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Multiplex Assay Kit for A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5) ,etc. by FLIA (Flow Luminescence Immunoassay)

ADMP-2; Aggrecanase-2

(Note: Up to 8-plex in one testing reaction)

Specificity

This assay has high sensitivity and excellent specificity for detection of A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5) ,etc. by FLIA (Flow Luminescence Immunoassay).
No significant cross-reactivity or interference between A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5) ,etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5) ,etc. by FLIA (Flow Luminescence Immunoassay) and the recovery rates were calculated by comparing the measured value to the expected amount of A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5) ,etc. by FLIA (Flow Luminescence Immunoassay) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 81-92 86
EDTA plasma(n=5) 86-93 89
heparin plasma(n=5) 92-105 102
sodium citrate plasma(n=5) 84-101 98

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of A Disintegrin And Metalloproteinase With Thrombospondin 5 (ADAMTS5) ,etc. by FLIA (Flow Luminescence Immunoassay) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 96-103% 79-96% 81-101% 86-103%
EDTA plasma(n=5) 90-97% 80-104% 80-97% 86-97%
heparin plasma(n=5) 85-103% 98-105% 80-91% 81-101%
sodium citrate plasma(n=5) 98-105% 79-98% 81-92% 79-89%

Stability

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
96-well plate 1 Plate sealer for 96 wells 4
Pre-Mixed Standard 2 Standard Diluent 1×20mL
Pre-Mixed Magnetic beads (22#:ADAMTS5) 1 Analysis buffer 1×20mL
Pre-Mixed Detection Reagent A 1×120μL Assay Diluent A 1×12mL
Detection Reagent B (PE-SA) 1×120μL Assay Diluent B 1×12mL
Sheath Fluid 1×10mL Wash Buffer (30 × concentrate) 1×20mL
Instruction manual 1

Assay procedure summary

1. Preparation of standards, reagents and samples before the experiment;
2. Add 100μL standard or sample to each well,
    add 10μL magnetic beads, and incubate 90min at 37°C on shaker;
3. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent A. Incubate 60min at 37°C on shaker;
4. Wash plate on magnetic frame for three times;
5. Add 100μL prepared Detection Reagent B, and incubate 30 min at 37°C on shaker;
6. Wash plate on magnetic frame for three times;
7. Add 100μL sheath solution, swirl for 2 minutes, read on the machine.

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Magazine Citations
PLoS ONE A Combinatorial Relative Mass Value Evaluation of Endogenous Bioactive Proteins in Three-Dimensional Cultured Nucleus Pulposus Cells of Herniated Intervertebral Discs: Identification of Potential Target Proteins for Gene Therapeutic Approaches Plosone: Source
Osteoarthritis and Cartilage Lack of anti-inflammatory and anti-catabolic effects on basal inflamed osteoarthritic chondrocytes or synoviocytes by adipose stem cell-conditioned medium PubMed: 26521751
Journal of Cellular and Molecular Medicine VIP and CRF reduce ADAMTS expression and function in osteoarthritis synovial fibroblasts Pubmed:26818776
Int Wound J. From varices to venous ulceration: the story of chronic venous disease described by metalloproteinases. Pubmed:26991748
The American journal of pathology Healthy and Osteoarthritic Synovial Fibroblasts Produce a Disintegrin and Metalloproteinase with Thrombospondin Motifs 4, 5, 7, and 12: Induction by IL-1β and Fibronectin and Contribution to Cartilage Damage. pubmed:27449198
PLoS One. Self-complementary adeno-associated virus serotype 6 mediated knockdown of ADAMTS4 induces long-term and effective enhancement of aggrecan in degenerative human nucleus pulposus cells: A new therapeutic approach for intervertebral disc disorders. pubmed:28207788
PLoS One Self-complementary adeno-associated virus serotype 6 mediated knockdown of ADAMTS4 induces long-term and effective enhancement of aggrecan in degenerative human nucleus pulposus cells: A new therapeutic approach for intervertebral disc disorders 10.1371:journal.pone.0172181
International Wound Journal From varices to venous ulceration: the story of chronic venous disease described by metalloproteinases pubmed:26991748
BMC musculoskeletal disorders Hemoglobin stimulates the expression of ADAMTS-5 and ADAMTS-9 by synovial cells: a possible cause of articular cartilage damage after intra-articular hemorrhage. pubmed:29137610
Journal of Clinical Medicine The Effects of TNF-α Inhibition on the Metabolism of Cartilage: Relationship between KS, HA, HAPLN1 and ADAMTS4, ADAMTS5, TOS and TGF-β1 Plasma … Pubmed:35407621
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