Development Of UPT Immunochromatographic Strip For Rapid Detection Of Brucella Antigen

Brucellosis caused by Brucella is one of the most important bacterial zoonoses endemic in the whole world. Brucella infects domestic and wild animals, leading to abortion and infertility,,causing great economic losses and posing great threat to human health. Furthermore,the potential use of these bacteria in biowarfare and bioterrorism increases their threats to humans. Therefore, Brucella attracts more and more attention today as one of the most serious pathogens associated with the public health and security problem.Brucellosis is epidemic in many areas,especially in developing countries. The rapid detection of Brucella for early diagnosis of Brucellosis is critical for control and prevention of this disease. The laboratory detection of Brucella is based on bacteriological,serological and nucleic acid-based methods. The bacterial culture is not well suitable for rapid detection of Brucella due to its highly infectious properties and the long isolation time. The serological methods include tube agglutination test and slide agglutination test, which may result in false positives and false negatives. ELISA has improved sensitivity and specificity, but until now, no specific diagnositic antigens are identified. Another limitation of these serological methods is that they are unable to differentiate sera of immunized animal from that of infected ones. The advantages of unnecessity of live bacteria manipulation and high specificity and sensitivity make PCR an appropriate method for Brucella detection. PCR can be used to identify Brucella and differentiate different species. The above mentioned methods played very important roles in Brucella detection and diagnosis. However, one common limitation of them is that they need to be performed in laboratory with professional quipments. And the detection need a long time to finish, and can not be performed on site. Immunochromatography is a technique that could be used for on site rapid detection. It does not need complicated equipment and can be easily performed in a short time, making it a very appealing method for pathogen detection. Up converting phosphor technology (UPT) is a new rapid quantification technology based on Up converting phosphor. Compared with conventional on site detection technology, UPT based immunochromatography has the advantages of high sensitivity and quantification capability.According to the host preferences, Brucella is classified into nine species: B. melitensis (from goats and sheep), B. abortus (from cattles), B. suis (from swines), B. ovis (from sheep), B. canis (from dogs), B. neotomae (from desert rats), Brucella pinnipedialis (from pinnipeds), Brucella microti (from the voles, Microtus) and Brucella ceti (from large sea animals, including whale, porpoise and dolphin). Of these species, B. melitensis, B. abortus and B. suis could infect humans. In the present study, the UPT was used to develop immunochromatography strip for rapid detection of Brucella, providing material for Brucella on site detection.Firstly, Brucella polyclonal antibodies were prepared. To develop antibodies with high titer, three types of antigens were selected:Brucella total proteins, inactivated bacteria and live bacteria. New Zealand rabbits were immunized respectively. At the 3rd immunization, the antibodies were monitored and found to be low. Therefore, for the 4th and 5th immunization, the live brucella were injected in veins. Sera were collected and antibody titers were tested. The antibody titers was improved and the higher ones were selected. For strip development, the embedding (Sample pad, combination pad, absorbent pad, sticky pad thickness and pore size analysis of materials and sizes and materials) and liquid material (UCP-SPA conjugates, the closure pad fluid samples, testing with reagents, the allegations with reagents) were screened. After optimization of the binding pad and reaction system of the analyzing membrane, the conditions for Brucella detection was determined. Then, the sensitivity, specificity, compatability, stability and repeatability of the strip were systematically analyzed. At last, simulated environmental and animal organ samples were used to evaluate the strip.Antibody titer monitoring showed that sera from inactivated Brucella and live Brucella had antibody titers of 1:32000 and 1:64000 respectively. Antibodies were purified for strip development. In our previous study, the enbeding material, embedding method, strip cutting, strip shape and liquid material were evaluated and the optimized procedures were determined, which was used in the present study directly. Glass fiber was chosen to make conjugation pad for its good performance on conjugation dissociation. Due to its hydrophilic property after addition of surface ative agent, nitrocellulose membrane was chosen as the analysis membrane. Glass fiber was used to make sample pad. After determination of the solid and liquid material, the capturing antibody and UCP labeled detection antibody and their combinations were determined. The A group antibody of 1mg/ml was used as capturing antibody, and the B group of 1mg/ml was used as the detection antibody. The ratio of sampling buffer to blocking buffers of 1:1 and the sampling volume of 140ul was found to be more sensitive. After optimization of the reaction system, the strip was developed. By using the serial dilutions of brucella, the detection cut-off was determined and the standard curved was made. The detection range is 10⁶-10¹⁰ CFU/ml, and the detection limit was 3.5×10⁴CFU. The T/C values of those bacteria closely related with Brucella were lower than the cut-off, indicating that the strip is specific. The strip could detect B. melitensis, B. abortus and B. suis, species that mainly infect human and animal, indicating that the strip is capatabile for different brucella species. The CV values from repeats of one test or different batches were lower than 12%, indicating good repeatability. The strips were place at 37℃and used for test at a 24h interval for 7 days, the CV means of T/C was 10.26%, indicating the strip stable at room temperature. Then, the strip kit was developed and their performance was evaluated by simulation sample detection. Environment, white powder and animal samples were simulated by soil, flour and mouse organs. The result showed that the strip kit could detect 2.8×10⁴～2.52×10⁶CFU. the strip could also detect brucella in infected mice spleens.In the present study, a new rapid quantitative detection immunochromatography strip for Brucella was developed. The strip showed good specificity, stability and repeatability, and could be used to detect Brucella in environmental samples and animal samples. From sample treatment to data analysis of the detection, 30 minutes is enough to complete the whole detection process, providing a new on site detection method for Brucella.