Clinical Assays for Proteins and Small Molecules (L6)

Question 1

What are the 3 general types of clinical assays?

Answer 1

1. Pathology
2. Genetics
3. Clinical chemistry

Question 2

What are the major electrolytes in the blood?

Which are the primary INTERCELLULAR cation and the primary EXTRACELLULAR cation?

Answer 2

K⁺,Na⁺,Cl^-,HCO3^-

K⁺ is the primary intercellular cation

Na⁺ is the primary extracellular cation

Question 3

What is a common protein found in the blood?

Answer 3

Albumin

Question 4

What forms of hormones are in the blood/urine?

Answer 4

All forms of hormones (peptide, steroid, thyroid)

Question 5

What are 3 things one would need to know about a compound or protein from a clinical perspective?

Answer 5

1. Detection
2. Quantization
3. Activity

Question 6

What clinical assay is used in identifying glycosylated hemoglobin (aka Hb A1C)

Answer 6

Ion exchange chromatography

Question 7

What 2 disorders require an ELISA test?

Answer 7

HIV, which requires a western blot to confirm results.

Hepatits C

Question 8

What are mono/polyclonal antibodies?

Answer 8

Monoclonal Ab: monospecific Ab’s that are made from a single type of immune cell

Polyclonal Ab: Ab’s secreted by several different immune (B cells) cell lineages

Question 9

What do Ion Selective Electrodes (ISE) measure?

Answer 9

Electrochemical potential which reflects pH

Question 10

Detection of creatine by rxn w/ picric acid in alkaline solution is an example of:

how does it work?

Answer 10

Non-enzymatic formation of chemical complexes.

Creatine binds to Picrate and the complex absorbs light at 510nm (red).

Question 11

How do enzyme-linked assays typically work?

Answer 11

They typically detect small molecules using enzymes that recognize the molecule as a substrate.

Question 12

At what angle and wavelength is light scattering typically measured at?

Answer 12

Right angles to the incident light and at the same wavelength.

Question 13

What do polystyrene beads do?

Answer 13

They covalently bind to antibodies or antigens and increase the intensity of light scattering.

Question 14

Why do polyclona antibodies directed against protiens aggregate but monoclonal antibodies don’t?

Answer 14

Polyclonal Ab’s aggregate due to recognition of multiple epitopes on the protein. The aggregation results in dectable light scattering.

Question 15

Monoclonal Ab’s don’t aggregate upon binding the antigen. Therefore they don’t scatter light. What is another alternative method for using them in an assay?

Answer 15

Enzyme-coupled competition assays.

Question 16

Describe the overall process for the ELISA.

Answer 16

Enzyme Linked Immunosorbant Assay (ELISA):

Uses a rabbit IgG that binds to HIV protein,

Then uses a goat IgG/horseradish perioxidase bound complex to bind to rabbit IgG/HIV complex. This will produce a colored product after a colorless substrate is added.

The amt of colored product formed in fixed time is porportional to amt of HIV envelope protein.

Question 17

What is the relationship b/w Cell homogenization, centrifugation, and cell fractionation?

Answer 17

Cell homogenization will disrupt cells to allow for isolation of proteins separated by centrifugation and fractionation.

Question 18

What are the different types of chromatography and by what basis do they separate?

Answer 18

1. Ion exchange chrom., charge
2. Size exclusion chrom., size
3. Affinity chrom., Ab/Ag binding

Question 19

Describe Ion exchange chromatography.

Answer 19

Separates on basis of charge. At an appropriate pH proteins will have either a net + or - charge. They will bind to oppositely charged beads in a column, which can then be eluted by salts (NaCl or KCl).

Question 20

Describe Size exclusion chromatography.

Answer 20

Separates on basis of size. Has polymers with pores of diameter that allow access to aqueous channels that pass through the bead. Proteins that are smaller will pass through pores that will slow them down, but large proteins will pass around them and elute first.

Question 21

What does Polyacrylamide gel electrophoresis (PAGE) do?

Answer 21

Allows detectioin of individual proteins even when mixed with other proteins.

Question 22

Describe SDS-PAGE.

Answer 22

Separates proteins by size. Proteins are denatured by SDS detergent, which binds to proteins and carries negative charge (so all proteins carry - charge). Proteins migrate towards cathod (+) in an e^- field.

Migration occurs through a polyacrylamide gel, where proteins are separated by size with small proteins migrating farther and more rapidly.

Question 23

Describe Isoelectric focusing (IEF) electrophoresis.

Answer 23

Separates protiens by net CHARGE. Proteins pass through a gel with an established pH gradient. Proteins will migrate toward the anode or cathode depending on their charge at that pH, and will stop at the location of their isoelectric point (pI), the pH where their net charge is neutral.

Question 24

What is 2D page?

Answer 24

Combination of SDS-PAGE and IEF-PAGE.

Question 25

Describe the process of using monoclonal and/or polyclonal antibodies along with SDS-PAGE to detect proteins.

Answer 25

Using SDS-PAGE separates proteins by size. A WESTERN BLOT can then be performed.

This is by transfering proteins from gel to a polymer sheet, then exposing the polymer sheet to radiolabeled specific antibody, and washing to remove unbound antibodies. Then using photographic film and devloping the polymer sheet one can see the protein band detected by the specfic antibody which bound to the protein.

Question 26

What does mondern mass spectrometry do?

Answer 26

Determines precise molecular weights using small amounts of protein.