Lab 4

• What is ELISA?

  • stands for enzyme-linked ImmunoSorbent Assay
  • serological test based on antigen antibody interactions
  • for pregnancy tests & HIV detections
• Purpose of Elisa in lab?
determine if specific antigen is present in given patient's "serum" sample

  • antigen = B. lievable
  • presence of B. lievable antigen = presence of specific chaplin protein
• Reagents:

  1. Rabbit primary antibody = IgG --> binds B. lievable chaplin protein
  2. Goat Secondary antibody --> binds Fc region (surface receptor) of rabbit IgG
  3. several 2º can bind to single 1º to enhance signal
  4. 3. Horseradish peroxidase (HRP) enzyme will bind to secondary complex
  5. Colorimetric detection
• What is in the wash?

  • Tween-20 = detergent that is included in wash buffer to remove unbound antigens or unbound antibodies from wells during wash
  • Tween-20 also in primary and secondary antibodies to prevent antibodies from binding nonspecifically to plastic wells
  • tween-20 NOT in serum to allow serum to bind to well and not get washed away (once bound, cannot be washed out)
• ELISA steps:

  1. bind serum proteins to well
  2. wash unbound protein
  3. add primary antibody
  4. wash out unbound antibody
  5. add secondary antibody
  6. wash out TWICE
  7. add HRP enzyme substrate
  8. observe color change
• Positive control: group that will receive treatment with known result, should show a change

  • will have purified bacterial chapelin antigen
  • show that protein of interest can be detected
• Negative control: group that doesn't receive any treatment, no change in experiment

  • will have protein not found in B. lievable
  • test aseptic technique = method to prevent contamination
  • ensures reagents used are free of contaminants
• ELISA mistake:

  • Mix up J & M --> false positive in M and false negative for J
  • M and + are only blues
• ELISA mistake:

  • forget to add 1º to J only --> false negative on J
  • only + is blue
• ELISA mistake:

  • Not adding 2ª antibody to J --> false negative on J
  • only + is blue
• ELISA mistake:

  • wash once instead of twice before adding HRP --> false positive M, H, and -
  • M, H, - will be blue alongside J and + (J already positive)
• ELISA mistake:

  • if negative control blue --> can't trust results
  • make new reagents to redo the assay and try again
• ELISA mistake:

  • if only J is positive --> can't trust results
  • make new reagents to redo the assay and try again