L6 - Measurement of drug concentration

Question 1

Define: Concentration

Define: Activity

Answer 1

Concentration:
Molar. Relies on knowing molecular weight of substance.

Activity:
Used when molecular weight is not known e.g. a protein. Amount of protein present can be expressed by ‘units of activity’ - in bioassay.

Question 2

Methods to determine conc of drug:

Answer 2

Physicochemical methods:

1. IR/UV spec
2. Mass spec

Immunoassays:

1. Depends on highly specific antigen and antibody interaction.
2. Radioimmunoassay (RIA)
3. Enzyme-linked Immunosorbent Assays (ELISA)

Bioassay:
2 x 2 bioassay

Question 3

Radioimmunoassay:

Answer 3

Amount of radioactivity is inversely proportional to amount of hormone in sample. Amount of hormone increases so less radioactivity.

1. Apply antibodies and then wash.
2. Apply known amount of radiolabelled hormone and then wash.
3. Count radioactivity (ones bound to antigen).

Question 4

ELISA:

Answer 4

Relies on antibody/antigen interactions. Uses primary and secondary antibodies. Secondary is attached to an enzyme. Enzyme converts colourless substrate to coloured product.
Used to detect presence of antigen (hormone e.g.) or an antibody (HIV test).
Amount of colour is proportional to amount of enzyme-linked secondary antibody and to the time substance is incubated for. Can be measured.

ELISA can be quantified by measuring colour density where amount of colour is proportional to the amount of secondary antibody and the time it is incubated for. Time should be fixed.

ELISAs are carried out in “96 well plates” where a few wells have known amount of substance.

Question 5

Bioassay:

Answer 5

Compare response produced by unknown sample to that of a standard preparation of known biological activity.

Add small and large volumes of standard to produce 20% and 80% response. Measure ratio of the two. Work out volume of test that gives 20% response and then see if increasing drug conc produces 80% response. Find M.

You measure M of standard curve and lines should be parallel. You check their potency and if M is calculated to be 10 then you need 10 more of test sample to achieve same response as standard. So if standard solution contains 1500 units per ml then test must contain 150 units per ml.

Question 6

ELISA to detect antigen (hormone):

Answer 6

Coat with antibody and wash. Hormones present so bind to primary antibody. Secondary antibody with enzyme binds to antigen and wash. Enzyme causes colour change of colourless substrate to coloured product.

No hormones then secondary antibody doesn’t bind and cause colour change.

Question 7

Non-competitive ELISA to detect antibody:

Answer 7

Incubate with antigen coating. Wash sample. If antibody present. It binds to hormone. Enzyme-linked secondary antibody binds to primary antibody and causes colour change.

No antibodies then secondary antibodies can’t bind to antigen and cause colour change.

Question 8

Competitive ELISA to detect antibody:

Answer 8

Incubate with antigen coating. Wash sample. If primary antibody present it binds to antigen. Secondary antibody can’t bind so no colour change - usually binds to antigen.

No primary antibody then secondary antibody can bind to antigen and causes colour change.