Radial Immunodiffusion Plates To Measure Human IgM In Serum, Plasma, Or Other Biological Fluids

February 28, 2017

Albert Einstein College of Medicine
Clinical Research
Pre-doctoral researcher

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Company:

The Binding Site Ltd.

Product Name:

IgM Radial Immunodiffusion Kit

Catalog Number:

RN012.3

We use The Binding Site's IgM RID Kits to quantify IgM in human plasma samples. Each kit comes with 3 RID plates, and each plate has 14 sample wells. The plates are filled with agarose gel containing antibody specific to IgM. Once the samples are added to the wells, IgM (the antigen) diffuses outward from the well and complexes with the IgM antibody in the agarose. The antigen-antibody complexes are visualized as a ring which can be measured and applied to a standard curve for IgM quantification in patient samples.

Experimental Design and Results Summary

Application

Human Immunoglobulin M Quantification

Starting Material

Human serum or plasma

Protocol Overview

Apply each sample and control (3 standards, 1 control sample) to a pre-formed well in the IgM specific agarose gel (plate samples in duplicate). The three standards and single control sample, all of which are included in the kit, are applied to generate and validate a calibration curve from which the concentrations in each human sample can be determined. During the 72 hour incubation, antigen-antibody complexes form and yield a precipitation ring. The diameter^2 from each standard is proportional to its IgM concentration in mg/L. The standard curve should be generated and validated. Subsequently, the experimental sample diameters can be measured, and the IgM concentrations can be determined using the standard curve. Precipitate ring measurements are best obtained using a ruler under magnification or an RID plate reader (if available).

Tips

Let each plate come to room temperature prior to applying samples, and ensure that all condensation has been wiped away/removed from the plate prior to sample application -- excess water could dilute your results, standards, etc. Research 'normal' IgM measures in your patient/sample population prior to using the assay to determine if any kind of dilution is appropriate.

Results Summary

First, convert all raw diameters to diameter^2. For each sample, enter the the diameter^2 value into the calibration curve equation to determine the IgM concentration. If a dilution was performed prior to plating, the concentration as determined by the calibration curve equation must be multiplied by the dilution factor. Quality control for results include minimum and maximum diffusion measures for the standards as well as for the control sample (all outlined by the manufacturer).

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

Simple protocol, relatively easy to optimize, reliable.

The Bad

Relatively expensive, not sold by many outside retailers -- must contact company for quote

The Bottom Line

If affordable, I highly recommend the IgM RID Kit for quantification of human IgM from plasma. The kit is incredibly simple to use and includes all of the reagents required to carry out the assay (excluding the patient samples). The short incubation time of 72 hours provides time efficient results, and each plate can be set up within minutes -- making the entire experiment very easy and nearly stress-free to perform.

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