| Bacterial endotoxin widely exists in air, soil, water and other environmental media and is intimately related with human health. After invaded by a certain dose of endotoxin at low immune function and or through wounds, human body can't deactivate endotoxin by producing plenty of humoral immunity. Extreme low amount of endotoxin can lead to a rise in body temperature and further cause some harmful syndromes such as pyogenicity, low white cell counts, Shwartzman Phenomenon, systemic inflammatory response syndrome (SIRS), endotoxemia, low blood pressure and multiple organ dysfunction syndrome (MODS), etc.Currently there are several test methods for endotoxin, e.g. , dometic rabbit method, limulus lysate test, ELISA and biosensor. Dometic rabbit method is seldomly used for its inability of quantification, high time consumption and low sensitivity. Limulus lysate test is golden standard for endotoxin test and widely used in clinical and environmental endotoxin researches with its celerity, convenience, high sensitivity and easy standardization. However, colored and high turbidity sample can affect test results and LAL preparation needs hunting of limulus, which is quite rared at present. Therefore, it is urgent to develop an alternate for limulus lysate test. ELISA has been applied to test endotoxin due to its high specificity for antigens and amplification of enzymes and able to eliminate interference of the color of sample. Its accuracy is lower than that of Limulus lysate test and thus rarely put into practical application. Biosenser is characterized by high measuring speed and sensitivity of physical device and capable of translating minor environmental change into measurable electrooptical signal. Current research of biosensor focuses on testing endotoxin with bioactivator.This research aims to establish a new testing method for endotoxin by polymyxin B and polyclonal antibodies against endotoxin and further develop an immunosenser based on adsorption of PMB.Part 1 the preparation of polyclonal antibody against E.coli endotoxin and the application in icELISAObjectives: To prepare rabbit polyclonal antiserum against E.coli endotoxin, to explore the sensitivity of ELISA in detection of purified E. coli endotoxin, to investigate the reaction of rabbit polyclonal antiserum with free and combined E. coli endotoxin. Methods: Purified E. coli endotoxin was used as the immunogen, the titer of rabbit serum was measured using rabbit ear blood every week, the heart blood was taken one week after the fifth immunization; the purified endotoxin was coated onto 96-wells ELISA plates, the quantitative rabbit polyclonal antiserum against endotoxin and the different concentrations of endotoxin were added at the same time, the indirect competitive enzyme-linked immunosorbent assay(icELISA) was established to detect endotoxin, the concentration of purification of E. coli endotoxin antigen and rabbit polyclonal antiserum were optimized by icELISA; the effectiveness of icELISA using rabbit serum or goat polyclonal antibody to detect purified, free and combined endotoxin was compared. Results: The endotoxin antiserum titer gradually increased with successive increased immunization dose. The serum titer was 1:6400 with OPD as the chromogenic substrate after the fifth immunization(1:16000 with TMB as the chromogenic substrate); the best parameters of icELISA were 100μg/mL of coating antigen on microplate and 1:500 dilution of rabbit polyclonal antibody The results showed the range of endotoxin detection was 0.1μg/mL～100μg/mL, which was in inverse proportion to the value of O.D and gived a linear regression equation: y = -0.1 ln (x) + 0.765, correlation coefficient R2 of 0.982, and the detection limit of 100ng/mL. Both of rabbit polyclonal antibody and goat polyclonal antibody could detect free and combined E. coli O157:H7 endotoxin, the titer was 1:16000 and 1:32000, respectively. Conclusions: The rabbit polyclonal antibody against endotoxin was successfully prepared, the titer of the rabbit polyclonal antibody was almost the same as the domestic antibody. The practicality of rabbit polyclonal antibody is more wider than goat polyclonal antibody, which could be used in detection of the purified, free and combined endotoxin.Part 2 the detection of endotoxin by sELISA using the conjunction of Polymyxin B (PMB) and polyclonal antibody against endotoxinObjectives: To detect endotoxin by sELISA using the conjunction of Polymyxin B (PMB) and polyclonal antibody against endotoxin (PMB-ET-pAB sELISA). Methods: The PMB was coated onto 96-wells ELISA plates, the different concentrations of endotoxin, endotoxin antiserum and enzyme-labelled antibody were added into each well after blocking. After chromogenic substrate was added, the value of O.D was measurement by microplate reader at A492nm. The consequence was compared with icELISA, established in part 1. Results: The best parameters of icELISA were 10μg/mL of PMB, 1:500 dilution of endotoxin antiserun and 1: 2000 dilution of donkey anti-rabbit lgG-HRP.The consequence of detecting endotoxin using the method of PMB-ET-pAB sELISA was as follow: the concentration of endotoxin from 0.1μg/mL to 100μg/mL was in direct proportion to the value of O.D and gived a linear regression equation: y = -0.094 ln(x) + 0.227, correlation coefficient R2 of 0.914 and the detection limit of 100ng/mL. Conclusions: The PMB-ET-pAB sELISA was established by the conjunction of polymyxin B (PMB) and polyclonal antibody against endotoxin, the detection line was consistent with icELISA of using polyclonal antibody only.Part 3 the immunosensor base on the adsorption of PMBObjectives: To establish the immunosensor based on the adsorption of PMB. Methods: The immunosensor was fabricated by adsorbing a certain concentration of PMB on the surface of cysteamine and glutaraldehyde modified gold electrode, the different concentrations of standard endotoxin, rabbit antiserum against endotoxin and enzyme labelled antibody were added on the surface of electrode after blocking, the substrate was PBS buffer (0.1M pH7.2) with the addition of 2μL H2O2 (30%) and hydroquinol(1.0mmol/L), the change of electrical signal was generated by reduction of hydrogen peroxide catalyzed by horseradish peroxidase (HRP)on the electrode surface, which was detected by cyclic voltammetry in the buffer. The relationship between endotoxin concentration and reduction peak current value was analyzed. Results: The best parameters for immunosensor were 10ug/mL of PMB,1:200 dilution of endotoxin antiserun and 1:100 dilution of donkey anti-rabbit lgG-HRP.The concentration of endotoxin from 0.001μg/mL to 100μg/mL was in direct proportion to the reduction peak current value and gived a linear regression equation: y = 1.386 ln(x) + 39, correlation coefficient R2 of 0.904 and the detection limit of 1ng/mL. Conclusions: The immunosensor was successfully prepared, and demonstrate the advantages of wide range of detection, highly sensitive and specific to certain endotoxin. |
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