Typical bands found on serum gel electrophoresis:

Transcription

1 Gel Electrophoresis LD Recognise EPG patterns typical of other body fluids including urine and CSF Identify patterns of changes including - Paraproteins - Hypogamma - Acute phase - Circulating immune complexes - Nephrotic syndrome - Liver disease - Pregnancy - Bisalbuminaemia - Alpha1 AT deficiency - Iron deficiency - Haemolysis Explain the principles and be able to interpret immunoasays that utilise the migration of proteins within a gel, such as - Counter immune- electrophoresis (CIEP) wrt ENA - Rocket electrophoresis (Including fused rockets) Typical bands found on serum gel electrophoresis:

2 Urine EPP: Normal glomerular filtrate contains traces of albumin and small MW proteins: α1 acid glycoprotein (40kDa) α1 microglobulin (27kDa) β2 microglobulin (12kDa) Retinol binding protein (20kDa) Normally absorbed by renal tubules Normal urine (when concentrated 100- fold): no bands or only small amount of albumin can be seen Glomerular proteinuria: Predominantly albumin, smaller amount of alpha and beta globulins Tubular proteinuria: α1, α2 and β microglobulins, some albumin (much less than in glomerular proteinuria) Non- selective (combined) proteinuria: Albumin, very large molecules such as IgG, often combined pattern in chronic renal disease, BJP may migrate in α,β or γ region CSF oligoclonal banding/isoelectric focussing: Oligoclonal bands positive in 75-90% of MS patients CSF IgG index positive in 70-80% of MS patients o CSF IgG index = (CSF IgG/CSF albumin)/(serum IgG/serum albumin) o A marker of relative synthesis of immunoglobulin in CSF o Normal for adults <0.64 Normally IgG in serum is distributed in a Gaussian fashion CSF IgG usually demonstrates restricted heterogeneity electrophoretic pattern therefore is not continuous.

3 Isoelectric focussing: agarose gel with ph gradient 3 to 10. Electrophorese, transfer to nitrocellulose membrane and blot, detect with anti- IgG conjugate. Four or more bands consistent with oligoclonal banding. The presence of bands in CSF but not serum is suggestive of intrathecal immunoglobulin synthesis. Isoelectric focussing is more sensitive than agarose gel electrophoresis for the detection of CSF oligoclonal banding. Paraproteins: IgG kappa: IgG lambda: IgA kappa: IgM kappa:

4 Luke Droney Hypogammaglobulinaemia Decreased gamma globulins Alpha- 1- antitrypsin deficiency (middle lane deficient)

5 Two peaks in alpha 1 region for heterozygotes. Acute phase Observed changes: Decreased albumin Increased alpha 1 region due to increased alpha- 1- antitrypsin Increased alpha 2 region due to increased haptoglobin and caeruloplasmin Increased beta region due to C3 Polyclonal hypergamma Presence of CRP or fibrinogen Fibrinogen: CRP:

6 May be present if sample collected/processed incorrectly (i.e plasma run), patient on anticoagulants. Remove by addition of thrombin to sample. Polyclonal hypergamma with beta/gamma bridging: Polyclonal hypergamma:

7 Luke Droney Bisalbuminaemia Bisalbuminaemia is a bifid albumin electrophoretic pattern due to the presence of alloalbumins that have either an increased or decreased electrophoretic mobility. Associated with: Genetic bisalbuminaemia (not pathologic) Acquired described in diabetes, Waldenstrom s, MM, sarcoidosis, nephrotic syndrome, pancreatitis, pancreatic pseudocyst, CKD Drugs: high dose penicillins

8 Nephrotic syndrome: Increase in alpha 2 region due to increased alpha- 2- macroglobulin and beta- lipoprotein (LDL) associated with decrease in albumin, decrease in gamma globulins. Liver disease/cirrhosis Beta- gamma block due to polyclonal hyper IgA in liver disease

9 Haemolysis Haemoglobin- haptoglobin complexes migrate in the alpha or beta fractions depending on the electrophoresis system. Band usually looks fuzzy.

10 Luke Droney

11 Luke Droney Anaemia Increased beta region due to increased transferrin. Pregnancy Increased beta- globulins due to increased transferrin Increased alpha1- globulins due to alpha1- lipoprotein Decreased albumin Decreased gamma globulins Circulating immune complexes Oligoclonal gamma banding CIEP Similar principle to Ouchterlony double- immunodiffusion. An electric field is applied to increase the rate of diffusion. - Principle o Formation of insoluble immune complexes where an antibody encounters the optimal concentration of antigen - Technique

12 o Buffered agarose gel (containing electrolytes) o Current applied across the gel forcing the antigen and antibody together o Antibodies are relatively neutrally charged and are carried by the flow of positively charged ions (principle known as electroendosmosis) o Protein/antigens are negatively charged and will migrate in the opposite direction towards the +ve o If the antigen and antibody are specific for each other, they combine to form a distinct precipitin line Rocket electrophoresis (Laurell Rocket Technique) Similar technique to radial immunodiffusion Electrophoresis is used to assist immunodiffusion i.e makes the assay faster than waiting for passive diffusion. Gel poured containing anti- sera. Wells punched in the gel for serum Electric current applied An immunoprecipitate forms (the rocket )

13 Concentration of the analyte is proportional to the height of the rocket (as it s proportional to the radius of the zone of immunoprecipitation in radial immunodiffusion). Fused rocket technique used to characterise elution fractions from a chromatography column. An aliquot of each fraction is placed in the wells and subjected to electrophoresis. The technique shows which fraction contains most of the protein of interest.