ELISA / dot blot Worksheet 26 points 1. What is the \"conjugate\" in \'an ELISA,
ID: 201756 • Letter: E
Question
ELISA / dot blot Worksheet 26 points 1. What is the "conjugate" in 'an ELISA, and what is its function? 2. Why do you need pure Ag bound to the plate in an indirect ELISA designed to detect Abs specific for that Ag from serum, but a Sandwich ELISA is able to use a mixed or dirty Ag preparation to detect the same specific Ab? 3. Suppose you are asked to determine whether a short lived virus "A" is endemic in a human population. Would you be better off using an ELISA that detected Ag or one that detected Ab? Why? 4. Use all of the reagents listed below to design an ELISA or dot blot to detect the presence or absence of E. coli bacteria in a water sample from a well. Number each step in proper order of use with binding to the plate or nitrocellulose as step 1. Draw it out if it helps. enzyme substrate Polyclonal Rabbit anti- E. coli (produced by immunization against whole bacteria) 2% milk solution (Blotto) HRP conjugated goat anti- rabbit IgG Well water Monoclonal mouse anti - E. coli (specific for bacterial surface Ag) 5. Why can't you use rabbit capture antibodies in question 4?Explanation / Answer
1.
Conjugates in ELISA are enzyme labeled antibodies. They are generally used in ELISA. They react specifically to sample analytes which are bound to a plate. Once the conjugate is added, then the substrate is added. Enzymes are conjugated with antibodies by forming a stable covalent bonds with enzyme. Examples of conjugates include horseradish peroxidase, alkaline phosphatase etc.
These are useful in detecting whether the target and the probe are attached/interacting, by using a secondary antibody which attaches to primary antibody.
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