Molecular Diagnostic II: Protein Techniques

Question 1

Improving on insulin

Answer 1

Lispro (Eli Lilly): insulin analogue. Faster acting, more readily absorbed than normal insulin, tighter glycemic control

Insulin aspart (NovoLog): behaves similarly to lispro

Insulin glargine (Sanofi-Aventis): longer acting analogue of insulin

Question 2

Antibody structure

Answer 2

Monoclonal antibodies (mAbs) can come from a hybridoma

Hybridoma: B cell (very specific) + myeloma (cancerous plasma cell that is immortal)

Specific for a single epitope generated by single clones of immortalized B lymphocytes

Heavy and light chains, antigen binding site (near Fab domain). The Fc domain is the support part

Question 3

Processing of insulin

Answer 3

Preproinsulin (signal peptide, C chain, A/B chain)

Proinsulin (C chain, A/B chain)

Insulin (A/B chain

Question 4

Epitope

Answer 4

is the specific site on the antigenic molecule

recognized by the Fab region (group of amino acids is recognized on antigen)

Question 5

B cells can make antibodies

Answer 5

True

Question 6

Polyclonal antibody vs monoclonal antibody

Answer 6

Monoclonal=specific for 1 thing

Question 7

Monoclonal antibody

Answer 7

short-lived antibody producing cell

B cell(antibody) + myeloma cell (immortal)

Question 8

ELISA: HIV

Answer 8

Looking for antibody

(Indirect: antigen coated well)

Can give false positives, so we use western to double check)

Question 9

Sandwich ELISA

Answer 9

Antibody coated well

Allowing both the detection and the quantitation of antigen

Question 10

ELISA: troponin

Answer 10

Looking for troponin T,I,C

Plate coated with antibodies (looking for antigen of Troponin T)

Troponin T and I ↑ in serum after myocardial infarction (escape b/c of damage)

Markers of acute ischemia along with myoglobin and creatine kinase

Question 11

ELISA: Hormones

Answer 11

Sandwich method used

Pregnancy test-free monoclonal anitbody for hCG is used

Sandwich formed by combination of capture antibody and free antibody when hCG is present, creating a color change

Question 12

Western blotting

Answer 12

Compare protein levels
Proteins separated by size

Steps:

1. Protein mix is separated by SDS (gives negative charge)
2. Transferred to nitrocellulose (b/c proteins are trapped in gel)
3. Blocked to reduce non-specific interactions
4. Add 1st antibody (against target protein)
5. Add 2nd antibody (against 1st antibody)

Detection using:

• HRP (horseradish peroxidase): turns brown
• Alkaline phosphatase (AP): dark blue/purple

Question 13

Proteomics

Answer 13

Study of structure/function of proteins

Analysis of protein expression and/or alteration

Post-translational Modifications

“Fishing expedition”-just looking at lots of stuff

knockout vs wild type

Question 14

2D gel electrophoresis

Answer 14

pH + size–> Use 3 colors: “young, old, standard”–>overlay images–>cut out spots–>mass spec–>break proteins into small fragments and bombard w/ electrons to ionize them–> cluster analysis