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Threshold Immunoligand Assay

The Threshold™ Immunoligand Assay (ILA) measures a broad range of analytes such as proteins, peptides, sequence specific DNA and micro-organisms for biopharmaceutical development and production.  Samples can range from fermentation supernatants, samples from a purification process, and other biochemical preparations to serum or other bodily fluids.  Assays can be developed using a sandwich or competitive format depending on the size and characteristics of the analyte to be measured.  The assay consists of four steps, labeling the antibody, the reaction stage, the separation stage and the detection stage.

Labeling the Antibody
ila-detection kit-labeling the antibody Using the ILA labeling Kit, antibodies are reproducibly labeled in about 2 hours by mixing them with N-hydroxysuccinimide esters of dinitrophenyl-biotin and fluorescein provided in the kit.  Becasue both haptens are chromogenic, photometric measurements are used to determine the number incorporated 
Reaction Stage

ila-detection kit-reaction stage

In the reaction stage, labeled antibodies, the sample solution containing the analyte of interest, streptavidn, and anti-fluoresceneine/urease conjugate are incubated together to form a reaction complex.   Since this binding occurs in solution, all molecules are maintained in their native conformation to assure accurate measurement of the analyte and 100% activity of the antibody.
Separation Stage

ila-detection kit-separation stage

The reaction mixture containing the reaction complex is filtered through a biotinylated membrane.  By leveraging the strong affinity streptavidin has for biotin, the reaction complex is captured and concentrated on the membrane. 
Detection Stage

ila-detection kit-detection stage

The membrane with the captured reaction complex is placed in the Threshold System which contains the substrate urea.  The urea is hydrolyzed by urease producing a pH change that is detected by the systems light-addressable potentiometric sensor.  Urease activity from up to eight different samples can simultaneouly measured on the Threshold system.

 

Using the ILA Labeling Kit, the following assays have been demonstrated.

The Threshold Immunoligand Assay Kit includes all of the reagents required to perform a Threshold System assay, except binding protein (such as antibodies) or DNA probes specific for the analyte of interest.  The kit includes Capture Reagent, Enzyme Reagent, membrane sticks, substrate concentrate, and appropriate buffers and washes.  A Universal Standard is also included, which can be used for training and trouble-shooting purposes without using the specific binding proteins and analyte.


R9003

Immunoligand Assay Detection Kit 192 determinations  includes: (1) Universal Standard Reagent,  (6) Enzyme Reagent,  (1) Capture Reagent,  (6) Threshold Blank Sticks,   (24) Threshold Sticks,   (1) Substrate,  (1) Assay Buffer Concentrate,  (1) Wash Buffer Concentrate  

R9002

Immunoligand Assay Labeling Kit  includes: 2 vials Biotin label (5 mg/vial),  2 vials Fluorescein label (5 mg/vial)