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Carl A. Ascoli, Ph.D. , CSO

2/29/2016

RID 101: Basic Principles of Radial Immuno-Diffusion

Everything you wanted to know about RID testing*But were afraid to ask*

Immunologicals Group

RID 101: Basic Principles

What Is RID??

RID is an acronym for a laboratory-based

testing procedure which stands for:

Radial Immuno-Diffusion.

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RID is typically used to

determine the quantity, or

concentration, of a specific

antigen (i.e. protein) in a

sample – usually serum or

plasma* – using antibodies.

‒ *Sample could be any “biofluid”:

serum, plasma, milk, saliva or

purified protein solution.

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What is RID Used For?

An antigen (either the specimen, calibrator,

or control) is applied to a cylindrical well cut

within an agar gel plate containing a

specific antibody.

The plate is then incubated at 22˚C for a

specified period of time (usually between

48 and 120 hours).

The antigen diffuses radially through the

agarose gel containing a mono-specific

antibody and an antibody/antigen complex

develops, as a precipitin ring.

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How Does RID Work?

Digital RID Plate ReaderJeweller’s Eyepiece

using a micrometer

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The precipitant ring that forms can be physically measured and is translated into a quantitative result.

Physical measurement can be made digitally or by eye..

How Does RID Work?

Calculating Results:

Linear relationship exists between

the ring diameter squared and

concentration of antigen.

By constructing a calibration curve

(i.e. a graph) using dilutions of

various calibration materials of

known concentrations, unknown

concentrations can be read off and

calculated.

Results are then used for measuring/determining the protein

concentrations contained in the test material.

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Data Analysis

Basic research laboratories in academia, private, government and biotech/ biopharma laboratories

Cell culture laboratories growing monoclonal antibodies

Laboratories wanting to quantify analytes beforepurification.

Clinical and Forensic Laboratories performing analytical measurements

Researchers needing to measure BSA in complex solutions.

Manufacturing facilities performing QA/QC checks on processes.

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Who Uses RID?

Analyte ELISA kits do quantify analytes like RID, but the process is more costly, more complicated and requires more complicated analysis.

Other companies market RID kits, but these kits are not as “user friendly” or optimized for cell culture production.

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What Competes with RID?

Other types of kits that measure analytes may be subject to interference caused by other analytes present in suspensions.

Other types of kits to measure analytes require specialized equipment to read or process the samples.

Easy to use.

Can measure an analyte without purification.

All encompassing, self-contained kit – pretty much everything required is included.

No special equipment/instrumentation is needed.

Sensitive and specific test results.

Over 2,000 peer reviewed citations confirm performance.

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Why Use RID?

RID Plates (14 well/tests per plate).

Calibration Material (lyophilized).

DI Water (for reconstituting Calibration Material).

Sample Diluent (for use with Calibration Material & test samples).

Package Insert / Instructions.

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What is in a RID Kit?

Calibrators are used as a quantitative reference – calibrators have a known protein concentration which will produce a specific precipitant ring diameter at this given concentration.

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Why are Calibrators Needed?

Calibrators are also used to create a “Calibration Curve” – by testing various calibrator dilutions on a RID plate (i.e. 100%, 75%, 50%, and 25%), a linear graph can be various dilutions. This graph is then used to calculate concentrations of unknown samples.

Both eliminate potential test errors due to water quality.

‒ #1 problem in test performance as recorded by Technical Services are those traced back to customers using poor water.

Ensures accuracy of test results.

Required component as stated within the “Instructions For Use”.

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Why Use the Provided Diluent

& DI Water?

Using reference materials known concentrations, tests are conducted to find the optimum volume/plate of antibody to give a specific ring diameter.

Liquid agar is mixed with measured antisera and poured onto the base RID plate via calibrated pump.

Plates are poured in a clean room to minimize risk of contamination.

When gel is cooled and set, wells are punched into plate and QC test plates are checked for the correct ring diameter using reference calibrator.

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Production of a RID plate

All finished components are tested together.

Calibrator should give correct ring diameter on plate (+/- 0.2mm).

Controls within +/- 10% of assigned value.

External standard/panel samples tested if available.

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Final QC Testing

A line of research-based RID kits focused on the detectionanimal proteins, namely Bovine Albumin & IgG and MouseImmunoglobulins (IgG & IgM) and the IgG sub-classes (IgG1IgG2 & IgG3).

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What Products are Available?

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Products

PRODUCT DESCRIPTION TESTS per KIT CATALOG NUMBER

Bovine Albumin NL-RID Kit 42 RN204.3

Bovine IgG LL-RID Kit 42 RL200.3

Bovine IgG NL-RID Kit 42 RN200.3

Mouse IgG1 NL-RID Kit 42 RN273.3

Mouse IgG2a NL-RID Kit 42 RN274.3

Mouse IgG2b NL-RID Kit 42 RN275.3

Mouse IgG3 NL-RID Kit 42 RN276.3

Mouse IgM NL-RID Kit 42 RN278.3

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Related Products

PRODUCT DESCRIPTION CATALOG NUMBER

Mouse TrueBlot® Western Blot Kit 88-8887-31

Anti-Mouse IgG γ1,2a,2b,3 Peroxidase Conjugated 610-403-C46

Bovine Serum Albumin, 30% Solution BSA-30

Blotto Immunoanalytical Grade B501-0500

Anti-Bovine IgG (H&L) Fluorescein Conjugated 601-4202

Protein A Purification CUST22M

Assay Development CUST35

For More Information

Our helpful Technical Service staff is ready 24/7 to provide assistance. https://www.rockland-inc.com/TechnicalService.aspx

More technical information can be found on our website at http://www.rockland-inc.com/radial-immunodiffusion-kits.aspx

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