Multiplex Assay Kit for Complement Component 5a (C5a) ,etc. by FLIA (Flow Luminescence Immunoassay)

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

Specificity

This assay has high sensitivity and excellent specificity for detection of Complement Component 5a (C5a) ,etc. by FLIA (Flow Luminescence Immunoassay).
No significant cross-reactivity or interference between Complement Component 5a (C5a) ,etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Complement Component 5a (C5a) ,etc. by FLIA (Flow Luminescence Immunoassay) and the recovery rates were calculated by comparing the measured value to the expected amount of Complement Component 5a (C5a) ,etc. by FLIA (Flow Luminescence Immunoassay) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 86-101 90
EDTA plasma(n=5) 96-105 102
heparin plasma(n=5) 79-101 89
sodium citrate plasma(n=5) 80-89 83

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Complement Component 5a (C5a) ,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 Complement Component 5a (C5a) ,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 Complement Component 5a (C5a) ,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) 98-105% 81-102% 79-94% 89-97%
EDTA plasma(n=5) 80-90% 82-89% 81-96% 81-98%
heparin plasma(n=5) 90-102% 79-99% 79-97% 81-93%
sodium citrate plasma(n=5) 89-96% 91-99% 89-103% 81-94%

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#:C5a) 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
Molecular Immunology Cathepsin D is released after severe tissue trauma in vivo and is capable of generating C5a in vitro ScienceDirect: S0161589011008297
Journal of Reproductive Immunology Elevated complement factor C5a in maternal and umbilical cord plasma in preeclampsia PubMed: 23415845
The Journal of Clinical Investigation Complement component 5 contributes to poor disease outcome in humans and mice with pneumococcal meningitis PubMed: PMC3195471
Journal of Antimicrobial Chemotherapy Efficacy of a synthetic antimicrobial peptidomimetic versus vancomycin in a Staphylococcus epidermidis device-related murine peritonitis model Pubmed: 23645587
Journal of Orthopaedic Research Conversion from external fixator to intramedullary nail causes a second hit and impairs fracture healing in a severe trauma model Pubmed: 23070742
SpringerPlus Therapeutic effects of sesame oil on monosodium urate crystal-induced acute inflammatory response in rats Pubmed: 24353977
American Journal fo Reproductive Immunology Evaluation of the Markers of Inflammation in the Umbilical Cord Blood of Newborns of Mothers with Thrombophilia Pubmed:25209155
Gynecol Obstet Invest Complement Split Products C3a/C5a and Receptors: Are They Regulated by Circulating Angiotensin II Type 1 Receptor Autoantibody in Severe Preeclampsia? PubMed: 26485247
Journal of Neuroimmunology Acute and prolonged complement activation in the central nervous system during herpes simplex encephalitis Pubmed:27235358
International Immunopharmacology Consistency and pathophysiological characterization of a rat polymicrobial sepsis model via the improved cecal ligation and puncture surgery Pubmed:26802602
Digital Repository Dosagem de frações ativadas do sistema complemento em empiema induzido em ratos 10183
Environmental Pollution Cadmium-induced immune abnormality is a key pathogenic event in human and rat models of preeclampsia pubmed:27511439
Cancer Letters Complement C5a/C5aR pathway potentiates the pathogenesis Q5 of gastric cancer by down-regulating p21 expression pubmed:29031586
Acta Paediatrica Serum complement factor 5a levels are associated with nonalcoholic fatty liver disease in obese children DOI: 10.1111/apa.14106
Scientific Reports Splenectomy modulates early immuno-inflammatory responses to trauma-hemorrhage and protects mice against secondary sepsis Pubmed: 30291296
Nutrients The Beneficial Effects of Lactobacillus plantarum PS128 on High-Intensity, Exercise-Induced Oxidative Stress, Inflammation, and Performance in Triathletes Pubmed: 30736479
Journal of NeuroVirology Intrathecal complement activation by the classical pathway in tick-borne encephalitis Pubmed: 30850976
PLoS One Sepsis causes right ventricular myocardial inflammation independent of pulmonary hypertension in a porcine sepsis model Pubmed: 31247004
Frontiers in Immunology C5a/C5aR Pathway is Critical for the Pathogenesis of Psoriasis Pubmed: 31447855
Phytomedicine Novel evidence that an alternative complement cascade pathway is involved in optimal mobilization of hematopoietic stem/progenitor cells in Nlrp3 …
Aging C5aR deficiency attenuates the breast cancer development via the p38/p21 axis Pubmed: 32669478
STEM CELL REVIEWS AND REPORTS A Novel Evidence That Mannan Binding Lectin (MBL) Pathway of Complement Cascade Activation is Involved in Homing and Engraftment of Hematopoietic Stem … Pubmed: 32406006
Modulation of C5a-C5aR interactions against murine mammary cancer cell line
FASEB J C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease Pubmed:35503088
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