. In this study, we established a novel microfluidic chip platform for the rapid diagnosis of malaria in clinical use.Methods1. A19-kDa fragment of the C-terminal of merozoite surface protein1(MSP1-19) and the F2fragment of a175-kDa erythrocytes binding protein (PfF2) were expressed in E. coli and purified. The microchips were fabricated using Polydimethylsiloxane (PDMS). The recombinant antigen MSP1-19/PfF2were captured by Protein A microspheres with anti-His-Tag antibody, and then injected into the microchannels of the chip. After blocking with5%BSA-PBS, the sera (1:100dilution) of patients infected by P. falciparum were injected into the microchannels. And then the FITC labeled rabbit anti-human antibody (1:100dilution) were injected into the microchannels, too. The fluorescence intensity was observed by a fluorescence microscope.2. Total mRNA of P. falciparum (strain FCR3) was extracted from its schizonts, and then reverse transcribed to cDNA, which used as a template for amplification of the Pfldh gene. The amplified Pfldh gene was then cloned into pQE-30vector, and transformed to E. coli M15cells. The expression of recombinant PfLDH was induced with IPTG at37℃for4hours. The protein was purified by Ni2+affinity chromatography and the enzyme activity of purified rPfLDH was measured by performing NBT colorimetric tests. BALB/c mice were immunized by purified rPfLDH intraperitoneally, and then the splenocytes of mice were used to create hybridoma cell lines. The specific antibodies secreted by hybridoma cells were screened by Hybridoma Technique. The selected mice monoclonal antibodies (McAb) against PfLDH were purified by using Affi-Gel Protein A MAPS Ⅱ Kit. The relative affinity and specitivity of McAb were measured by ELISA and Western blot. The cross activity of McAbs reacting with human blood proteins was also detected. The dissociation constant equilibrium (KD) of McAb were mesured by Octet system of ForteBio company.Results1. The recombinant MSP1-19and PfF2were expressed and the purity were both more than95%. The volume of serum used for serological detection based on the microfluidic chip technology was less than1μL and the amount of the other reagents was also greatly reduced. The time of detection could also be limited to less than1hour and the results were consistent with ELISA.2. The Pfldh gene of P. falciparum (FCR3) was amplified by RT-PCR and sequenced. The sequence was completely identical with that of3D7strain. An expressing plasmid was constructed and the recombinant PfLDH was expressed and purified. McAb against PfLDH with high specitivity were selected successfully. The positive hybridoma cell lines were1A1D9,1C3G4,1F1C4,1G6D10,2F4F7,3D10D10,4A6F3,4G11B4,5G10B4,5C2E8and6F2D9, and the antibodies secreted by1C3G4,4A6F3,5G10B4and6F2D9could recognize linear epitopes of PfLDH. The ELISA results showed the McAb had no cross activity with human blood proteins. The relative affinity of McAb binding PfLDH was1C3G4>6F2D9>5C2E8,1F1C4>5G10B4>4G11B4>4A6F3>1A1D9. The KD of1A1D9,1C3G4,1F1C4,5C2E8and6F2D9were2.04×10-10,7.88×10-12,2.68×10-11,2.56×10-11and9.21×10-11, respectively.Conclusion Serological diagnosis of malaria based on the microfluidic chip technology showed its unique advantages over conventional platform such as integration, higher throughput, lower reagent consumption, and it could be useful for epidemiological studies, for the sera screening of blood donors from regions where the malaria is not endemic and for the diagnosis of infected individuals. The gene of lactate dehrdrogenase of P. falciparum FCR3strain was cloned successfully, and the rPfLDH with high enzyme activity was highly expressed in E. coli. The acquirement of anti-PfLDH monoclonal antibody laid the foundation for the detection of malaria based on the microfluidic chip system.