The Goat/Sheep C5a (Complement Component 5a) ELISA Kit from AssayGenie is a cutting-edge tool for precise measurement of C5a levels in goat and sheep serum, plasma, cell culture supernatant and other biological samples. This assay delivers exceptional sensitivity and specificity, ensuring accurate quantification across diverse sample types and concentration ranges. C5a is a potent anaphylatoxin released when complement component C5 is cleaved during activation of the classical, lectin or alternative complement pathways. It drives neutrophil and monocyte chemotaxis, mast cell degranulation, increased vascular permeability and pro-inflammatory cytokine release, making it a key marker of complement activation in studies of infection, inflammation, immune response and veterinary disease.
Equipped with this ELISA kit, researchers can delve deep into the intricate mechanisms governed by C5a and gain valuable insights into its involvement in different biological pathways. Unleash the potential of your goat and sheep research with the AssayGenie Goat/Sheep C5a (Complement Component 5a) ELISA Kit empowering innovation and discovery in the realm of molecular biology.
Serum, Plasma, Cell Culture Supernatant, Cell/Tissue Lysate, Other Biological Fluids
Storage:
2-8°C for 12 months.
Linearity:
Sample
1:2
1:4
1:8
Serum (n = 5)
88-103%
87-101%
87-94%
EDTA Plasma (n = 5)
84-101%
84-96%
83-99%
Heparin Plasma (n = 5)
82-97%
83-89%
82-96%
Recovery:
Matrix
Recovery Range (%)
Average (%)
Serum (n = 5)
89-99
93
EDTA Plasma (n = 5)
85-105
91
Heparin Plasma (n = 5)
86-100
95
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Step
Procedure
1
Reagent & Plate Preparation: Equilibrate TMB substrate to room temperature for at least 30 minutes. Set standard, test sample and control (zero) wells on the pre-coated plate and record their positions.
2
Standard, Sample & Control Loading: Aliquot 100 µl of standard solutions into the standard wells, 100 µl of properly diluted sample into test sample wells, and 100 µl of Sample Dilution Buffer into the control (blank) well.
3
First Incubation & Wash: Seal the plate and incubate at 37°C for 90 minutes. Aspirate the liquid and wash the plate 2 times with Wash Buffer.
4
Biotin-labelled Antibody & Wash: Add Biotin-labelled Antibody working solution to each well and incubate at 37°C for 60 minutes. Wash the plate 3 times, allowing the buffer to stay in the wells for 1-2 minutes per wash. Add SABC working solution and incubate at 37°C for 30 minutes, then wash 5 times.
5
Color Development: Add 90 µl of TMB substrate into each well and incubate at 37°C in the dark for 10-20 minutes.
6
Stop Reaction & Reading: Add 50 µl of Stop Solution and measure absorbance at 450 nm in a microplate reader immediately.
Sample Type
Protocol
Serum
Allow whole blood to coagulate at room temperature (2 h) or 2-8°C overnight. Centrifuge at 1000 × g for 20 min and collect the supernatant. Store or use immediately.
Plasma
Collect in anticoagulant tubes (EDTA, citrate, or heparin), mix gently, and centrifuge within 30 min at 1000 × g, 2-8°C for 15 min. Store or assay as needed.
Tissue Homogenate
Homogenize tissue in 50 mM Tris + 0.9% NaCl + 0.1% SDS (pH 7.3) with protease inhibitors (e.g. 1 mM PMSF). Avoid NP-40, Triton X-100 and DTT. Disrupt by ultrasonic homogenization or freeze-thaw cycles, centrifuge at 5000 × g for 5 min and collect the supernatant.
Cell Culture Supernatant
Centrifuge at 2500 rpm for 5 minutes at 2-8°C and collect the clarified cell culture supernatant.
Cell Lysate
Lyse cells in lysis buffer with protease inhibitor (e.g. 1 mM PMSF) on ice for 30-60 minutes, or by ultrasonic treatment. Centrifuge at 10,000 rpm for 10 min at 2-8°C and collect the supernatant.
Other Sample Types
For more information about how to process other sample types, (e.g., body fluids, breast milk & more), please contact our Tech Support Team at techsupport@assaygenie.com.
Component
Quantity
Storage
48T
96T
ELISA Microplate (Dismountable)
8×6
8×12
Place the test strips into a sealed foil bag with the desiccant. Store for 1 month at 2-8°C; Store for 12 months at -20°C.
Lyophilized Standard
1 vial
2 vials
Place the standards into a sealed foil bag with the desiccant. Store for 1 month at 2-8°C; Store for 12 months at -20°C.
