| Talaromyces marneffei(TM)is the only temperature-dependent biphasic fungus in the genus Talaromyces,which can cause infection of T.marneffei,and it is regionally distributed in Southeast Asia and southern China.The majority of patients are HIV-infected populations.In recent years,non-HIV-infected patients with T.marneffei infection have gradually increased.Due to its lack of specificity in clinical symptoms and imaging manifestations,laboratory tests of T.marneffei infection currently rely mainly on culture or histopathological diagnosis,but the culture takes a long time,has low sensitivity,and pathological tissues are not easy to implement.Serological testing of other fungal antigens lacks a stable,approved kit that can be used in clinical testing,which leads to long diagnosis of TM infection and high mortality.Mannoprotein 1 protein(Mplp)is a TM specific antigen with abundant expression and can be used for laboratory diagnosis of TM infection.In this laboratory,rabbit polyclonal antibodies(PAb)specific to Mp1p and 18 monoclonal antibodies(MAb)that recognize different epitopes were prepared.A dual-antibody sandwich ELISA system with multiple antibodies and monoclonal antibodies was established.After clinical evaluation,it has certain sensitivity(75%)and specificity(99.4%).However,it is unclear whether it can be used for early detection,and because rabbit polyclonal antibodies are used as capture antibodies,batch differences are easy to produce.Therefore,in this study,we used monoclonal antibodies for a variety of free combinations,and optimized them to mix monoclonal antibodies.Instead of polyclonal antibodies,an optimized TM Mp1p antigen capture ELISA system was established and its role in the early diagnosis of TM infection was discussed.This study consists of three parts:Part ?:Optimization of Mp1p antigen capture ELISA detection systemBased on the preparation of 18 monoclonal antibodies in the previous stage,this study combined the analysis of binding epitopes of different MAbs in the results of previous experiments,indirect immunofluorescence to identify the strength of binding of MAb to TM cells,and the immunohistochemical experiments of MAb and intra-infected tissue.As a result of the cell wall binding strength of T.marneffei,different antibodies were identified among 18 MAbs and had strong binding reaction with the natural antigen of T.marneffei,and they were freely combined and matched to select the best mixed monoclonal antibody.The combination is used as a capture antibody,and one of the antibodies is selected as a detection antibody for pairing.The mixing ratio,coating concentration,and detection antibody concentration of each monoclonal antibody in the coated antibody are optimized to establish a polyclonal antibody,establish a double-antibody sandwich ELISA system with sensitivity similar to or more sensitive than the original multi-antibody monoclonal antibody system.The results showed that the best results were obtained by using four monoclonal antibodies(M4,M6,M8,and M14)for mixed coating.The coating concentration was 10 ?g/ml,and the optimal coating ratio was 4:3:2:1,the best detection antibody is still M12-Biotin,which is the same as that of the original polyclonal antibody and monoclonal antibody detection system.The working concentration of detection antibody(M12-Biotin)is 1:1000 dilution,and the working concentration of Streptavidin-HRP is 1:1000.Part ?:the performance evaluation of the optimized ELISA detection system for the capture of Mp1p antigen of T.marneffei by double antibody sandwich methodBased on the previously established multiple antibody-mAb paired detection system and the optimized mixed monoclonal antibody-mAb paired detection system in the first part,in order to avoid the difference of Mp1p antigen levels in different isolates,we selected 4 clinical isolates TM in The culture supernatants prepared with different initial spore concentration and different culture time were simultaneously tested for recombinant Mp1p to determine the difference in sensitivity between the two groups of different detection systems.At the same time,the culture supernatants of other fungi common to clinical infections are detected to determine whether there is a cross-reaction with other fungal infections.The results showed that the levels of Mp1p secreted by different clinical isolates of TM strains were different,and neither of the two detection systems could detect the culture supernatant when the initial culture concentration was lower than 1 × 105/ml,which may be related to the low-level secretion of Mp1p during TM morphological transformation.However,the system was more sensitive after the optimization,and the minimum detectable supernatant was 1 × 105/ml for 3 days.Moreover,when testing the culture supernatant of 4 strains,the optimized system showed a higher A450 value,and it could detect 160-fold diluted culture supernatant,while the original system could only detect 40-fold diluted culture supernatant.The detection of recombinant protein Mp1p gradient dilution showed that both systems can detect Mp1p antigen at 125 pg/ml and have the same sensitivity for detecting recombinant protein.Simultaneously,the specificity of the two systems is similar,and there is no cross-reaction with the culture supernatants of other common clinical fungi(Candida albicans,Cryptococcus neoformans,Aspergillus fumigatus,Aspergillus flavus,Aspergillus terreus,Penicillium citrinum)Part III:Evaluation of Preliminary Clinical Application of ELISA System Optimized by Double Antibody Sandwich Method to Capture Mp1p Antigen of T.marneffeiWe further explore the value of the optimized detection system in the early diagnosis of T.marneffei infection through the detection of clinical serum samples.Serum of 202 normal persons and 53 cases of other fungal infections collected by the Department of Laboratory Medicine of Zhujiang Hospital(1 confirmed Candida infection,5 confirmed proven Aspergillus infection,7 probable Aspergillus infection,36 possible Aspergillus infection,confirmed 1 case of spore infection,1 case of confirmed pulmonary mucormycosis,and 2 cases of confirmed cryptococcosis).The specificity of the detection system was evaluated,and the sensitivity of the detection system was evaluated with 20 sera from 5 patients with confirmed TM infection.The results showed that the optimized system and the original system were used to simultaneously detect the serum of 202 normal persons diluted 10-fold,and the critical value was established.Further testing the sera of 53 patients with other fungal infections to verify the specificity of the detection system.The test results were all negative,suggesting that there is no cross-reactivity with other common clinical fungal infections,and the specificity is 100%(255/255).Subsequently,20 sera of diagnosed TM-infected patients were tested.The results of the two systems were the same,and 19 of them were positive,with a sensitivity of 95%(19/20),the positive predictive value(PPV)of both test systems was 100%,and the negative predictive value(NPV)was 99.6%.Analysis of 5 confirmed cases of TM found that the optimized ELISA detection system could produce a clinical report 2-10 days earlier than the culture method,suggesting that the system could be used as an auxiliary method for early diagnosis of TM infection laboratories.After evaluation of clinical specimens,it is suggested that the optimized mixed monoclonal antibody-MAb detection system has similar sensitivity and specificity as the original multiple antibody-MAb detection system.However,the raw materials of the ELISA system can use monoclonal antibodies instead of polyclonal antibodies,simplifying the process and reducing batch differences.The conclusions:1.Based on the original antibodies,a double-antibody sandwich(4 monoclonal antibodies mixed with 1 monoclonal antibody as the detection antibody)system was successfully established,and the system was optimized to provide a basis for early diagnosis of TM infection;2.Compared with the original detection system,the TM supernatants of different sources,different concentrations and different time culture supernatants are evaluated.The optimized system can detect Mplp secreted by different strains and is more sensitive.And the detection of recombinant Mp1p has the same sensitivity and no cross reaction with other fungal culture supernatants;3.The optimized detection system after clinical patient serum evaluation has similar sensitivity and specificity to the original detection system and has better performance.It is reported to the clinic 2-10 days before the culture method and can be used for the auxiliary diagnosis of clinical TM infection. |
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