150 2nd journal report

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ELISA

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  • CPB2:lethal for miceand for variouscell linesand causes hemorrhagic necrosis when injectedinto intestinal loops of guinea pigsIn toxinotype A not directly involved in virulence
  • 150 2nd journal report

    1. 1. Development of an antigen-capture enzyme-linked immunosorbent assay for Clostridium perfringens beta2-toxin in porcine feces and the neonatal piglet intestine Guevarra; Olivar; Santos; Tan; Virata
    2. 2. Clostridium perfringens •third most common cause of food poisoning though it can sometimes be ingested and cause no harm • Generally affect the health of your livestock. Killing them and preventing their usage as commodity.
    3. 3. Clostridium perfringens •5 toxinotypes (A-E), A being most important in neonatal pigs •In toxinotype C, beta2-toxin (CPB2) is involved in the virulence • 2 forms: CONSENSUS and ATYPICAL • Expressed in ALL toxinotypes • Believed to be MARKER for presence of toxinotype A
    4. 4. Methodology Purification of CPB2 protein Preparation of Monoclonal Antibodies Development and optimization of ELISA polyclonal capture antibody (rabbit anti-rCPB2)E. coli D5α
    5. 5. Development and Optimization of ELISA • Assessment of the optimal concentrations allowing antigen-antibody interaction • Optimization of incubation conditions • Selection of blocking buffers • Selection of coating buffers
    6. 6. Extraction Protocols Intestines Stool Sample Centrifugation at 4 C at 17,500 x g for 30-60 min until clear supernatant is collected Centrifugation at 17,500 x g until clear supernatant is collected
    7. 7. Assessment of the optimal concentrations allowing antigen- antibody interaction •captured CPB2 detected was between 2.0 and 3.5 ng/ml •pAb diluted 1:2,500 for 2 hr at 37°C followed by overnight at 4°C was optimal for coating the wells
    8. 8. Development and Optimization of ELISA ELISA Components Used in Experiment Antibody rabbit anti-rCPB2 Antigen Beta2-toxin Enzyme-linked Antibody horseradish peroxidase (HRP)-labeled monoclonal detection antibody (mouse anti-rCPB2 mAb-HRP) Colorless substrate 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) Wash buffer phosphate buffered saline [PBS], 0.05% Tween 20, 0.5% fish skin gelatin Blocking buffers PBS, 0.05% Tween 20, 2% BSA Coating buffers PBS Positive color GREEN intensity measured at 405 nm OPTIMUM condition: incubation of pAb diluted 1:2,500 for 2 hr at 37°C followed by overnight at 4°C was optimal for coating the wells and that a 1:5,000 dilution of mAb–HRP incubated for 1 hr at room temperature was optimal for detecting captured antigen.
    9. 9. Fight against proteases Agent Action amidinophenyl-methanesulfonyl fluoride INHIBITOR, binds with serine protease inhibitor mix INHIBITOR Processing at 4 C Slows down activity of protease pH effects Changes ionic interaction of proteases *Before any procedures done, dead tissue samples were “SPIKED” (incubated at -70 C) to allow CLUMPING of different proteins **Freezing yielded best results since it was able to detect the toxin at low concentrations and it provided the least “noise”.
    10. 10. Conclusion •With appropriate sample preparation, antigen- capture ELISA can detect beta2-toxin in the intestinal content and feces of neonatal piglets.

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