Columbia Weather Forecast, MO (65202)

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MFI offers many advantages over ELISA. Among them are: increased sensitivity and specificity, better reproducibility, faster throughput of samples, the ability to assay for up to 100 different antigens and control preparations in a single well (multiplexing), and, most importantly to you, the ability to perform ALL primary testing on only 0.2 μl of undiluted serum.

MFI stands for Multiplex Fluorescent Immunoassay. The technology is based both on bead-based immunoassay and flow cytometry. Each purified RADIL antigen or control preparation is covalently linked to one of 100 different types of polystyrene beads, which vary slightly in the intensity of their color. If IgG antibody to a particular antigen is present, it will bind to the antigen on a specific bead and will then be detected by subsequent binding of goat anti-mouse antibody conjugated to a fluorochrome, R-phycoerythrin. The reader channels single beads through a dual laser detector which simultaneously determines both the bead type by the internal dye combination and the fluorescent intensity associated with each individual bead. The fluorescent intensity associated with each of 100 individual beads of each type are used in the determination of each MFI value.

The RADIL has performed comparative, side-by-side testing of thousands of individual results from hundreds of samples. The overall correlation between MFI and ELISA is greater than 99.5% for both mouse and rat samples. In general, MFI is more sensitive than ELISA and is less prone to false positive results.

MFI is the RADIL’s new state-of-the-art primary screening assay for rodent serology. The secondary confirmatory testing technique will continue to be the indirect fluorescent antibody (IFA) test. RADIL has added Western blot (WB) as the tertiary testing technique for the ultimate in sensitivity and specificity.

The cost of your serologic testing using the new MFI technology and RADIL’s smart SEROLOGY approach will not increase over traditional ELISA testing!

MFI requires only 0.2 μl of undiluted serum (1.0 μl of 1:5 diluted serum) regardless of the number of tests requested. This means that survival-bled rodents can now be comprehensively tested.

To allow for potential secondary and tertiary confirmatory testing, we recommend that a minimum of 20 μl of undiluted (100 μl of diluted serum) be submitted for each sample. With the advent of MFI technology, insufficient serum volumes have become a thing of the past.

The units of MFI (Multiplex Fluorescent Immunoassay) are also called MFI (median fluorescent intensity)! The reader determines this value, which can range from less than 10 to up to 32,667 by calculating the median signal value of 100 beads for each agent and control bead set. Positive MFI results will be reported as ‘+’ with a value between 1 and 33. This numerical value represents the MFI value rounded off to the nearest 1,000. For example, a reported MFI value of ‘+7’ means that an MFI value of between 6,500 and 7,499 was measured. You may either see your results in this format or you may opt for the numerical value after the + sign to be omitted.

Wherever available, dilutions of known positive serum for mice and rats were used as the “gold standard” to assure assay performance. In addition to this type of sample, we also used samples from experimentally infected rodents and clinical samples from well-characterized outbreaks to validate the performance of MFI.

We have carefully chosen five control preparations that represent a wide spectrum of proteinaceous materials and we have established statistical parameters for determining if a given serum sample binds non-specifically to one or more of these preparations. If that occurs and we also detect binding to antigens, we will report a result of NA for that particular antigen, just as we currently do in ELISA. Non-specific binding will always occur to some extent in serologic testing. However, it has been our experience that MFI is less prone to this problem than ELISA. Of 30 mouse samples which yielded NA by ELISA and/or NF by IFA for two or more tests, 28 samples (93%) yielded valid results using the new MFI technology. Thus, we anticipate MFI will yield a higher percentage of testable serum samples.

Over 600 random negative mouse samples and 275 random negative rat samples (as determined by primary ELISA and secondary/tertiary testing as required) representing over 21,000 individual data points for all the agents and controls were used to calculate baseline values for MFI. The baselines were set at the mean plus 5 standard deviation units for each agent / species combination and rounded off to the nearest 25 MFI units. For example, the mouse MHV MFI baseline was calculated to be 373 and then rounded up to 375 (n=638). Any value under 375 will be reported as negative, any value between 375 and twice that value, 750, will be reported as borderline (*) and automatically retested by IFA, while any value greater than 750 will be reported as positive (+1 to +33).

While mean positive to negative ratios of ELISA-tested samples are typically about 10:1, mean MFI positive to negative ratios are generally 50:1 and greater. This means that from a statistical standpoint, it is much easier to differentiate between the negative and positive signals on MFI versus ELISA.

Thus, MFI testing should yield fewer equivocal results than traditional ELISA.