Method of Serum Antibody Concentration Determination by Indirect ELISA

The need for accurate antibody concentration measurement has become increasingly crucial in various fields, including drug and vaccine development, as well as infectious disease diagnostics. To this end methods often rely on indirect ELISA assays, where the concentration of serum antibodies is determined by comparing it to a standard. These standards are typically sourced from individuals confirmed to have the disease and are quantified in arbitrary units.

By comparing OD (optical density) responses from individuals with the standard, the relative potency of each serum can be determined. Usually, concentrations higher than a predetermined cut-off indicate incidence of the disease. However, the actual concentrations can be used to assess various aspects of the immune response, such as the severity of infection or the effectiveness of vaccination.

Material:

96 well ELISA plate; PBS; 0.05M Carbonate Buffer ; Coating sample (this is typically the antigen or a subunit of it); Tween 20; Sera; 2ndry antibody (HRP-conjugated); TMB; 2N H2SO4; Antibody Standard

Equipment:

Multichannel micropipette; Plate shaker; ELISA Reader

Day 1:

Coat

Fill each well with 100ul of the coating sample diluted in PBS or carbonate buffer and incubate plates.

A 5ug/ml dilution is usually sufficient to fully coat the well. Lower amounts may also be used but testing is required.
PBS is a safe choice as a diluent. However, in cases where improved binding is required, dilution of the sample in carbonate buffer may give better binding and responses. Note that some samples are not stable in the alkali pH of this buffer.
Typically, the outer wells in the plate are not used but are filled with buffer/diluent throughout the plate. Edge wells can heat up or cool down faster than the center ones, affecting the reactions that take place during the ELISA. Hence their results can be unreliable.
Cover the plate with a protective film or a lid to avoid surface evaporation.
Incubation is typically performed for >12h at 2-8°C. In some cases, excessive incubation (for example >20h) may result in coating sample aggregation and invalid results.
For samples that are more difficult to bind (such as polysaccharides) increased temperatures may be used.

Day 2:

Wash x1

Wash each well with 300ul wash buffer to remove unbound proteins.

PBS can be used as a wash buffer. Alternatively, PBS+ Tween 0.005% can be used. For a more effective wash buffer, use PBS +150mM NaCl + Tween 0.005%.
Each wash consists of filling each well with 300ul buffer followed by emptying the contents of the plate in the sink or a suitable waste container.
When working with crude sample, you may want to incubate the plate in wash buffer for 30 -60 seconds.
Make sure that the wells are completely emptied out as the remaining solution can dilute the reagents in the next steps. Tap the plate on water absorbing paper (paper towels will do).
Do not allow the well to remain dry as this may result in protein unfolding and invalid results.

Block

If required, block wells with 300ul blocking buffer to cover empty spaces and incubate plates.

The type of blocking agent used depends significantly on the source of the sample that is being assayed. For example, using skim milk for human sera usually results in a high background.
Try to choose a blocking agent that is dissimilar from the type of sample being assessed.
For many applications, blocking is not necessary as tween can act as a blocking agent. For this purpose, Tween must be added to all the buffers throughout the assay (except coating). Use of PBS +150mM NaCl + Tween 0.005% is recommended.
Other that Tween, typical blocking agents include 2% BSA, 2% Skim Milk, 5% Fish Collagen and 5% Fetal calf serum.
It should be noted that using a blocking agent, especially in high concentrations can reduce the responses in ELISA.
Shaking is not necessary for most assays but will increase repeatability and consistency of the results significantly. An ELISA plate shaker is ideal, but any other kind of shaker can work. A plate shaker at 400rpm is considered optimal.
An incubation period of 2 hours in room temperature is enough, however times as low as 30minutes can be used. Times less than this may result in increased edge effects.

Wash x2

Primary Antibody (Sera)

Create a serial dilution of the Sera. Add 100ul of each dilution to the wells and incubate.

Using dilutions under 50 is not recommended as it might result in the prozone effect.
The first assay is typically used to identify a dilution range where the results decrease in a steady, predictable way (linearly). To achieve this, stronger dilution steps might be used, like diluting the sample 1:4 or 1:10. Different types of samples may require different starting dilutions.
Typically, a 1:2 dilution step should suffice, but other dilutions is possible.
A duplicate assay of each dilution is preferred.
The assay standard is added here and is treated as another sample. Usually, extra dilutions and replications are used for the standard to create better reliability. Due to limitations in standard availability, a secondary internal standard is usually used and the concentration of this secondary standard is evaluated by comparison with the initial standard.
An incubation period of 2 hours in room temperature is enough, however times as low as 30minutes can be used. Times less than this may result in increased edge effects and inefficient protein-protein interaction.
Shaking is not necessary for most assays but will increase repeatability and consistency of the results significantly. An ELISA plate shaker is ideal, but any other kind of shaker can work. A plate shaker at 400rpm is considered optimal.
In case of high background, increasing the incubation temperature (for example to 30°C) allows the decrease of time and may help reduce the noise.

Wash x3

Secondary Antibody

Add 100ul of diluted sera to the well and incubate.

The recommended dilution for the secondary antibody is usually provided by the manufacturer.
The first assay may be used to test a few different dilutions and ensure optimum responses.
Shaking is not necessary for most assays but will increase repeatability and consistency of the results significantly. An ELISA plate shaker is ideal, but any other kind of shaker can work. A plate shaker at 400rpm is considered optimal.
An incubation period of 2 hours in room temperature is enough, however times as low as 30minutes can be used. Times less than this may result in increased edge effects and inefficient protein-protein interaction.

Wash x4-5

Visualization

100ul of TMB is added to each well. Upon formation of a blue color, 2N H2SO4 is used to stop the reaction and create a yellow color. This is recorded via a plate reader at 450nm.

It is better to protect the plated from light during TMB treatment.
The differences between wells are more prominent when stopped.
Incubation with TMB usually requires 3-20 minutes depending on the assay parameters.
Stopped plates should be read as soon as possible to avoid changes in color and loss of resolution.

Data analysis

The results of the assay for each sample are displayed as “OD vs log dilution”. This should present a sigmoidal curve. The relative potency between the sample and standard is evaluated using 4-parameter logistic fit.

Various software can be used for this. Examples include PLA 3.0, Gene5, Bioassay Assist
Each graph should be tested for non-linearity and parallelism with the standard curve.
Results are presented as relative potency and the 95% CI. The suitable CI depends on the purpose and type of the study but typically should not exceed 30%-300% of the relative potency.

By following this method, one can perform and analyze a typical indirect ELISA. This method may be also applicable to any type of indirect ELISA with a few minor adjustments.

Pleease feel free to comment or ask any questions.

Preprint

Preprint

Method of Serum Antibody Concentration Determination by Indirect ELISA

Method of Serum Antibody Concentration Determination by Indirect ELISA