The most sensitive method for the detection of oligoclonal bands (OCB) is isoelectric focusing (IEF). The principle involves the separation of proteins (IgG's) in serum and cerebrospinal fluid (CSF) pair using agarose gel electrophoresis followed by passive transfer onto nitrocellulose membrane. The separated IgGs are then detected directly by horseradish peroxidase labelled anti-human antibody.
GEL SOLUTION: 0.3g agarose, 3.6g sorbitol, 27ml of 10% glycerol and microwave to dissolve.
CASTING GEL: AT 65°C add 2ml (pH 3-10) & 0.5ml (pH 8-10.5) pharmalyte to molten agarose (65-70°C), mix and cast at 65°C.
HYSTERESIS: Keep the casted gel at 4°C in a moist chamber for at least 30 min.
SAMPLE PREPARATION: Serum diluted at 1:400 & 5µl loaded. Vol. (µl) of CSF loaded = (2.5/total protein).
RUNNING THE GEL: Blot the gel with nitrocellulose membrane (NCM). Position the electrode wicks at 7cm apart (1M NaOH (-ve), 0.05M H2SO4 (+ve)) on the gel and then the application strip 2cm from +ve electrode. Remove application strip after 20mins. The gel is electrophoresed at 1250 volt/hour.
BLOTTING: Pre-blot gel for 10 sec with NCM and discard it. Blot with another NCM piece for 30min.
BLOCKING: Dry the blot. Incubate in 2% marvel/saline for 30minutes and quick rinse in tap water.
PRIMARY ANTIBODY: Anti-human IgG (gamma chain) peroxidase, ~1:200 dilution in 0.2% marvel/saline and incubate for 60mins.
WASH/TAP WATER/SALINE: Rinses in tap water followed by 5 min in saline.
DEVELOPMENT OF THE BLOT: To 50mL of 50mM sodium acetate (pH 5.1), add 20mg tablet of A.E.C. dissolved in 2.5ml D.M.F/methanol and 30µl of H2O2, (20-30mins).
WASH/TAP WATER: Rinses in running tap water, dry and store in dark.
For Interpretation of oligoclonal bands. See real examples of most commonly recognised patterns of OCB elsewhere on this site.