A Luminex Multiplex assay is a bead-based assay employed widely at every step of the drug discovery process. Due to its multiplexing capacities, the Luminex Multiplex cytokine assay can quantify up to 100 analytes in complex biological matrices. This ability has made them the primary choice of an assay in drug discovery research. However, with such advanced instruments, scientists must be an expert in Luminex testing to troubleshoot potential assay issues. Below are nine signs that prove you’re an expert in Luminex Multiplex assay.
1. Not adjusting the temperatures and incubation times mentioned in the assay manual
All assays are optimized thoroughly for both temperatures and incubation period. The protocol provided with the kit manual is optimally validated. Therefore, assay developers do not guarantee performance if protocols are altered.
2. Avoiding reagents from different Luminex multiplex assay kits
Assay components are unique to individual assays. Researchers can only interchange the calibrator diluent, assay diluent, and other assay components when they have the same lot and part number. Hence, researchers must never use assay components from different sources or lots.
3. Deciphering acquisition problems and error messages
Identifying the sources for acquisition problems and error messages is critical for any Luminex multiplex bead assay expert. Some possible sources of error messages are incompatible instruments, incorrect probe height, clogged sample probe, incorrect instrument setting, and instruments out of calibration.
4. Troubleshooting low microparticle count
Most often, instruments out of calibration may result in low microparticle count. An expert in Luminex XMAP bead assays should verify and calibrate the assay to solve the issue. Regular calibration ensures that the Luminex assay produces accurate measurements. Ideally, an expert will run the assay within a week of calibration. Besides, verifying the assay on the day of the assay run is highly recommended.
5. Identifying poor sensitivity or low fluorescence intensity signal
An expert will confirm the standard reconstitution volume from COA or the standard value card. Incorrect reconstitution will provide erroneous results. Hence, an expert will thoroughly follow the protocol for all lyophilized reagents present in the assay kit.
6. Evaluating out-of-range sample readings
When a sample does not contain the analyse of interest or has out-of-range values, it can cause problems in sample readings. Thus, an expert will evaluate the kit instructions to confirm whether any issue exists with the dynamic concentration range or if the target analyte is missing from the sample solution.
7. Poor precision
Interfering components, especially in complex biological matrices, can lead to poor assay precision. Also, An expert will confirm the presence of interfering substances by evaluating gel separators, additives, or metabolites in the study matrix. Besides, they will run a linearity test and spike/recovery assessment to troubleshoot issues related to poor precision.
8. Interpreting high background signal
Incorrect assay buffer or accidental spiking of blank wells can raise the assay background signal. An expert will only use recommended calibrator diluent for standard or samples. Moreover, they will take extra precautions to avoid spiking of assay wells.
9. Issues about microparticle aggregation
Samples with hyperlipidemic or hemolyzed matrices can aggregate microparticles. Also, These microplates often do not mix thoroughly in the sample matrix. Hence, an expert researcher will follow the protocol to the point and ideally use a plate shaker before analysis.
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