| Malaria is a parasitic disease spread by the Anopheles and causes serious harm to human health. Latest WHO data reveals that as at 2014, there were 66 million people worldwide deaths caused by malaria and its related diseases, out of which 90% of such cases occurred in Africa, most of whom are children under five years of age. The effect of the prevalence of malaria in endemic areas on the health and life of people has been devastating and serves as a serious impediment to the development of such societies. The WHO therefore recommends that all suspected malaria patients be immediately tested with rapid and accurate diagnostic method to enable proper use of anti-malaria drugs, so as to avoid drug resistant strains of the Plasmodium species. This would essentially help to control disease progression in patients, and drastically reduce mortality rates. Plasmodium falciparum is the most dangerous form of the malaria causing agents. Plasmodium falciparum secrets a unique water-soluble biomarker which is the Plasmodium falciparum Histidine-rich protein 2(Pf HRP2).This study aims at exploring the Pf HRP2 as a biomarker in the diagnosis of malaria. The research content and results are as follows:(1) Naturally occurring Pf HRP2 protein extraction and recombinant protein expression: In-vitro cultures of Plasmodium falciparum strains were established and the naturally occurring HRP2 proteins were extracted from the supernatant of these cultures. Recombinant HRP2 proteins were also overexpressed in bacteria cells. In order to obtain a more concentrated antigen, the supernatant of the established cultures were collected and the infected red blood cells were lysed to release the cytoplasmic antigens present in the red blood cells after which affinity chromatography was used to purify the secreted HRP2 antigen. Pf HRP2 gene sequence was also constructed into plasmids carrying bacteriophage T7 promoters for further expression into the p ET-30a(+) vector. The Pf HRP2 sequence was inserted behind the bacteriophage T7 promoter for transcription and translation signalling to enable the use of the host cell T7 RNA polymerase for its translation. The result of the experiment showed the correct construction of the sequence into the expression vector forming the p ET3-a(+)/HRP2 plasmid. This recombinant plasmid was further used to transform competent BL21(DE3) cells for overexpression of soluble protein showing good immunogenic properties, as those extracted from the cultured Plasmodium falciparum, after subjecting it to Pf HRP2 rapid diagnostic test(RDT) and Western blotting analysis. Both the naturally occurring and recombinantly expressed Pf HRP2 antigens were used to immunize mice in the production of high affinity Pf HRP2 monoclonal antibodies for the development of sensitive Pf HRP2 point-of-care testing(POCT) of Plasmodium falciparum.(2) Preparation and identification of Pf HRP2 specific monoclonal antibody:This study explores the principles and techniques in the preparation of monoclonal antibody to optimize the existing immunization protocol and the mode of screening to enhance the selection of highly sensitive antibodies. BALB/C mice were immunized with the recombinant HRP2 protein. The positive clones were screenedwith both the naturalprotein and recombinant protein and the selected clones were later injected into mice for ascite preparation and purification. Four stablehybridoma cell lines secreting anti- Pf HRP2 monoclonal antibody(3A5,1F6, 1C10,5C7) were obtained. Out of the fourhybridoma clones, one was of the Ig M subtype and the other 3 were of the Ig G1 subtype. Antibody affinity of these clones were greater than 10-7,indicating ahighaffinity of these monoclonal antibodies for Pf HRP2 protein.Western blotand RDTresults show that these four antibodies reacted favourably with Pf HRP2 protein.(3) Peptide sequence and phage peptide library screening of HRP2 MAb-antigen bindingepitopeexperiment:HRP2 is a kind ofpolymorphicprotein. In order to explorethe epitopes involved in the binding of the four MAbs(3A5, 1F6, 1C10, 5C7) with HRP2 antigens, peptide sequencescanningmethod and twelve random phage peptidelibraries forepitopeanalysis and identification were used.Experimental results showed that therecognition site of the MAbs include both linear and conformational epitopes positions.The amino acid sequences that were identified include: HHAH, AT(S/L)DHHAAN(S)HH and ADHH.The 1F6 MAb has a conformationalpeptide sequence at its epitope, and cannot be identify using a peptide scan, however, its epitope site was accurately recognized among the twelvephage peptide libraries used. The effect of the various MAb epitopes in the selection of the antibody pairs for used in POCT kits was further investigated to determine the most precise and best combination of these antibodies for use in detecting Pf HRP2 amino acids.(4) Establishment and evaluation of HRP2 immunofluorescence rapid diagnostic technique. In this study, fluorescent nanoparticles were used to label different sites of the Pf HRP2 MAbs through a double antibody capture sandwich method. Using fluorescence spectrophotometer, the fluorescent signals were transformed into electrical signals for detection and evaluation on a computer screen display. This method establishes a novel rapid Pf HRP2 immunofluorescence POCT detection method and reagents for diagnosis of malaria. Experimental results showed that the limit of detection was about 25 / μL parasite density, a significant improvement in its sensitivity than most conventional immunochromatographic RDT reagents. In the development of this reagent, precision, stability, cross-reactivity, and the overall relative performance of this test showed that these indicators met the standard requirement for universally accepted RDTs. Clinical trials in parallel and multi-center study showed a positive predictive value of 100% and a negative predictive value of 99.6%, and an overall sensitivity of 99.7%.This study underlines the use of Pf HRP2 protein as a biomarker in the diagnosis of malaria. The study comprises the extraction of naturally occurring HRP2 protein in cultures, the expression of recombinant antigens, monoclonal antibodies preparation and an in-depth analysis of the various epitopes involved in HRP2 antigen-antibody system for the establishment of immunofluorescence detection platform. Innovation embodied in this study includes: firstly, the introduction of new immunization strategies and screening methods, preparation of four strains with high affinity and specific anti Pf HRP2 protein including Ig M and Ig G subclasses of different monoclonal antibodies; secondly, the use of peptide sequence scanning and phage display technologies to identified Pf HRP2 epitopes cluster, for the establishment of next generation sensitive, specific and accurate and real-time detection reagents or other diagnostic methods or ideas for future investigation; thirdly, the establishment of a novel high sensitivity immunofluorescence detection method and corresponding Pf HRP2 reagent, for Pf HRP2 marker detection that would eventually replace low sensitive and ineffective diagnostic methods currently used for malaria diagnosis... |
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