Multiplex Assay Kit for Cyclophilin A (CYPA) ,etc. by FLIA (Flow Luminescence Immunoassay)

PPIA; CyP-A; CYPH; Peptidylprolyl Isomerase A; Peptidyl-Prolyl Isomerase A; Peptidyl-prolyl cis-trans isomerase A; Cyclosporin A-binding protein

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

Specificity

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

Recovery

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

Matrix Recovery range (%) Average(%)
serum(n=5) 90-101 97
EDTA plasma(n=5) 82-96 90
heparin plasma(n=5) 84-102 94

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Cyclophilin A (CYPA) ,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 Cyclophilin A (CYPA) ,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 Cyclophilin A (CYPA) ,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) 87-96% 78-91% 78-88% 95-105%
EDTA plasma(n=5) 91-101% 97-105% 81-101% 96-104%
heparin plasma(n=5) 80-103% 97-104% 84-92% 85-92%

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#:CYPA) 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
Atherosclerosis EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation PubMed: 19758589
Biomarkers Cyclophilin-A: a novel biomarker for untreated male essential hypertension Pubmed: 24164594
Cardiovascular Diabetology Plasma level of cyclophilin A is increased in patients with type 2 diabetes mellitus and suggests presence of vascular disease Pubmed:24502618
International Journal of Biological Sciences Decrease in Plasma Cyclophilin A Concentration at 1 Month after Myocardial Infarction Predicts Better Left Ventricular Performance and Synchronicity at 6 Months: A Pilot Study in Patients with ST Elevation Myocardial Infarction Pubmed:25552928
Medicine Cyclophilin A in Ruptured Intracranial Aneurysm PubMed: 26426668
Mol Cell Biochem PPIA rs6850: A> G single-nucleotide polymorphism is associated with raised plasma cyclophilin A levels in patients with coronary artery disease PubMed: 26702934
Medicine (Baltimore) Urinary Cyclophilin A as a New Marker for Diabetic Nephropathy: A Cross-Sectional Analysis of Diabetes Mellitus PubMed: 26496315
International Journal of Cardiology Cyclophilin A modulates bone marrow-derived CD117(+) cells and enhances ischemia-induced angiogenesisvia the SDF-1/CXCR4 axis. Pubmed:27057951
Clinica Chimica Acta Novel findings of secreted cyclophilin A in diabetic nephropathy and its association with renal protection of dipeptidyl peptidase 4 inhibitor pubmed:27823952
Cardiovascular Diabetology Cyclophilin A enhances macrophage differentiation and lipid uptake in high glucose conditions: a cellular mechanism for accelerated macro vascular disease in diabetes mellitus. pubmed:27809851
Clinical Science Metformin attenuates effects of cyclophilin A on macrophages, reduces lipid uptake and secretion of cytokines by repressing decreased AMPK activity Pubmed:29382697
Journal of Molecular Histology Cyclophilin a increases CD68 cell infiltration in rat experimental periodontitis Pubmed:29318411
Free Radical Research Cyclophilin A and CD147 associate with progression of diabetic nephropathy Pubmed: 30572748
The Kaohsiung Journal of Medical Sciences Association of cyclophilin A level and pulse pressure in predicting recurrence of cerebral infarction Pubmed: 31670477
Cells. Cyclophilin A/EMMPRIN Axis Is Involved in Pro-Fibrotic Processes Associated with Thoracic Aortic Aneurysm of Marfan Syndrome Patients Pubmed: 31936351
JAMA Neurology APPENDIX A: Relationship between cyclophilin A levels and matrix metalloproteinase-9 activity in cerebrospinal fluid of cognitively normal apolipoprotein E4 carriers … Pubmed: 24030206
Menoufia Medical Journal Urinary podocalyxin and cyclophilin A: markers for early detection of type 2 diabetic nephropathy
Toxins Evaluation of the Therapeutic Effects of Protocatechuic Aldehyde in Diabetic Nephropathy 34437430
Cell Death & Disease Proteomic analysis reveals USP7 as a novel regulator of palmitic acid-induced hepatocellular carcinoma cell death Pubmed:35732625
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