BET testing USP85 an Introduction
- 1. ENDOTOXIN TESTING USP 85 Amrutha Raiker • Introduction to bacterial endotoxins • Types of Endotoxin tests • Procedure
- 2. INTRODUCTION TO ENDOTOXIN • Bacterial Endotoxins are Lipopolysaccrides(LPS), cause Fever and Diseases • Endotoxins bind with limulous amoebocyte lysate to form clot, principle for bacterial endotoxin test.
- 3. Bacterial Endotoxin Test Gel Clot Method •Based on Gel Formation Turbidometric method development of turbidity after cleavage of endogenous substrate Chromogenic method Development of colour by cleavage of synthetic peptide chromogen complex
- 4. • Limit test • Semi Quantitative Gel Clot • End Point Turbidometric • Kinetic Turbidometric Photometric I: Turbidometric method • End point Chromogenic • Kinetic Chromogenic Photometric II: Chromogenic Method DIFFERENT KINDS OF BET
- 5. Set UP • BET Kit • Heating Block Reconstitute CSE vial • Standard Preparations • Dilution Calculations Sample Preparations • MVD • Lysate Calculations PROCEDURE OF BET
- 7. CONFIRMATION OF LYSATE SENSITIVITY CSE Dilutions +Lysate Incubation Results
- 8. LYSATE SENSITIVITY (D) 50µl of 2,1,0.5,0.25 λ of CSE and negative (0) 50µl of Limulus Amebocyte Lysate(LAL) reagent water 100µl of lysate Incubate 1 hour 37 Firm gel Positive and no intact gel negative result. Lowest concentration should be negative
- 9. LYSATE SENSITIVITY • Geometric Mean End Point Concentration= Antilog (Se/f) Where, Se is Sum of log endpoint concentration f is number of replicate test tube (usually 4) • λ measured is EU/ml • Value should not be less then 0.5λ and more then 2 λ • Thus confirm labelled sensitivity
- 10. MAXIMUM VALID DILUTION (MVD) Maximum Valid Dilution = Endotoxin Limit × Product Concentration Lysate Sensitivity Entotoxin Limit = threshold human pyrogenic dose of endotoxin/ kg of body weight maximum recommended human dose of product / kg of body wt.
- 11. EXAMPLE FOR CALCULATION OF MVD • The limit for a vaccine could be 5EU/kg of body • For a 70kg person the EU limit would be 5*70=350units of EU • Therefore according to the formula MVD will be 11,666.666 • Thus it can be diluted so many times.
- 12. PRODUCT INTERFERENCE FACTOR Sample BET water MVD/4 CSE Lysate Negative Product Control 50µl 50µl 4 λ50µl 2 λ 50µl 1 λ 50µl 0.5λ 50µl 50µl Positive Product Control NA 50µl 4 λ50µl 2 λ 50µl 1 λ 50µl 0.5λ 50µl 100µl Positive Water Control 50µl NA 4 λ50µl 2 λ 50µl 1 λ 50µl 0.5λ 50µl 100µl Negative Water Control 100µl NA NA 100µl
- 13. BET TEST RESULTS Sample Control Standard Endotoxin LAL Reagent Water Product Lysate Results Negative Water Control Not Applicable 100µl Not Applicable 100µl Negative Positive Control 50µl of 4ʎ 50µl Not Applicable 100µl Positive Product Positive Control 50µl of 4ʎ Not Applicable 50µl 100µl Positive Product Not Applicable 50µl 50µl 100µl Negative
- 14. CHROMOGENIC ENDOTOXIN QUANTIFICATION A small volume of the sample (10μL) is combined with the Limulus Amebocyte Lysate, and endotoxins in the sample activate the proteolytic activity of Factor C. When the chromogenic substrate is added, the activated protease catalyzes the cleavage of p- nitroalinine (pNA), resulting in yellow color that can be quantitated by measuring the absorbance at 405nm (A405) and extrapolating against a standard curve. A standard curve is created using the E. coli endotoxin standard included with each kit to calculate endotoxin levels as low as 0.1 EU/mL, where one endotoxin unit/mL (EU/mL) equals approximately 0.1ng endotoxin/mL of solution www.thermofisher.com
- 15. TURBIDOMETRIC QUATIFICATION OF ENDOTOXIN • This assay is performed in a 96-well plate allowing many samples to be processed at one time • Kinetic Turbidimetric Assay is measured at the 340 nm wavelength and has a sensitivity range from 0.01 to 100.0 EU/ml. http://www.lonza.com
- 16. TURBIDOMETRIC QUATIFICATION OF ENDOTOXIN • A sample is mixed with the reconstituted LAL reagent, placed in the incubating absorbance plate reader, and automatically monitored over time for the appearance of turbidity • In the presence of endotoxin, the lysate will begin to gel, causing the solution to become cloudy or turbid • The time required for the change is inversely proportional to the amount of endotoxin present. • The concentration in unknown samples can be calculated from a standard curve.
- 17. REFERENCES • www.thermofisher.com • http://www.lonza.com • Ryan, J. Endotoxins and Cell Culture. Corning Technical Bulletin. (2008).