Name: multiplexed fluorometric immunoassay testing methodology and troubleshooting
Uploaded: 2011-12-12T13:00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting at JoVE.com

The research presented here was supported by Charles River.

<ol>
	<li>Jacobson, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+validation+of+diagnostic+assays+for+infectious+diseases.">Principles of validation of diagnostic assays for infectious diseases.</a> Manual of Standards for Diagnostic Tests and Vaccines. , 8-15 (1996).</li><li>Wright, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+standards+for+test+methods+and+reference+sera+for+diagnostic+tests+for+antibody+detection.">International standards for test methods and reference sera for diagnostic tests for antibody detection.</a> Rev. sci. tech. Off. int. Epiz. 17 (2), 527-533 (1998).</li><li>Pepe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Statistical Evaluation of Medical Tests for Classification and Prediction. , (2003).</li><li>Barr, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+interpretation+of+diagnostic+tests+for+feline+immunodeficiency+virus+infection.">Comparison and interpretation of diagnostic tests for feline immunodeficiency virus infection.</a> J. Am. Vet. Med. Assoc. 199, 1377-1381 (1991).</li></ol>

All authors work for Charles River RADS, a major provider of animal serology testing services and reagents.

The MFIA testing process is highly efficient, requiring less equipment and smaller sample and reagent volumes than traditional singleplex tests. The functionality of the multiplex system gives the user the flexibility to screen simultaneously for multiple strains or serotypes of common agents in laboratory rodents (i.e. Coronavirus, parvoviruses etc.). This also enables us, to design customized bead panels based on the area of interest (ie. specific virus family) and is adaptable to screening other types of biomolecules including cytokines and other biomarkers. In addition, it allows us to incorporate several internal control assays to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. A tissue control bead detects non-specific binding of serum immunoglobulin and the &alpha;Ig bead control confirms that serum has been added and contains a sufficient immunoglobulin concentration. Another control bead, coated with serum species immunoglobulin, demonstrates that the labeled reagents and assay reader are functioning properly. Other commercially available multiplex format serologic assays (ie. MicroArrays, ImmunoComb) may not offer this same level of result confirmation.
The ability to narrow down the possible source of assay failure prior to retesting can help an investigator save on time and materials. Critical aspects of the MFIA should be confirmed before repeating a failed sample. Since the assay is conducted at ambient temperature it should be verified that the laboratory temperature is approximately 27&deg;C&plusmn;2&deg;C, higher temperatures can lead to lower than expected scores for controls and samples. Washing is perhaps the most critical step in the assay. Insuring that the test plate is properly washed, blotted and resuspended can eliminate the vast majority of sampling errors due to low bead count (due to aggregated beads) or insufficient reagent addition (lost by wicking out of test plate filter bottom). We routinely count 25 beads per assay (agent) and have found no statistical difference in the results by counting higher number of beads which will also lead to a longer read time for the plate.
We have performed comprehensive validation studies of MFIA on several species of commonly used laboratory animals (Mouse, Rat, Hamster, Guinea Pig and Rabbit) to demonstrate diagnostic accuracy, reproducibility, and ruggedness by testing large numbers of known positive and negative serum samples, and comparing their ELISA, IFA and MFIA results. The detection limits (i.e., standard immune serum titration endpoints) of MFIA were comparable to, and in some cases surpassed, those of corresponding ELISA. Diagnostic specificity, measured with SPF rodent sera, exceeded 99%; the overall correspondence between ELISA and MFIA performed on known positive and known negative sera was greater than 95%. In summary, these results proved that multiplex MFIA is a good alternate to the singleplex ELISA, and is suitable for its intended use, i.e. in routine serosurveillance of laboratory animal colonies.

multiplexed fluorometric immunoassay testing methodology and troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Watch this Scientific Journal Video about Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting at JoVE.com

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

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