| The purpose of research is the establishment of a competitive enzyme-linked immunosorbent assay for fungal 1,3-β-D-glucan,and use as clinical research.The main content includes preparation of glucan immunogen,preparation,Purification,screening as well as enzyme labelling of polyclonal antibody,development,optimization and performance verification of the competitive enzyme-linked immunosorbent assay(ELISA).Firstly,the study developed 7 types of immunogen,which included bo-vine serum albumin-conjugated laminarin,carboxymethyl pachyman and carboxymethylated curdlan(1,3-β-D-glucans)by using chemical coupling methods.Two types of Aspergillus fumigatus were cultured for spores and fungal extracts.Secondly,each immunogen was used to immunize 3 male New Zealand white rabbits.After the sixth immunization when the antiserum titers remained unchanged,whole blood was collected to obtain antiserum.High titer antiserum that had weak cross-reactivity was separated,and the highest titer polyclonal antibody Ab-3B was labelled with horseradish peroxidase(HRP)and purified.Finally,the study was established a competitive ELISA and optimized antigen coating,fluid handling,concentration of enzyme labelled antibodies as well as the standards.After immunization,among antiserums,the titers of antiserums for 3 types of glucan-BSA conjugates have exceeded 1:64,000 and showed little cross-reactivity with other fungal polysaccharides.The antiserum obtained from immunization of carboxymethylated curdlan-BSA conjugate had the highest titer with over 1:128,000.Enzyme labelled antibody HRP-Ab3B was obtained with the protein concentration of 0.629 mg/mL and the antibody titer of 1:8,000.The performance of competitive ELISA for fungal 1,3-β-D-glucan was validated.The coefficient of variation for repeated tests was<15%,with a linear detection range of 3.125-200 pg/mL and detection limit of 2.130 pg/mL.The recovery range for spiked 100,25 and 6.25 pg/mL serum antigen was 86.9-115.3%.The stability of various components of competitive ELISA could be maintained for at least 10 days at 37℃.The detection system could effectively detect low concentrations of Aspergillus fumigatus,Candida albicans and high concentration of Cryptococcus neoformans from serum samples.Meanwhile,the system itself demonstrated little interference against 5 types of pathogenic bacteria,including Mycobacterium tuberculosis,Escherichia coli,Salmonella,Klebsiella and Staphylococcus aureus.With carboxymethylated curdlan used as a coated antigen and enzyme labelled antibody HRP-Ab3B used as a detection antibody,a competitive ELISA successfully developed for detection of invasive fungal disease.The whole ELISA detection process could be quickly completed within 2 hours. |
