Overview

The Rapid-Lenti HIV-1 p24 ELISA quantifies the HIV-1 capsid protein p24 in viral stocks, lentiviral pseudoparticles, and culture supernatants. It is a sandwich ELISA optimized for speed, sensitivity, and linear dynamic range. For core background on HIV-1 structure and p24 biology, see NCBI Bookshelf (NIH) (link), NCBI Gene LGALS3 is unrelated; consult HIV-1 gag/p24 entries in GenBank (NIH) (link) and PubMed method literature (NIH/NLM) (link). General laboratory biosafety guidance is available from CDC (link) and OSHA (link).

RUO disclaimer: This content and kit are for Research Use Only (RUO). Do not use for diagnostic procedures. Follow institutional biosafety approvals.

Principle of the assay

1. Capture: Microplate wells are pre-coated with anti-p24 capture antibodies (gag capsid specific).

2. Bind: Samples/standards containing p24 bind to the capture layer.

3. Detect: A biotinylated or directly conjugated anti-p24 detection antibody binds a distinct epitope.

4. Signal: HRP/TMB development yields an optical density read at 450 nm with 570–620 nm reference.

For concise immunoassay fundamentals: NCBI Bookshelf (NIH) (link), MedlinePlus lab testing primer (NLM/NIH) (link), and MIT OpenCourseWare (edu) (link).

Typical kit contents & storage

• Pre-coated 96-well plate (break-apart strips)

• Recombinant HIV-1 p24 standard (lyophilized)

• Sample/assay diluent, wash buffer (10×/20×)

• Detection antibody, HRP conjugate

• TMB substrate, stop solution

• Plate sealers and IFU

General handling of chemicals: EPA hazardous waste guidance (link). Reference metrology and materials: NIST SRM program (link).

Intended samples (research)

• Lentiviral production supernatants (second/third-generation systems)

• Pseudotyped HIV-1 particles and VLPs

• Cell lysates (validated internally)

For handling potentially infectious materials, review CDC BSL guidance (link) and NIH biosafety resources (link).

Quick protocol (fast workflow)

1. Equilibrate reagents to RT (20–25 °C). Plan a plate map.

2. Prepare standards: 2–3-fold serial dilutions spanning expected range (e.g., 3–5 logs).

3. Load 100 µL/well of standards, blanks, QCs, and diluted samples in duplicates/triplicates.

4. Incubate (rapid format, e.g., 30–60 min; follow IFU) → wash 3–5×.

5. Add detection Ab, incubate → wash.

6. Add HRP conjugate, incubate → wash.

7. Add TMB, develop to target OD (≤1.5 top standard) → stop with acid.

8. Read at 450 nm with 570–620 nm reference; export CSV.

9. Analyze using 4PL/5PL with appropriate weighting (1/y or 1/y²).

Curve-fitting references: NCBI Bookshelf statistics chapters (link), NIDA research stats primers (NIH) (link).

Calibration, units, and conversion

• Report p24 results as pg/mL (primary) or convert to capsid molecules/mL using molecular mass assumptions.

• For inter-lab comparability, document standard lot, dilution scheme, and curve parameters.

• Consider periodic verification with traceable materials where available (see NIST SRM, concept only: link).

Data standards and sharing: NIH Data Management & Sharing (link).

Analytical validation (lab-level)

Range & LLOQ

• Define functional range based on back-calculated standards with total error within spec.

• Establish LLOQ where accuracy is 80–120% and %CV ≤ 20%.

Precision

• Intra-assay %CV: multiple replicates within a plate.

• Inter-assay %CV: different days/operators/reagent lots.

Accuracy/Recovery

• Spike-recovery in matrix at low/mid/high (target 80–120%).

• Parallelism across serial dilutions to confirm matrix equivalency.

Specificity

• Verify minimal cross-reactivity to non-HIV retroviral proteins and host proteins.

Foundational guidance (general bioanalytical concepts): FDA (method validation best practices, conceptual) (link), and NLM research reporting resources (link).

Plate map & QC

Include blank, STD A–G, QC-L/M/H, and sample replicates. Maintain Levey–Jennings charts to track drift and lot shifts (plotting with ImageJ/Fiji: NIH, link). Maintain equipment verification logs; see NIST metrology principles (link).

Data analysis (4PL/5PL)

• Prefer 4PL; use 5PL for asymmetric curves.

• Weighting (1/y²) often stabilizes residuals at high ODs.

• Flag and treat outliers with a pre-specified rule (e.g., Grubbs), documented a priori.

• For samples above top standard, dilute into assay diluent and re-run within range.

For statistical grounding: NHLBI research methods (NIH) (link) and PubMed methods reviews (NIH/NLM) (link).

Biosafety and handling

Work with lentiviral supernatants under approved conditions. Review: CDC biosafety (link), NIH lab safety (link), OSHA laboratory safety (link). Waste management per EPA (link) and institutional EHS policies (example resources: Stanford EHS (edu) link, Berkeley EH&S (edu) link).

Applications (research)

• Lentiviral titer tracking during vector production and concentration steps.

• Process optimization (MOI targeting, transduction workflows).

• Stability studies (freeze-thaw, storage at 4 °C/−80 °C).

• Comparative pseudotyping (envelope variants, producer cell lines).

Background on HIV-1 life cycle and gag processing: NCBI Bookshelf (NIH) (link) and GenBank sequence resources (NIH) (link).

Troubleshooting

• Weak signal: short incubations, inactive HRP/TMB, cold reagents → verify times/temps; use fresh substrate.

• High background: insufficient washing, plate drying, contaminated buffers → increase wash cycles, maintain humidity, replace diluent.

• Non-parallelism: matrix effects → increase dilution, add blockers, validate alternate diluent.

• Hook effect at very high p24 → pre-dilute samples and re-measure.

• Edge effects: fully equilibrate plates, avoid drafts; seal consistently.

University practical guides: Cornell (edu) (link), University of Arizona (edu) (link), UC Berkeley (edu) (link).

Documentation & compliance checklist

• Institutional SOP and IBC approvals (HHS policy portal: link).

• Training/competency records; equipment calibration logs.

• Full run records (raw ODs, plate map, curve, QC results).

• Data retention and sharing per NIH DMS (link).

Frequently asked questions (SEO-friendly)

Q1. Can I use the p24 ELISA to estimate functional titer?
Use p24 as a proxy for particle load; correlate empirically with infectious units determined by a cell-based assay. Keep separate specifications. See PubMed method papers (NIH/NLM) (link).

Q2. What weighting improves curve stability?
Heteroscedastic ELISA data typically benefit from 1/y² weighting. Validate against residual plots. See NCBI Bookshelf stats (NIH) (link).

Q3. How many standards are ideal?
≥7 points plus blank across 3–4 logs of concentration supports robust 4PL/5PL fits; verify back-calculated recovery. See NIST concepts (link).

Reference links (.gov / .edu)

1. NCBI Bookshelf (NIH) — https://www.ncbi.nlm.nih.gov/books/

2. GenBank (NIH) — https://www.ncbi.nlm.nih.gov/genbank/

3. PubMed (NIH/NLM) — https://pubmed.ncbi.nlm.nih.gov/

4. MedlinePlus Labs (NIH/NLM) — https://medlineplus.gov/lab-tests/

5. CDC Labs — https://www.cdc.gov/labs/

6. OSHA Laboratory Safety — https://www.osha.gov/laboratory-safety

7. EPA Hazardous Waste — https://www.epa.gov/hw

8. NIST Standard Reference Materials — https://www.nist.gov/srm

9. NIH Data Management & Sharing — https://sharing.nih.gov

10. NLM Research Reporting/Data Sharing — https://www.nlm.nih.gov/NIHbmic/nih_data_sharing.html

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