High Sensitive ELISA Kit for Complement Component 5a (C5a)

Specificity

This assay has high sensitivity and excellent specificity for detection of High Sensitive Complement Component 5a (C5a).
No significant cross-reactivity or interference between High Sensitive Complement Component 5a (C5a) and analogues was observed.

Recovery

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

Matrix Recovery range (%) Average(%)
serum(n=5) 85-93 89
EDTA plasma(n=5) 79-93 87
heparin plasma(n=5) 78-99 81

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level High Sensitive Complement Component 5a (C5a) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level High Sensitive Complement Component 5a (C5a) 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 High Sensitive Complement Component 5a (C5a) 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) 95-102% 89-98% 90-98% 82-96%
EDTA plasma(n=5) 89-98% 80-96% 98-105% 97-105%
heparin plasma(n=5) 91-105% 80-104% 98-105% 87-98%

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
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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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 …
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