High Sensitive ELISA Kit for Terminal Complement Complex C5b-9 (C5b-9)

MAC; Membrane Attack Complex

Specificity

This assay has high sensitivity and excellent specificity for detection of High Sensitive Terminal Complement Complex C5b-9 (C5b-9).
No significant cross-reactivity or interference between High Sensitive Terminal Complement Complex C5b-9 (C5b-9) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant High Sensitive Terminal Complement Complex C5b-9 (C5b-9) and the recovery rates were calculated by comparing the measured value to the expected amount of High Sensitive Terminal Complement Complex C5b-9 (C5b-9) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 84-101 90
EDTA plasma(n=5) 93-101 97
heparin plasma(n=5) 85-105 95

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level High Sensitive Terminal Complement Complex C5b-9 (C5b-9) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level High Sensitive Terminal Complement Complex C5b-9 (C5b-9) 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 Terminal Complement Complex C5b-9 (C5b-9) 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) 82-96% 89-96% 81-105% 98-105%
EDTA plasma(n=5) 80-105% 81-105% 88-102% 79-101%
heparin plasma(n=5) 97-104% 95-105% 94-101% 99-105%

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
Journal of Biomedical Materials Research Part A Effect of thiol functionalization on the hemo-compatibility of PLGA nanoparticles Wiley: source
The Journal of Clinical Investigation Complement component 5 contributes to poor disease outcome in humans and mice with pneumococcal meningitis PubMed: PMC3195471
Colloids and Surfaces B: Biointerfaces Glucosylated polymeric nanoparticles: A sweetened approach against blood compatibility paradox ScienceDirect: S0927776513001720
Perfusion The effect of normovolemic modified ultrafiltration on inflammatory mediators, endotoxins, terminal complement complexes and clinical outcome in high-risk cardiac surgery patients Pubmed: 23429100
Nephrology Dialysis Transplantation The efficacy of recombinant human soluble thrombomodulin for the treatment of shiga toxin associated hemolytic uremic syndrome model mice Pubmed:25694534
journal of neuroinflammation Adjuvant treatment with dexamethasone plus anti-C5 antibodies improves outcome of experimental pneumococcal meningitis: a randomized controlled trial PubMed: 26272468
J Thromb Haemost Thrombin‐activatable fibrinolysis inhibitor influences disease severity in humans and mice with pneumococcal meningitis PubMed: 26340319
Digital Repository Dosagem de frações ativadas do sistema complemento em empiema induzido em ratos 10183
J Neuroinflammation.  Mannose-binding lectin-associated serine protease 2 (MASP-2) contributes to poor disease outcome in humans and mice with pneumococcal meningitis PMC5234106
Cancer Letters Complement C5a/C5aR pathway potentiates the pathogenesis Q5 of gastric cancer by down-regulating p21 expression pubmed:29031586
Effects of immunoadsorption combined with membrane filtration on complement markers–Results of a randomized, controlled, crossover study
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