Proteins & Tumor Markers Flashcards
1
Q
Electrophoresis
A
- Movement of charged molecules/particles in a liquid medium under the influence of an electrical field
- Used to separate proteins, mainly serum*, CSF, urine and hemoglobins
- Sample applied at cathodic (-) end of alkaline gel and voltage applied –> negatively charged proteins migrate based on net charge/size
- Proteins stained and visualized, quantified by densitometer
2
Q
Electrophoresis Media
A
- Most common is agarose gel –> polysaccharide from seaweed, neutral so separation based on mainly charge, low endosmosis
- Polyacrylamide gel –> used in IEF & nucleic acid separation, not charged = no endosmosis, particles separated by size (crosslinking fibers), medium has neurotoxic monomers (issue if made in-house)
3
Q
Electroendosmosis
A
- Support has fixed negative charges (OH-) with adjacent immobile (+) ions –> Stern Potential
- When voltage applied, solvent flows towards cathode
- Mobile ions (Zeta Potential) with low to no-charge migrate with solvent toward cathode, strong negative charges ions remain fixed to medium (or migrate slowly)
- Migration flow dependent on distance from fixed charges (less flow as farther away)
4
Q
Hemoglobin Electrophoresis
A
- Run on both alkaline and acid gels to separate Hgb variants
ALKALINE: A / F / S,D,G / C,E,O,A2
ACID: F / A,D,G,O,A2,E / S / C
- separation also done by HPLC or capillary electrophoresis
5
Q
Capillary Electrophoresis
A
- Process run in long capillary tube with each end in inlet/outlet buffer; spec (lamp&detector) set up to detect fractions
- Good heat dissipation allows for very high voltage
- Rapid separation, no staining or densitometry, highly automated
- Very high endosmosis
6
Q
Isoelectric Focusing (IEF)
A
- Ampholyte mixture buffer creates linear pH gradient along gel
- Proteins migrate to isoelectric point (pI) = pH at which protein has no net charge
- Refocusing of proteins that try to diffuse (due to gradient they are sent back to pI) allows for sharp bands
- Used for CSF oligoclonal banding (MS)
7
Q
Western Blot
A
- not used much clinically
- proteins separated by MW in gel electrophoresis
- transferred to nitro-cellulose membrane “blotting”
- Sample Ab’s bind to immobilized target proteins, washed and stain using labeled Ab
8
Q
Albumin
A
- Only protein quantified by ep, others need nephelometry/turbidimetry
- Most abundant protein in plasma (60%), maintains Colloid Osmotic Pressure (COP) and carrier for many molecules
- Hyperalbuminemia: not significant other than dehydration
- Hypoalbuminemia: increased loss (NEPHROTIC SYNDROME, gi, burns), decreased production (liver failure, malnutrition)
- Analbuminemia (little to no albumin) and bisalbuminemia (two peaks, dimeric protein)
- Being bound to bili/drugs may alter mobility
9
Q
Pre-Albumin
A
- Not a form or precursor of albumin
- Migrates before albumin on gel (seen in CSF gels, not always in serum gels)
- aka Thyroxine-binding protein (TBPA), transthyretin
- Sensitive marker of nutritional status (measured by neph/turb)
10
Q
Alpha 1 Proteins
A
- A1-Antitrypsin (AAT): protease inhibitor that binds/inactivates trypsin, deficiency associated w/ pulmonary emphysema & neonatal hepatitis, SPEP screening needs IEF to determine phenotype (ZZ & SZ high risk)
- A1-Acid Glycoprotein (AAG): APR, rarely measured
- A1-Fetoprotein (AFP): main fetal plasma protein, measured in amniotic fluid/maternal serum to screen for fetal abnormalities (neural tube defect), hepatic tumor marker
11
Q
Alpha 2 Proteins
A
- A2-Macroglobulin (A2MG): Largest non-Ig, increased in nephrotic syndrome- replaces lost albumin to maintain COP (not cleared, increased synthesis in liver), rarely measured
- Ceruloplasmin: Cu binding protein (8 Cu per molecule), plasma redox reactions (ferritin -> transferrin), screen for Wilson’s Dz
- Haptoglobin: binds Hgb irreversibly in intravascular hemolysis to prevent Fe loss (or bacteria use), low levels indicate hemolytic dz
12
Q
Beta Proteins
A
- Transferrin: Iron transport protein, increased in IDA (also pregnancy, estrogen therapy), decreased in inflammation/malignancy/liver dz, NEGATIVE APR
- C3/C4 Complement Proteins
13
Q
Complement Proteins
A
- Classical Pathway (C1-C4): Immune complexes recognized by C1, C2/C4 activate C3 (MAC)
- Alternative Pathway (C3, factor B/D, properdin): activated by bacteria, yeast, or endotoxins –> foreign surfaces
- C3 in both pathways, C4 classic only
14
Q
Complement Deficiency
A
- C2/C4**: Autoimmune Dz
- C3**: infection, encapsulated bacteria; low in all complement situations
- C5-C9: persistent Neisseria infection
- C1 Inhibitor: Hereditary Angioedema (HAE)–> can cause subcutaneous edema in laryngeal/bronchial/GI tissues, can be life-threatening, low C4
- CH50 = Total complement assay not preferred as large individual deficiencies may not be detected
15
Q
Immunoglobulins
A
- IgG: 2 heavy chains + 2 light chains, produced by plasma cells, long term immunity (or chronic dz), crosses placenta
- IgM: pentamer linked by J peptides, produced by plasma cells as first-line immunity and persists for 6-12months (acute dz)
- IgA: monomer or dimer, circulatory and secretory (tears, sweat, saliva etc), cleared by liver
NOT ON SPEP: - IgE: allergies
