What is it?

Types

Main Components

What is an immunoassay?

An immunoassay is a method used by many different fields of science to determine the presence of an analyte in a sample. The method is based on the interaction between the antibodies and the analyte, and the results can be reported in concentrations, positive/negative or greater than/less than thresholds.

Immunoassays are used in academic, pre-clinical and clinical research, clinical diagnosis and in supporting technologies in many different fields of science such as pharmaceuticals, veterinary, forensic, military and food sciences.

Commercially available assays are designed and optimized for specific analytes, sample types, sample volumes and ranges. The protocol contained with the product should be strictly followed, without deviation, to obtain the most accurate and reproducible results.

Types of Immunoassays

Immunoassays report results in either a qualitative or quantitative manner. Qualitative results are reported as positive/negative or greater than/less than thresholds, while quantitative results are calculated concentrations. Assay results are achieved through either a direct or competitive interaction.

Direct Assay

The analyte present in the sample is measured directly using two antibodies for capture and detection. The analyte will be captured by the antibody adhered to the plate and detected using a second antibody conjugated to a detection molecule. The detection molecule will display a signal that can be measured. The level of signal produced and the analyte concentration are proportional.

Competitive Assay

The analyte present in the sample is measured directly using two antibodies for capture and detection. The analyte will be captured by the antibody adhered to the plate and detected using a second antibody conjugated to a detection molecule. The detection molecule will display a signal that can be measured. The level of signal produced and the analyte concentration are proportional.

Main Components of an Immunoassay

Antibodies

Assays use the kinetics of the antibody:antigen interaction to generate a standard curve using known values to interpolate unknown values.

Standards/Calibrators

Standards/Calibrators are a set of known concentrations from which a curve can be generated. They contain specific concentrations of analyte in a matrix similar to the sample type recommended for the assay. The signal values obtained from the analyte:antibody interactions in the standards allow an algorithm for a curve to be calculated. The sample results are interpolated based on this algorithm as the interactions between the analyte in the sample and the antibodies should relate to the interactions in the standards.

Substrates

  • Enzymatic: an enzyme is conjugated (chemically attached) to the antibody. A substrate is necessary to bind with the enzyme and produce the signal.
  • Colorimetric: the enzyme converts a substrate to produce a colored reaction product.
  • Fluorescent: the enzyme converts a substrate to a reaction product that fluoresces when excited by light.
  • Luminescent: the enzyme converts the substrate to a reaction product that emits photons of light.
  • Radioactive: radio isotopic tracers are conjugated to the antibody. Gamma or beta scintillation detection equipment is used to detect the radioactivity in Counts Per Minute (CPM).

Immunoassays

FAQs view all
Q. What kind of sample type can be used with the kit I purchased?

A. Each type of kit has been validated for the sample types indicated in its protocol. All other sample types would have to be validated internally. Please contact ALPCO's product support team for further information regarding possible testing of other sample types performed by the product users.

Q. Is this kit cross reactive with another species?

A. Each type of kit has been validated for the species indicated in the protocol. Although cross reactivity may exist, validation of other species would need to be performed internally. Please contact ALPCO's product support team for further information regarding possible species validation performed by the product users.

Q. How many standards and controls do I run?

A. Each kit will include at least one set of standards (or one vial of standard to be diluted) and in most cases one or two controls. Standards and controls (if included) must be run each time the assay is performed and it is highly recommended to run all standards/controls/and samples in duplicate.

Q. Can I use reagents from other kits or lots?

A. No. Do not substitute components from other kits or lots.

Q. Can I modify incubation times or temperatures?

A. No. Please do not deviate from the protocol.

Q. Can I use an expired kit?

A. No. The kit should not be used if it is past the expiration date specified on the kit label.

Q. Can I use a kit that has been left at room temperature?

A. Please be sure that the kit and its components are stored as indicated in the kit insert. For additional information about kit stability at room temperature please contact ALPCO's product support team.

Q. Can I skip running the control(s) on my assay?

A. No. If a control(s) is provided always be sure to run the control(s) with each assay. The concentrations of the controls should fall within the range specified on the certificate of analysis.

Q. Can I wash my plate with a multichannel pipette?

A. We do not recommend the use of a multichannel pipette. Wash buffer must be dispensed with adequate and equal force to properly wash the wells. We recommend the use of a Statmatic wash nozzle www.tricontinent.com/products/statmatic-i/ or an automated plate washer. Please refer to the Learning Center tab of the Resources section of the website for a video displaying proper washing technique or click here.

Q. Is it necessary to make a layout of my samples?

A. It is extremely important to know where your standards, controls and samples are located on the plate so that results are generated appropriately. Click here for a downloadable platemap. It also helps to label the strips on the plate (use small tabs at each end) in the event that strips become loose while decanting.

Q. Why is the washing step so important?

A. Correct washing of the plate is a very critical and important aspect of running any successful ELISA. Consistency in washing the plate is essential. Washing the plate too rapidly or too slowly, incomplete washing or aspirating, and allowing the wells to sit dry are all factors that will affect the precision of the assay. We do not recommend the use of a multichannel pipette for this purpose. Multichannel pipettes do not apply enough pressure to thoroughly remove the unbound material and this will result in higher background noise. A video displaying proper washing technique is available in the Learning Center section of the website or click here.

Q. How many times can I freeze and thaw my samples?

A. Recommended freeze/thaw cycles vary based on kit. We recommend to avoid repeated freeze and thaw cycles unless otherwise indicated in the manual. If the collected sample amount allows for it, prepare and freeze aliquots to eliminate or reduce freeze/thaw cycles.

Q. Can I use my own "homemade" buffers?

A. Each kit contains diluents and buffers that are formulated to closely match specific sample types. The manufacturer will not guarantee the performance of the kit if components that were not provided with the kit are used to run the assay.

Q. What should I do if I run out of diluent?

A. Enough reagents are provided to run the entire kit when the protocol is followed. If your type of study requires a greater dilution, calculate the amount of diluent needed prior to setting up the assay and additional diluent may be available for purchase. Please contact customer support for information on the availability of individual components.

Q. My controls are out of range. Is my data invalid?

A. The concentration of the controls must be within the range specified in the certificate of analysis. If the controls are out of range the assay may be invalid. Also, make sure the curve fit recommended in the protocol has been used. If more than one recommendation is provided use the regression method that best fits the standard data points.

Q. I didn't get good values from my standard curve. Can I use the example values from the protocol?

A. No. Never use example values to calculate your assay results. Sample concentrations should be generated from the standard/calibrator values obtained with each assay.

Q. My samples generated OD values out of the standard range. Can I still calculate concentrations?

A. Only sample values that fall within the range of the standard curve can be used. Values outside of this range are generally not linear and can lead to incorrectly extrapolated results.

Q. My standard curve is fine but I didn’t get a signal with my samples. Why?

A. The sample may contain the analyte but it is undetectable based on sensitivity of the kit. The matrix of the sample may also be masking the detection. Ensure that the dilutions have been made as stated in protocol and review the procedure to ensure all reagents were added with the correct volume and in the correct order.

Q. How many samples can I run?

A. ALPCO now offers a tool to help with calculating the number of samples that can be run in a kit. Once you review the protocol to determine the number of standards, controls, and blanks that may be required for the specific kit you plan to run click here to go to the Sample Calculator.

Q. What is reference wavelength?

A. A reference wavelength is a secondary wavelength used for correction (normalization) of the principal wavelength OD values. It helps to account for imperfections in the plate.

Q. Why is 450nm the most common measure wavelength?

A. The measurement wavelength is determined by the substrate used in each kit. The most common one is TMB. It produces a blue color measurable at a wavelength of 650nm. It can be used in end point assays by stopping the reaction with either 1M phosphoric acid or 1M sulfuric acid. A yellow reaction product is formed upon acidification that is measurable at 450 nm.

Q. What is the difference between measurement wavelength and reference wavelength?

A. The main difference between measure wavelength and reference is that the first one has been chosen to read the absorbance produced by the substrate and the second one (reference) to detect imperfections in the plate.

Q. Can I read the plate without having a reference wavelength?

A. Yes, you can. However, the use of a reference wavelength may improve the sensitivity of the assay.

Tips & Tricks view all
Poor precision between duplicates (high CV values)
Automated plate washer is not functioning properly and is not washing all wells in the same manner:
Inadequate plate washing:
Problem with the multi-channel pipette during the addition of the conjugate or substrate:
Carry over from high and low samples:
Poor pipetting technique:
Poor standard curve, does not match certificate of analysis
Inadequate plate washing:
Poor pipetting technique:
Inadequate mixing of reagents:
Inadequate shaking of plate:
Wrong volume of reagents added to the well:
Low or inconsistent ambient temperature:
All wells turned bright blue
Automated plate washer is not functioning properly and is not washing all wells in the same manner:
Inadequate plate washing:
Contamination of the substrate with enzyme conjugate:
High background OD readings (Blank or 0 STD values are too high)
Automated plate washer is not functioning properly and is not washing all wells in the same manner:
Inadequate plate washing:
Old or contaminated wash buffer:
Contamination of the substrate with enzyme conjugate:
Wrong conjugate dilution:
Wrong filter used in the plate reader:
Elevated incubation or ambient temperatures:
Unexpected results
Improperly prepared sample:
Omission of a reagent:
Dilution error:
Sample IDs mixed up:
Wrong curve fit used:
No color development or very low OD readings
Wrong filter used in the plate reader:
Inadequate mixing of reagents:
Inadequate shaking of plate:
Reagents contaminated or added in the wrong order:
Assay incubation times not followed correctly:
Improperly prepared sample:
Low ambient temperature:
Control out of range
Reagent stability issue:
Standard curve issue:
Incorrect reconstitution volume used:
Wrong curve fit used:
Tools & Videos view all
Tools
Plate Shaking Techniques Instructions for proper plate shaking with an ELISA assay
Sample Calculator “How many samples can I test on my 96 well ELISA plate?
Videos
Plate Washing Technique Video Recommendations for hand washing and drying an ELISA plate
Pipetting Tips, Reverse, Forward Mode Video Pipetting Tips, Reverse, Forward Mode Video

Related Resources

Technical Literature