The following technology platforms are employed for our assay services or products:

Luminex Bead-Based Multiplex Assay

Our multiplexing immunoassays with bead suspensions are based on xMAP (Multiple- Analyte Profile) technology developed by Luminex Corp.

 

Luminex bead-based multiplexing sandwich immunoassays for protein biomarkers employ a suspension of microsphere sets in a 96-well microplate where each bead set represents an individual immunoassay. The microspheres are 6.5-µm paramagnetic beads functionalized with carboxyl groups on the bead surface for covalent attachment of capture antibodies. The beads are internally encoded by fluorescent dyes and individually addressed to a specific assay in the multiplexed tests. This technology uses a proprietary dying process to create up to 500 unique dye mixtures which are used to identify an individual bead. The fluorophore-coded beads function as bar codes for individual analyte. Each bead set can be coated with a capture antibody specific to one analyte. During a bead-based sandwich immunoassay, captured analyte on the bead surface is bound by a biotinylated detection antibody and streptavidin-phycoerythrin (S-PE). Multiple analytes in a single aliquot of serum, plasma, cell culture supernatant, cell lysate and tissue extract, etc. are determined quantitatively and simultaneously with a Luminex bead reader. Within the bead reader, one laser excites the beads’ internal dyes, which identify each bead set by its fluorescent color code, while a second laser excites the reporter dye (PE) captured during the assay. Tens to hundreds of readings are made for each bead set. The bead reader classifies each bead according to its predefined map region by means of digital signal processing. The magnitude of the reporter-derived signal is in direct proportion to the amount of analyte bound. Analyte concentrations of the multiplex immnuassays are typically determined by 5-parametere logistic regression algorithm with analysis of the median fluorescence intensity readings of each 8-point calibrator curve.

 

Ultra-Sensitive Bead-Based Assay

Our ultra-sensitive immunoassays are established upon Quanterix’s Simoa (Single Molecule Array) technology to detect low abundance protein targets.

 

During ultra-sensitive single molecule array (Simoa) sandwich immunoassays, fluorescently distinct paramagnetic beads (2.7 µm diameter) are coupled with a capture antibody. Conventional bead-based sandwich immunoassay approach is applied but single immunocomplexes labeled with enzyme (streptavidin-β-galactosidase) are formed on the bead surface. When samples containing extremely low concentrations of analytes are tested, the ratio of analyte molecules (and the resulting immunocomplexes) to beads is small (<1) and the percentage of beads that contain a labeled immunocomplex follows a Poisson distribution - beads carry either a single immunocomplex or none. It is impossible to detect these low numbers of enzyme labels using conventional detection technology, because the fluorophores generated by each enzyme diffuse into a large assay volume (typically greater than 0.1 mL). It takes hundreds of thousands of enzyme labels to generate a fluorescence signal above background.

The very low concentrations of enzyme labels on the bead surface can be detected by loading beads (diameters of 2.7 µm) into an array of 216,000 wells (diameters of 4.5 μm and depths of 3.25 μm) and confining the fluorophores generated by individual enzymes to extremely small volumes (~50 femtoliter). Beads are sealed with oil to ensure only one bead in a well. Beads carrying a single enzyme-labeled immunocomplex generate a high local concentration of fluorophores in the confined well. By acquiring time-lapsed fluorescence images of the array using standard microscopic optics, it is possible to differentiate beads associated with a single enzyme molecule (“on” well) from those not associated with an enzyme molecule (“off” well). At low concentrations of proteins, when the ratio of enzyme labels to beads is less than 1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of “on” or “off” beads (digital regime). At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog mode for fluorescence measurement of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label can be achieved.

In ultra-sensitive single molecule array (Simoa) multiplex sandwich assays, different capture antibodies are coupled with fluorescently distinct paramagnetic beads. Multiple analytes in a single aliquot of serum, plasma, cell culture supernatant, cell lysate and tissue extract are determined quantitatively and simultaneously with Quanterix’s HD-1 Simoa Analyzer.

Since Simoa platform enables low analyte concentrations to be determined digitally rather than by analogue signals, it is termed a digital immunoassay. The digital nature of the Simoa technology allows an average of 1000× sensitivity increase over conventional assays with CVs <10%. This ultra-sensitivity assay platform can be completely automated with multiplexing and custom assay capability.

 

Microplate-Based Sandwich ELISA

Our conventional microplate-based immunoassay is based on a solid phase sandwich enzyme linked immunoassay (ELISA) method. Samples, calibrators and controls are added to the wells coated with an antibody specific to a target. After incubation step, the target in the samples binds to the capture antibody on the plate well. After wash step using an ELISA Plate Washer, anti-target detection antibody conjugated with horse radish peroxidase (HRP) is incubated in wells and binds to the target. Unbound target and HRP conjugate are washed off by wash buffer. Upon the addition of the substrate, the intensity of color with an ELISA plate reader is proportional to the concentration of target in the samples. Analyte concentrations are typically determined by 4-parametere logistic regression algorithm with analysis of the median optical density readings of protein standard curve.

Positive and negative controls on a plate allow for assay quality assurance. The tests are end-point measurements.

 

Microplate-Based Competitive ELISA

Another conventional microplate-based immunoassay is based on a solid phase competitive enzyme linked immunoassays. A primary antibody is captured by an secondary antibody coated on a microplate. A constant concentration of biotinylated target (tracer) and varying concentrations of unlabeled target in samples compete for binding specifically to the primary antibody. Therefore, the concentration of tracer-bound primary antibody is inversely proportional to the target concentrations in samples. Captured biotinylated tracer is subsequently bound by streptavidin-conjugated horseradish peroxidase (HRP). After washing away the unbound components, TMB is added as a substrate for the HRP. Finally, the enzymatic reaction is terminated by adding an acidic stop solution. The intensity of color with an ELISA plate reader is inversely proportional to the concentration of target in the samples. Analyte concentrations are typically determined by 4-parametere logistic regression algorithm with analysis of the median optical density readings of protein standard curve. Positive and negative controls on a plate allow for assay quality assurance. The tests are end-point measurements.

 

Endotoxin/LPS Measurement

Our Endotoxin/LPS test is a quantitative, kinetic assay for the detection of Gram negative bacterial Endotoxin in a variety of samples including serum and plasma.

 

A sample is mixed with the reconstituted Limulus Amebocyte Lysate (LAL) reagent, placed in the photometer or 96-well microplate and automatically monitored over time for the appearance of turbidity. The time required before the appearance of turbidity (Reaction Time) is inversely proportional to the amount of endotoxin present. That is, in the presence of a large amount of endotoxin the reaction occurs rapidly; in the presence of a smaller amount of endotoxin the reaction time is increased. The concentration of endotoxin in unknown samples can be calculated from a standard curve.

 

Proenzyme  Coagulase
 


                            

Coagulogen  Coagulin
 


 

 

Gram negative bacterial Endotoxin catalyzes the activation of a proenzyme in the LAL. The initial rate of activation is determined by the concentration of Endotoxin present. The activated enzyme (Coagulase) hydrolyzes specific bonds within a clotting protein (Coagulogen) also present in LAL. Once hydrolyzed, the resultant coagulin self-associates and forms a gelatinous clot. The turbidimetric Endotoxin/LPS assay measures the increase in turbidity (optical density) that precedes the formation of the gel clot.

How is the LAL-Based Endotoxin/LPS testing performed for serum/plasma?

We have built up extensive experiences in performing Endotoxin/LPS testing for serum/plasma samples. Preliminary sample preparation is required for serum and plasma. If testing plasma, we use “platelet rich plasma” obtained from whole blood by centrifuging at low speeds to remove the white and red blood cells. Serum samples, whole blood processed to remove fibrogens, coagulants, whole blood cells, etc., can also be tested in the same manner as plasma.

 

 

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