What is the difference between immunoassay (Immune assay) and immunoblot (Immunoblotting)?

Differences Between Immunoassay and Immunoblot

Immunoassays and immunoblots are distinct laboratory techniques that differ primarily in their ability to identify specific proteins - immunoblots can identify individual proteins based on molecular weight, while immunoassays detect the overall presence of antibodies without distinguishing specific protein targets.

Immunoassay (ELISA/EIA)

Definition and Principles

• Immunoassays are screening tests that detect the overall antibody response (typically IgM and IgG) to antigens 1
• Most commonly performed as Enzyme-Linked Immunosorbent Assay (ELISA) or Enzyme Immunoassay (EIA)
• Can also be performed as Immunofluorescence Assay (IFA) 1

Key Characteristics

• Provides quantitative values of relative antibody concentration compared to controls 1
• Uses objective cutoff values to determine positive/negative results 1
• Often uses whole-cell sonicate preparations or specific antigens (like VlsE or C6 peptide) 1
• Highly automatable and suitable for high-throughput screening 1
• Cannot distinguish between specific proteins that antibodies are targeting 1

Applications

• Used as first-tier screening tests in diagnostic algorithms 1, 2
• Sensitivity ranges from 85-100% and specificity around 79-95% depending on the assay 1
• Commonly used in serodiagnosis of infections like Lyme disease and H. pylori 1

Immunoblot (Western Blot)

Definition and Principles

• Immunoblotting (Western blot) involves electrophoretic separation of proteins followed by transfer to a membrane and detection with antibodies 3, 4
• Detects antibodies against specific proteins based on their molecular weight 1
• Can identify individual protein targets rather than just overall antibody response 4

Key Characteristics

• Combines gel electrophoresis for resolving proteins with immunodetection 4
• Requires precise internal controls for semi-quantitative evaluation 1
• Available in whole-cell antigen formats or recombinant antigen formats 1
• More labor-intensive and time-consuming than immunoassays 3
• Higher specificity but may have lower sensitivity than some immunoassays 1

Applications

• Used as confirmatory tests after positive immunoassay results 1, 2
• Allows identification of specific immunoreactive proteins (e.g., CagA, VacA in H. pylori) 1, 5
• In Lyme disease, requires specific band patterns for positive interpretation (e.g., ≥2 of 3 specific bands for IgM, ≥5 of 10 specific bands for IgG) 2
• Can detect as little as 1 ng of antigen 3

Key Differences

1. Separation vs. Detection:

   • Immunoblot: Separates proteins by molecular weight before detection 3
   • Immunoassay: Detects antibodies without separating individual proteins 1

2. Specificity and Information:

   • Immunoblot: Identifies specific proteins/antigens being recognized 1
   • Immunoassay: Provides overall antibody response without identifying specific targets 1

3. Workflow and Complexity:

   • Immunoblot: More complex, multi-step process requiring technical expertise 3
   • Immunoassay: More easily automated and standardized 1

4. Diagnostic Algorithm Position:

   • Immunoblot: Typically used as a confirmatory test 2
   • Immunoassay: Usually employed as a screening test 1

Clinical Implications

• Immunoassays and immunoblots are not truly independent tests - there's correlation between their results 6
• In two-tiered testing (e.g., for Lyme disease), immunoblot should be considered supplementary rather than purely confirmatory 6
• Immunoblotting has shown higher specificity than immunoassay in some studies (e.g., for H. pylori) 5
• For certain applications like detecting CagA or VacA antibodies, immunoblots may be more informative than standard immunoassays 1, 5

Common Pitfalls

• False-positive immunoassay results can occur due to cross-reactivity with other antigens 5
• Immunoblot interpretation requires expertise to distinguish specific from cross-reactive bands 1
• Persistent antibodies after successful treatment can lead to false-positive results in both techniques 1, 2
• Low pretest probability can lead to misleading results regardless of technique 2

By understanding these differences, clinicians can better interpret laboratory results and select the appropriate testing strategy based on the clinical context and diagnostic needs.