Majee, S. B.Jadhav, S. V.2017-10-092017-10-092013http://krishikosh.egranth.ac.in/handle/1/5810032554The present study was undertaken to extract and characterize sLPS from standard smooth strains of B.abortus strain 544 and B. abortus strain 19 to be used as antigen in the indirect ELISA. Smooth Lipopolysaccharide (sLPS) from reference strains Brucella abortus 544 and B. abortus S19 were extracted by i) hot sodium dodecyl sulfate (SDS) extraction and proteinase K digestion method and ii) Tri-reagent method. The sLPS were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and silver staining after periodate oxidation. Purity of extracted sLPS was checked by HPLC and Carbocyanine colorimetric (CC) assay respectively. This sLPS was then used for standardization of Indirect Enzyme linked immunosorbent assay (i-ELISA) by checkerboard titration and subsequently for diagnosis of brucella antibodies in sera samples from clinical cases of brucellosis in buffaloes and from the abattoir and results were compared with those obtained with commercially available c-ELISA kits, STAT and RBPT. Out of the two methods used for extraction of sLPS, the sLPS obtained by Hot SDS-Proteinase K method showed less contamination with nucleic acid and proteins when compared with the sLPS of E.coli (Sigma) and sLPS (Tri reagent). The yield of sLPS from B. abortus 544 was 11mg/15 g wet weight of cells and B. abortus S-19 was 10mg/12 g wet weight of cells and that of sLPS obtained by Tri-reagent method (B. abortus 544 gave 5mg/25mg dry weight of cells and B. abortus S-19 gave 7 mg/46mg dry weight of cells). Tri-reagent method yielded good quantity of sLPS from low amount of Brucella abortus cells. SDS PAGE analysis of sLPS obtained from both the methods displayed smearing pattern on the gel. The molecular weight of the slowly migrating components that represented intact LPS (i.e., lipid A, core, and O-linked sugars) was approximately from 30kDa to 70kDa. The fast-migrating bands (10kDa) in all lanes represent lipid A without O-linked sugars. The nanoparticles layered with sLPS also showed a similar pattern without any change in the purity or characteristics of the sLPS. The purity of sLPS was assessed by HPLC and CC assay. The chromatogram of sLPS of B.abortus 544 and strain 19 extracted by both the methods showed a sharp peak suggesting very low content of impurities when compared with commercially available E.coli LPS. However, when the normalized spectra was observed the sLPS of S19 showed a sharper peak with less tailing as compared to sLPS of S544 that showed multitude peaks and tailing effects suggestive of impurities. The results of CC assay revealed a positive regression coefficient between the absorbance and the concentration of sLPS in the range of 0.8 to 0.9 for sLPS of B.abortus S-19 and strain 544 respectively. An i-ELISA was developed using sLPS as antigen and using negative and positive sera each from buffaloes. i-ELISA was standardized by checker board method which revealed that the minimum concentration of antigen that showed a positive result was 0.1 ug and the range of antibody dilution was 1:200. The results of 132 buffalo sera screened by RBPT, STAT, c-ELISA and i- ELISA indicated that 74(56.06 %), 120(90.90 %), 73(55.30 %) and 111(88.8 %). samples were positive for brucellosis respectively. STAT detected higher proportion of positive animals than that of RBPT whereas i-ELISA developed in this study detected 111 (88.8 %). Considering c-ELISA as the gold standard test the sensitivity and specificity of RBPT, STAT and i-ELISA were found to be 98.63 %, 97.26 %, 90.54 % and 96.61 %, 16.94 %, 15.69 % respectively whereas proportion of agreement between the c-ELISA, RBPT, STAT and i-ELISA were 97.72 %, 61.36 % and 60 %. Considering i-ELISA as the gold standard test the sensitivity and specificity of RBPT and STAT were found to be 62.16 %, 91.07 % and 14.28 %, 14.25 % respectively whereas proportion of agreement between the i-ELISA, RBPT and STAT were 53.6 % and 83.2 %. To conclude, the sLPS extracted from B.abortus strain 19 was more superior than that of strain 544 and was employed as antigen in i-ELISA that proved to be good screening test for Brucellosis. However, standardization of the assay using lower concentration of the antigen and various dilutions of the conjugate will eliminate the errors due to high background values.ennullThesis