HIV Ag/Ab Combo ECLIA Test Methodology
Test Principle and Mechanism
The HIV Ag/Ab combo electrochemiluminescence immunoassay (ECLIA) simultaneously detects HIV-1 p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in a single test using electrochemiluminescent technology on automated platforms. 1, 2
Core Technology
The ECLIA uses a sandwich immunoassay format where the sample is incubated with biotinylated capture antibodies and ruthenium-labeled detection antibodies that bind to HIV antigens and antibodies present in the specimen 2
Streptavidin-coated microparticles capture the immune complexes, which are then magnetically separated and transferred to a measuring cell where voltage application triggers electrochemiluminescent emission measured by a photomultiplier 2
The assay employs recombinant antigens and synthetic peptides representing HIV-1 group M, group O, and HIV-2 epitopes to ensure broad reactivity across viral subtypes 3, 2
Specimen Requirements and Processing
Sample Collection
The test requires serum or plasma collected in standard blood collection tubes with EDTA, citrate, or heparin as acceptable anticoagulants 1, 4
A minimum volume of 100 μL of specimen is typically required for automated ECLIA platforms 1
Pre-Analytical Considerations
Specimens should be centrifuged at no greater than 400g for 3-5 minutes to separate serum or plasma from cellular components 1
Samples can be stored at refrigerator temperatures (4-10°C) for up to 24 hours before testing, or frozen at -70°C for longer-term storage 1
Testing Algorithm and Workflow
Initial Screening
The specimen is automatically pipetted by the analyzer and mixed with reagents containing biotinylated HIV antigens (for antibody detection) and labeled anti-p24 antibodies (for antigen detection) 2
After an 18-minute incubation period at 37°C, the reaction mixture undergoes magnetic separation and washing steps 2
The electrochemiluminescent signal is measured and compared to a cutoff value determined by calibration; results above the cutoff are considered reactive 2
Repeat Testing Protocol
All initially reactive specimens must be retested in duplicate using the same ECLIA assay 1
A specimen is considered repeatedly reactive only if at least one of the duplicate retests is reactive 1
Confirmatory Testing Pathway
Repeatedly reactive specimens by HIV-1/HIV-2 ECLIA must be tested by HIV-1 Western blot or another licensed supplemental test for confirmation 1
If the HIV-1 Western blot result is positive, this confirms the presence of HIV antibodies and no further testing is needed unless HIV-2 risk factors are present 1
If the HIV-1 Western blot result is negative or indeterminate, an HIV-2-specific EIA should be performed, followed by HIV-2 supplemental testing if positive 1
For specimens with indeterminate Western blot results, HIV RNA nucleic acid testing (NAT) should be performed to detect acute infection 1, 5
Performance Characteristics
Sensitivity
Fourth-generation ECLIA assays demonstrate 100% sensitivity for established HIV-1 and HIV-2 infections across diverse viral subtypes including groups M, O, and rare genotypes 3, 2, 6
The assay can detect seroconversion at the same bleed or one bleed earlier compared to third-generation antibody-only assays due to p24 antigen detection capability 2
In acute/early HIV infection, ECLIA detects approximately 90.9% of cases, with false negatives occurring primarily in individuals with viral loads <400 copies/mL or those on antiretroviral therapy 3, 6
Specificity
Clinical specificity ranges from 96.7% to 100% depending on the population tested and pre-screening protocols 2, 6
False-positive rates of 0.2% to 3.3% have been reported, with higher rates in low-prevalence populations 7, 2
Window Period Reduction
The addition of p24 antigen detection reduces the diagnostic window period by approximately 5-7 days compared to third-generation antibody-only assays 4, 2
However, a window period of approximately 2-4 weeks still exists between initial infection and detectable p24 antigen or antibodies 1, 5
Quality Control and Validation
Internal Controls
Each ECLIA run must include negative and positive controls to ensure assay validity 1
The run is considered valid only when all controls fall within acceptable ranges with no false positives in negative controls or false negatives in positive controls 1
Calibration Requirements
The assay requires periodic calibration using manufacturer-provided calibrators to establish the reactive cutoff value 2
Calibration frequency varies by platform but typically occurs every 28 days or after reagent lot changes 2
Clinical Interpretation Caveats
False-Negative Considerations
Antibody tests cannot rule out infection that occurred less than 6 months before testing, as HIV antibody is detectable in ≥95% of patients within 6 months of infection 5
Acute HIV infection within the first 2-4 weeks may yield negative ECLIA results despite active viral replication 5, 7
Immunosuppressed patients or those on antiretroviral therapy may have undetectable p24 antigen levels leading to false negatives 6
False-Positive Considerations
Never disclose a positive HIV diagnosis without confirmatory Western blot or supplemental testing, as false positives occur with screening tests 5
Cross-reactivity with other conditions (autoimmune diseases, recent vaccinations, pregnancy) can cause false-positive screening results 7
Specimens with signal-to-cutoff ratios close to the threshold have higher likelihood of being false positives and require careful confirmatory testing 7
Special Populations
Infants Born to HIV-Positive Mothers
For infants <15 months of age, definitive diagnosis must be based on virologic testing (HIV DNA or RNA PCR) rather than antibody testing due to maternal antibody transfer 5
ECLIA antibody results in this population reflect maternal rather than infant infection status 5
HIV-2 Considerations
HIV-2 testing should be considered in persons from endemic regions (West Africa, Portugal, France, India) or their sexual partners 5
Testing for HIV-2 is recommended when clinical evidence of HIV disease exists but HIV-1 antibody tests are negative 5
If HIV-1 Western blot shows an unusual indeterminate pattern (gag plus pol bands without env bands), HIV-2-specific testing should be performed 1