| The purpose of research is the establishment of a competitive enzyme-linked immunosorbent assay for fungal 1,3-?-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-?-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-?-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. |
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