Definition Of The Method For The Detection Of Serological Of Gallstone Caused By Nanobacteria Infection In Rabbit By Dot Immunoenzyme Filtration Assay

Nanobacteria (Nanobacteria, NB) are supper microorganisms discovered by Kajander in 1989, a Finnish scientist. They are spherical or rod-shaped with the wall. They usually show bundle, the diameter of which is from 80 to 500 nm. They can produce the biofilm containing apatite, with a strong anti-heat, anti-γradiation ability and the ability to resist antibiotics. They are filterable through 100 nm pore size sterile filtration. They have unique ability of forms biomineralization. Some literatures suggested that it is associated with gallstones, kidney stones, atherosclerosis and several bone calcification diseases or sclerosis in recent years. It has got great concerns of science field.At present, the main test methods of nanobacteria are as follows:immunofluorescence and immunohistochemical detection with anti- nanobacteria monoclonal antibody; scanning electron microscopy and transmission electron microscopy observation.Objectives:The purpose of this study is to produce polyclonal antibody through the isolation and culture nanobacteria from cholelithiasis patients bile and immune animal, and explore the optimization of the experimental conditions to establish primary diagnostic method detecting nanobacteria antibodies in animal serum by dot immunoenzyme filtration assay.Methods:1. Six gallbladder bile samples were obtained from gallstone patients without acute cholecystitis symptoms and without antibiotic therapy before laparoscopic surgery. With aseptic manipulation,the nanobacteria were cultured in bile samples, and DMEM was cultured and Hydroxyapatite was cultured as negative control culture simultaneously.2. The turbidity of nanobacteria culture was tested by digital turbitimeter, and the curve was drawed to reflect the nanobacteria growth.3. Cholelithiasis patients bile culture were observed by scanning electron microscope,and then they were analyzed with EDX. And the Nb culture were observed by transmission electron microscopy.4.A drop of nanobacteria slice was stained by indirect immunofluorescence staining (IIFS), VON KOSSA staining and Hoechst 33258 staining were performed separately as described in references.5. Purificationh nanobacteria antigen was finished, and the total protein concentration (174.2μg/ml) through quantized by coomassie brilliant blue to preserve.6. Immune rabbitsThree rabbits were injected in nanobacteria(volume dose 200μg/ml) containing Freund's adjuvant and they were injected with nanobacteria (volume dose 50μg/ml) containing incomplete adjuvant two weeks later to strengthen. Then nanobacteria polyclonal antibody was produced, and its effect was determined by ELISA.7. Definition of the method for the detection of nanobacteria nanobacteria polyclonal antibody by dot immunoenzyme filtration assayThe solution of rabbit anti nanobacteria polyclonal antibody is diluted by using the doubling sequential dilution method to varying concentration ranging from1:6400～1:51200; nanobacteria antigen is diluted to a concentration of 1:10～1:160 while the universal polymerized HRP-Anti Ms/Rb IgG is diluted to a concentration of 1:10～1:40. Following that, the reaction concentration of corresponding antigen and antibody at the individual test points is determined with matrix titration method. The same determination work is made at each test point repeatedly for 6 times. Through gross inspection, all those in forms of brown spots or in circles can be identified as positive, and those that cannot be identified are regarded as negative. And each test is made with negative contrast. The optical density (OD) of each positive reaction point is measured with the Motic Med 6.0 digital medical image analyzing system (A) for determining the mean OD value at each point. Through data analysis, selection is made of the optimum reaction combination of the titers of the nanobacterial antigen and the universal polymerized HRP-Anti Ms/Rb IgG. After the determination of the titers of the two kinds of antibodies, the antigens at different solution concentrations are measured. Based on the data obtained and the analysis of the variation of OD value in relation to the content of antigen, a standard curve can be plotted for deriving the regression equation and correlation coefficient.8. Detection of specificity with dot immunoenzyme filtration assayThe reaction combinations of the best nanobacterial antigen titer and the universal polymerized HRP-Anti Ms/Rb IgG titer were used to test the positive and negative specimens serum with different concentration by dot immunoenzyme filtration assay.With the sensitivity, specificity, positive required value, negative required value appraised, the rabbit serum of each test group was tested.The best rabbit serum dilution was detected through sensitivity, specificity and false positive rate .The reaction of the best nanobacterial antigen titer was combined with the reaction of the universal polymerized HRP-Anti Ms/Rb IgG titer to finish the specificity test of immunological assay. With the sensitivity, specificity, positive required value, negative required value appraised, the rabbit serum of each test group was tested.Results:1. All the six cholelithiasis patients bile samples was cultured in DMEM with 10% FBS under mammalian cell culture conditions (37℃,5%CO2) and the nanobacteria detected positive.2. The cholelithiasis patients bile culture as tiny particles could be observed making Brownian movement near the bottom of the culture vessel by using phase inverted microscope. During the cause of culture, the changing of pH value was not obvious. The sub-cultured nanobacteria take the same characteristic as the original.3. Measurement each 5 days, the turbidity of Nb culture group growth as rise tendency detected by digital turbidimeter in 4 week. DMEM, hydroxyapatite and inactived Nb cultured control group without marked change.4. Identification of nanobacteriaObservation with transmission electron microscope, nanobacteria revealed 80~350nm particles. They appeared bacillus or coccus in shape, as clusters by attaching to each other with bilfilm-like material.Elements of calcium, phosphor, aluminium, silicon and sulfur could be detected by EDX microanalysis, and calcium-phosphate ratio of nanobacteria was 1.62, which was similar to that of hydroxyapatites 1.66.Calcified shell of nanobacteria could be stained as black dots by Von KOSSA method.Indirect immunofluorescence staining indicated that nanobacteria labeled fluorescent antibody produced the green fluorescence in fluorescence microscope.Stained with Hoechst 33258, nanobacteria showed reaction to the dye by producing the characteristic blue fluorescence, which indicted that DNA exist in nanobacteria.5. Rabbit ant polyclonal antibody was tested as 56.25mg/ml by the ELISA.6. The selection of best reaction conditions in testing Nb polyclonal antibody by dot immunoenzyme filtration assay is achieved by square titration. The best combined titers of antigen and antibody were as follows: Nb antigen 1:20 (8.71μg / ml), universal polymerized HRP-Anti Ms/Rb IgG are concentration 1:10, which is the concentration of this experiment. Polyclonal antibody was diluted further, and the standard curve indicating the relationship between the OD value and antigen content was established. Then the regression equation was obtained:Y=0.0832X+0.0745. The correlation coefficient was: r = 0.876(P < 0.05).The highest dilution radio of Nb polyclonal antibody was 1:12800,and could be detected the lowest antibody concentration was 68.59ng/ml.7. The reaction combinations of the best nanobacterial antigen titer and the universal polymerized HRP-Anti Ms/Rb IgG titer were used to test the positive and negative specimens serum with different concentration by dot immunoenzyme filtration assay. The best rabbit serum dilution was detected as 1:6400.8. The result of used the reaction combinations of the best nanobacterial antigen titer and the universal polymerized HRP-Anti Ms/Rb IgG titer test the rabbit serum of each groups by dot immunoenzyme filtration assay. The results of gallstones were found successfully in specimens tested by dot immunobinding assay were as follows: 40 positive, 56 negative; the results of the specimens by ELISA of nanobac were as follows: 38 negative, 58 positive. Matching Chi-square test of the test results was finished, and the methods were used to test the correlation of (χ2=88.055,P=0.001). Both had really good diagnostic consistencies. ( kappa value 0.957) .Conclusions:1. Nanobacterial infections exist in the bile of gallstone patients. Under physiological conditions, hydroxyapatite mineralization shell can be produced on the surface of nanobacteria with tiny size and slow growth.2. Antiserum can be produced through immune animals because of the immunogenicity of nanobacteria.3. Dot immunoenzyme filtration assay was initially established to test nanobacteria antibody in rabbit serum. Some evaluate index was as follows: sensibility: 96.55%;specificity:82.09%;Youden index:0.7867;total effective rate:86.46%;positive required value:70.00%;negative required value:98.21%.