| Legionella is an endotrophic and gram-negative bacteria, which can intrude into human alveolar macrophages, other macrophages, lung epithelial cells and the protozoa in aquatic environment. In macrophages, Legionella destructed the transmission of small bubble, prevented phagosome-lysosome fusion, prevented the meaning of the sterilization of endosome and lysosome, established its own unique copy of the body and swallowed up full of host cells, finally leaded to the host cell death. Legionella is widespread in natural water and soil, which can also be long-term survival in the artificial environment of water, such as more than one year in tap water, two to four months in distilled water. In recent years, water sampling results showed that the detection rate of Legionella is above 60 percent in both inside and outside buildings. Artificial water system has been the primary means of transmission, which indicates that with the globalization of the industrial development, Legionella will become a major threat to human health. At present, Legionella have been identified 43 kinds, 65 serotypes. Legionella pneumophila (LP) is an important etiologic agent related to human diseases, which has been found 15 serotypes. LP is responsible for 80~85% of reported cases of Legionella pneumonia.Legionnaires'Disease is an acute bacterial respiratory disease, due to inhalation of aerosol containing Legionella, pontiac fever and Legionella pneumonia as the main performances. LD is easy outbreak, about 1 / 3 of Community-acquired pneumonia (CAP) is caused by Legionella. Most of LD patients'clinical manifestations are similar to other respiratory infections, such as cough, fever, muscle-ache and chest X-ray revealed that infiltration of the lung tissue, which may lead to misdiagnosis and missed diagnosis. The resulting mortality rate, ranging from 25% to 40%, can be lowered if the disease is diagnosed rapidly and appropriate antimicrobial therapy is instituted early. Rapid and accurate identification of etiologic agent is the key to control the disease.Since 80 to 85 percent of the Legionella pneumonia is caused by LP, so a rapid detection of LP is particularly important. There are many ways to detect the infection, bacterial culture is the gold standard for detection of infection, but the harsh training conditions and more time-consuming can not conducive to rapid diagnosis. WHO has recommended other methods, such as direct fluorescent antibody (DFA), indirect fluorescent antibody (IFA), test-tube agglutination test (TAT), micro-agglutination test (MAT), enzyme immunoassay (EIA) and DNA probes, and so on. Specimens for testing for patients are respiratory specimens (sputum, lung BALF and living tissue, etc.) or paired serum samples (acute phase and recovery phase). One of the clinical symptoms of LD patients is the less phlegm, BALF and lung biopsy are traumatic and difficult to be patient and LD patients'serum antibody levels can achieve the level of detection after three to six weeks, which restrict early clinical diagnosis.Research showed that the majority of patients can discharge the LP O-polysaccharide antigen with thermal stability and anti-trypsin activity in their urine after 1 to 3 days infected, the concentration is higher than 30 to 100 times in their serum. Therefore, the urine samples'test of patients with soluble and specific antigen of LP can quickly and accurately identify pathogens.The detective methods of LP urine antigen have DFA, radioimmunoassay (RIA) and EIA, and so on. Being the lower sensitivity of DFA, radioactive of RIA, time-consuming of EIA, above tests are not recommended clinical application. In recent years, gold immunochromatographic assay (GICA) has been applied in the rapid detection of LP and achieved certain results. The main principle is that the anti-LP specific antibody is coated on NC membrane to capture LP antigen in urine samples and then use the same antibody tags colloidal gold, the tagged antibody and the captured antigen appear precipitation zone in chromatography which is visible to the naked eye. Compared with other methods, the technology have more advantages, such as high sensitivity and specificity, simple samples handling, non-specialized equipment and non-training of personnel, and so on, so it's a ideal method for emergencies .There have been some colloidal gold immunochromatography test reagent products for sale. Currently, the Binax NOW Legionella Urinary Antigen Test is be used in many countries, it allows for early diagnosis of L. pneumophila serogroup 1 infection through detection of a specific soluble O-polysaacharide antigen present in urine of patients with Legionnaires'Disease,but the kit does not apply on the detection for other LP serotype. There is no similar domestic products.In the 15 standards types of serum, L. pneumophila serogroup 1 is responsible for 80%~90% of reported cases of L. pneumophila infection. O-polysaacharide antigen of L. pneumophila serogroup 5 is common to multiple serogroups of L. pneumophila, According to double antibody sandwich method, we use rabbit-anti-L. pneumophila serogroup 1 antibody and rabbit-anti-L. pneumophila serogroup 5 antibody to develop two immuo-colloidal gold tests respectively. Main works listed below as:1,Preparation and identification of polysaccharide antigen of L. pneumophila Bacteria of LP1 ~ 7,9 and 10 were cultured on yeast agar buffer activated carbon (BCYE) for three to five days at 37℃, 5%CO2. Protein-free polysaccharide antigens were obtained after harvest in cells, extraction, deproteinization, dialysis and other steps. Their immunogenicities were verified by ultraviolet spectrophotometer full-wavelength scanning and Western blotting.2,Preparation and identification of rabbit anti-LP1 antibodies and rabbit anti-LP5 antibody Rabbit anti-LP1 and anti-LP5 antibodies were purified after rabbits were immuned with antigens isolated as described above. The purities of both antibodies were above 80% and the titer of blood serum 1:32 tested by double antibody sandwich assay.3,Development of colloidal gold immunochromatographic assay kit The size of colloidal gold particles in the kit was 25nm. The optimal concentrations for antibodies were 30μg/ml and the sensitized concentrations of NC membrane were 5 mg/ml. Laboratory evaluation showed that the kit using anti-LP1 antibodies only recognized LP1 antigen and detection limit was as low as 10 ng/ml. These results were verified by using the Binax NOW Legionella Urinary Antigen Test kit. However, the kit using anti-LP5 antibodies recognized all antigens isolated in our report. The sensitivities for each serogroups were as listed: LP2~5, 0.1μg/ml; LP1,7,9 and 10, 1μg/ml; LP6, 10μg/ml. The preliminary immunological data indicate the assay kits using both antibodies have the potential to be used in diagnosis of Legionnaires'Disease, which deserve further clinical evaluation.
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