Western blot

Principle

• Proteins (antigens) are separated by size using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred electrically onto a nitrocellulose or PVDF membrane.
• In diagnostic immunoblots, the membrane strip carrying separated antigens is incubated with patient serum; specific antibodies bind to individual antigen bands.
• Bound antibodies are detected with an enzyme-labelled anti-human immunoglobulin and a chromogenic or chemiluminescent substrate, producing visible bands.
• Band position (molecular weight in kDa) identifies which antigens the patient's antibodies recognise.

Specimen

• Serum or plasma.

Procedure

• Commercial kits supply ready-made antigen strips.
• Block strips (e.g. milk protein or BSA) to prevent non-specific binding.
• Incubate with diluted patient serum; wash.
• Add enzyme conjugate (alkaline phosphatase or peroxidase anti-human IgG or IgM); wash.
• Add substrate; stop reaction; dry and compare bands with the kit reading template and control strips.

Interpretation

• HIV (historic confirmatory test): positive when at least two of p24, gp41 and gp120/160 bands are present; no bands is negative; other band patterns are indeterminate. CDC algorithm has replaced it with an HIV-1/HIV-2 differentiation immunoassay and NAT.
• Lyme disease (second tier): IgM positive with at least 2 of 3 bands (23/24, 39, 41 kDa), used only in the first 30 days of illness; IgG positive with at least 5 of 10 specified bands.
• Indeterminate results require repeat testing or NAT after an interval.

Quality control and pitfalls

• Include strong positive, weak positive and negative controls in every run.
• Indeterminate patterns may occur in early seroconversion, pregnancy, autoimmune disease or after vaccination.
• Visual reading is subjective; densitometric or scanner reading improves consistency.
• Time-consuming and costly compared with immunoassays.

Clinical use

• Confirmation of screening results in infections such as Lyme disease and HTLV-1/2; research protein detection. Line immunoassays using recombinant antigens are a related format (e.g. ENA profiles).