Introduction

A multiplex immunoassay simultaneously measures multiple analytes in a single assay procedure. Fluorescent assays utilize a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. There are two types of equipment that are used for this analysis: filter fluorometers and spectrofluorometers. Filter fluorometers use filters to isolate the incident light and fluorescent light. Spectrofluorometers use diffraction grating monochromators to isolate the incident light and fluorescent light. The patented AssayLite™ Multiplex Fluorescent Immunoassay uses different fluorescent colors as markers for each protein or antibody to generate a multiplex fluorescent immunoassay.

AssayLite™ Fluorescent Multiplex Kits

Assaypro multiplex fluorescent assay is based on a none array-based platform which can more accurately measure complex biomarkers in human fluids using patented technology. The biomarkers tend to bind to each other in the samples. The section below illustrates the potential problems with other array-based platform multiplex assays on the market.

Assaypro fluorescent multiplex assay kits have been validated against ELISA and other methods to ensure the measurement is comparable to ELISA performance. During the validation process, kits have been evaluated for CV, sample performance, cross-reactivity, dilution linearity, kit stability, etc.

Each AssayLite Multiplex kit includes:

• Antibody coated Microplate

• Sealing Films: Each kit contains 3 precut, pressure sensitive aluminum sealing films that can be cut to fit the format of the individual assay.

• Standard

• Fluorescent Probe (color includes: Red, Blue, Orange, Yellow)

• Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml)

• Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml)

• Stabilizing Solution

Key features of AssayLite Multiplex Kits:

• 96 strip-well plate similar to 96 strip-well plate using in ELISA kit

• Plate handling equipment in ELISA kit system can be used for this multiplex kits

• Using low cost fluorescent microplate readers

• Low cost with high throughput through put

• It is as accurate as single format ELISA kit

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Potential problems with other array-based platform multiplex assays currently available on the market when proteins form a complex

Multiplex immunoassay kits currently available on the market are an array-type of multiplex. This type of multiplex divides different capture antibodies into an array. The difference between manufacturers on this type of assay is the support for the array. Some examples of current arrays include bead based arrays, biochip arrays, 96 well spot arrays, glass chip arrays, etc. For all of these, the assay mechanism is similar. All the antibodies in the array can bind to the sample loaded to the well with the array antibody. After binding to different antibodies, the detection antibody links to the fluorescent or luminescent probe and will report the signal from each antibody in the array. This type of system will work well when the desired protein does not form a complex with other proteins, such as cytokines. However, if the protein acts not only as a single form, but also as a complex form in the sample, it will create a number of problems if using this type of assay, especially when using this type of technology in measuring blood proteins. There are many proteins that bind to one another in plasma or other bodily fluids, such as PAI-1, PAI-1/tPA complex, and PAI/uPA complex.

Figure below illustrates potential problem with other array-based platform multiplex assays currently available on the market when proteins form a complex

Step 1: Three different types of antibodies are coated on the support surface (bead, BioChips, glass slide, 96 well microplate etc.) as an array. Step 2: Antibodies bind to its targeted protein and protein complex. Step 3: Different biotinylated antibodies are added. Step 4: Antibodies with different colored fluorescent probe binds to its targeted protein and the specific fluorescent signal reported by the fluorescent plate reader. The signal is reported by fluorescent color which has two antibody a, two antibody b, and two antibody c (translate into two protein A, two protein B, two protein C in the samples).

How AssayLite™ Fluorescent Multiplex Kit handles complex proteins in samples

Step 1: Three antibodies uniformly coated to the microplate. Step 2: Antibodies will bind to respective targeted proteins and protein complexes. Step 3: Different antibodies labeled with different fluorescent colors are added to the wells. Step 4: Antibodies with different colored fluorescent probes bind to its targeted protein and the specific fluorescent signal reported by the fluorescent plate reader. The signal is reported by fluorescent color which has two antibody a, two antibody b, and two antibody c (translate into two protein A, two protein B, two protein C in the samples).

Mutiplex Assay Comparison (includes ELISA):