ELISA assay

Introduction¶

First described by Eva Engvall and Peter Perlmann in 1971, the enzyme-linked immunosorbent assay (ELISA) is a widely used analytical biochemistry assay. ELISA uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. The ELISA test is the first screening test commonly employed for HIV and was approved for use on March 2, 1985.

Scientific Background¶

Although HIV antibody tests are the most appropriate for identifying infection, alternate technologies can contribute to an accurate diagnosis, assist in monitoring therapy response, and predict disease outcomes. Viral isolation, nucleic acid tests to detect viral RNA, and tests to detect p24 antigen can demonstrate virus or viral components in blood, verifying infection. These methods are highly specific, and a positive result confirms infection. However, each has limitations and their use must be tailored to proper testing situations. Tests for viral nucleic acid require sophisticated technology and well-trained personnel.

The HIV antigen test is currently used for screening blood for transfusion and is appropriate for several other testing situations. It offers the advantages of simplicity and cost-effectiveness for verifying infection but is less than perfect.

The p24 antigen assay measures the viral capsid (core) p24 protein in blood, which is detectable earlier than HIV antibody during acute infection. It occurs early after infection due to the initial burst of virus replication and is associated with high levels of viremia, during which the individual is highly infectious. When antibodies to HIV become detectable, p24 antigen is often no longer demonstrable, likely due to antigen-antibody complexing in the blood. When detected, p24 antigen is highly specific for infection.

The goal of this study is to identify compounds that can inhibit HIV replication in latently infected cells.

Materials and Methods¶

A microplate is prepared containing a blank, positive and negative controls, the standards, and the samples.

Controls¶

For confidence in ELISA results, three experimental controls are required for comparison: a positive control, negative control, and standard control. Both the positive and standard controls are known to contain the protein or peptide of interest.

The positive control confirms the procedure is performing as intended.
The negative control is known to not contain the biomolecule of interest and adds validity to any positive results.
The standard control is necessary for quantification. It contains a known quantity of the target molecule and provides the information needed to make a standard curve.

Standards¶

To determine the levels of p24 antigen in latently infected cells, an HIV-1 antigen standard is diluted to prepare a series of eight standards of varying concentrations (0.0 to 200 ng/mL).

Samples¶

For this study, two cloned cells chronically infected with HIV-1 (U1 and OM10.1) are used. The cells are incubated with different compounds to test.

Principle¶

The optical densities at 490 nm and 650 nm are measured for the microplate, and the ratio of both measures is calculated. These values are then corrected with the mean of the blank wells' optical densities.

A standard curve is generated using a linear graph, plotting the concentration of the HIV-p24 antigen standard (ng/mL) on the X-axis versus the mean optical densities for each standard on the Y-axis. Each standard is added in duplicate wells.

The optical density values of the unknown samples are interpolated in the standard curve to determine their concentration using linear regression.

If the value of the unknown sample is higher than the value of the highest standard, the sample must be diluted and the entire neutralization procedure repeated.

Prerequisites¶

An existing run template, created using the Design Mode, following the tutorial here.

Files to use:

Run Creation¶

To create a new run, click on the New Run button.
Select Mode as Execution Mode.
Give a Run Name.
Select Run Template: the one saved in 3.9 (Publisher Selection) of Design Mode, for instance.
Go to File Reading step.

File Reading¶

The plate format to use is already set: 96 (8x12).
The reader named (Analyst GT (TAB)) is already fixed.
Upload the file 05-Elisa_Raw_Data.
Go to Raw Data File step.

Raw Data File¶

Number of Measurements per Plate is already mapped: 2.
You cannot modify the Readout Alias (fixed to READOUT490 and READOUT620)
Transformation Ratio 490/650 is already specified
Go to Plate Mapping step.

Plate Mapping¶

The Control Layout selected in Design Mode is already applied to all plates.

Go to Well Validation step.

Well Validation¶

Visualization template selected in Design Mode is already applied.
You can change this template if needed.
For QC Validation, invalidate points following "Business rules".

Example 1: Plate ZScore interval

Plate_ZSCORE < -1.5 or Plate_ZSCORE > 1.5. Select and invalidate the points out of this scope.

Example 2: Z' factor interval

The Z' factor is an indicator of the statistical data quality for a bioassay.

A Z' factor between 0 and 1 is ideal.
A Z' factor between 0.5 and 1.0 is an excellent assay.
A Z' factor between 0 and 0.5 is marginal.
A Z' factor less than 0 means that the signal from the positive and negative controls could overlap, making the assay not very useful for screening purposes.

Select aberrant wells in the visualization and click on the Well Validation step: Invalidation button button. Multiple selection is allowed by holding the Ctrl key.

Tip

The space occupied by the visualization can be optimized to your liking by clicking on the full screen icon icon and the icon.

Sample Mapping¶

Repeat Step 6 from Design mode in order to map Samples, Doses, and Experimental conditions.

Go to Sample Analysis Settings step.

Sample Analysis Settings¶

Groups are automatically created (1000 groups).
Basic Statistic analysis is already applied.
Go to Sample Analysis step.

Sample Analysis¶

The template specified in Design mode is applied.

See Step 8 of Mode design as reference.

For hits selection, select points (hits) in visualization and add them to a list (click on the trolley icon). This selection can be exported in CSV format (export button).
Lock your Analysis (padlock symbol).
Go to Publisher step.

Publish Data¶

Depends on the Publisher defined in the run template.