Multiplex Assay Kit for Pyruvate kinase isozymes M2 (PKM2) ,etc. by FLIA (Flow Luminescence Immunoassay)

M2PK; PKM; M2-PK; PKM1; CTHBP; OIP3; PK3; PKM; TCB; THBP1; Pyruvate kinase muscle isozyme; Thyroid hormone-binding protein 1; Cytosolic thyroid hormone-binding protein; Pyruvate Kinase, Muscle

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

Specificity

This assay has high sensitivity and excellent specificity for detection of Pyruvate kinase isozymes M2 (PKM2) ,etc. by FLIA (Flow Luminescence Immunoassay).
No significant cross-reactivity or interference between Pyruvate kinase isozymes M2 (PKM2) ,etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Pyruvate kinase isozymes M2 (PKM2) ,etc. by FLIA (Flow Luminescence Immunoassay) and the recovery rates were calculated by comparing the measured value to the expected amount of Pyruvate kinase isozymes M2 (PKM2) ,etc. by FLIA (Flow Luminescence Immunoassay) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 92-101 97
EDTA plasma(n=5) 88-96 93
heparin plasma(n=5) 97-104 101
sodium citrate plasma(n=5) 85-92 89

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Pyruvate kinase isozymes M2 (PKM2) ,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 Pyruvate kinase isozymes M2 (PKM2) ,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 Pyruvate kinase isozymes M2 (PKM2) ,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) 93-101% 80-99% 96-104% 82-90%
EDTA plasma(n=5) 78-95% 89-103% 80-94% 95-102%
heparin plasma(n=5) 95-103% 81-96% 93-101% 80-95%
sodium citrate plasma(n=5) 98-105% 85-94% 90-101% 96-103%

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#:PKM2) 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
Cancer Research Antigen Presentation by Dendritic Cells in Tumors Is Disrupted by Altered Metabolism that Involves Pyruvate Kinase M2 and Its Interaction with SOCS3 AACR: 70189
Molecular BioSystems Abnormal levels of heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in tumour tissue and blood samples from patients diagnosed with lung cancer Pubmed:25483567
Tumour Biol M2 isoform of pyruvate kinase (PKM2) is upregulated in Kazakh’s ESCC and promotes proliferation and migration of ESCC cells PubMed: 26404132
Oncotarget Identification of a serum biomarker panel for the differential diagnosis of cholangiocarcinoma and primary sclerosing cholangitis Pubmed:29707118
Journal of Neurotrauma Acteoside improves muscle atrophy and motor function by inducing new myokine secretion in chronic spinal cord injury Pubmed: 30318996
Cell Reports Secreted Pyruvate Kinase M2 Promotes Lung Cancer Metastasis through Activating the Integrin Beta1/FAK Signaling Pathway Pubmed: 32049010
Chrysin serves as a novel inhibitor of DGKα/FAK interaction to suppress the malignancy of esophageal squamous cell carcinoma (ESCC)
Cancer Cell International Bufalin Induced Mitochondrial Dysfunction Promotes Apoptosis of Glioma Cells by Regulating Annexin A2 and DRP1 Proteins
Cancer Cell Int Bufalin induces mitochondrial dysfunction and promotes apoptosis of glioma cells by regulating Annexin A2 and DRP1 protein expression 34376212
Life (Basel) Putative Association between Low Baseline Gene Expression in the Peripheral Blood and Clinical Remission in Rheumatoid Arthritis Patients Treated with Tofacitinib 34947916
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