Goat Platelet-Derived Growth Factor-BB (PDGF-BB) ELISA Kit

This assay employs a two-site sandwich ELISA to quantitate PDGF-BB in samples. An antibody specific for PDGF-BB has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and anyPDGF-BB present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for PDGF-BB is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of PDGF-BB bound in the initial step. The color development is stopped and the intensity of the color is measured.
 

The Platelet-Derived Growth Factor (PDGF) family consists of proteins derived from four genes (PDGF-A, -B, -C and -D) that form four disulfide-linked homodimers (PDGF-AA, -BB, -CC, -DD)and one heterodimer (PDGF-AB). These proteins and the related VEGF family proteinsshare the conserved PDGF/VEGF homology domain characterized by a pattern of highly conserved cysteine residues, which form the cysteine knot motif. PDGF-B is synthesized as a pre-pro-protein, which has a signal peptide, a mature region, an N- terminal pro-peptide, and a C-terminal extension. The C-terminal extension contains a conserved cell retention motif that confers retention of the secreted PDGF-BB within the pericellular space. Most cells produce both PDGF-A and B chains. The individual chains are assembled stochastically into disulfide-linked inactive homodimeric or heterodimeric precursors in the endoplasmic reticulum.
 

Reagents	Quantity	Reagents	Quantity
Assay plate (96 Wells)	1	Instruction manual	1
Standard (lyophilized)	2	Sample Diluent	1 x 20 mL
Biotin-Conjugate (concentrate 100 x)	1 x 120 μL	Biotin-Conjugate Diluent	1 x 12 mL
Streptavidin-HRP  (concentrate 100 x)	1 x 120 μL	Streptavidin-HRP Diluent	1 x 12 mL
Wash Buffer (concentrate 25 x)	1 x 20 mL	Substrate Solution	1 x 10 mL
Stop Solution	1 x 6 mL	Adhesive Films	4

 

This assay has high sensitivity and excellent specificity for detection of Goat PDGF-BB. No significant cross-reactivity or interference between Goat PDGF-BB and analogues was observed.
 

Matrices listed below were spiked with certain level of recombinant Goat PDGF-BB and the recovery rates were calculated by comparing the measured value to the expected amount of Goat PDGF-BB in samples.

Sample Type	Number	Recovery range (%)	Average(%)
Serum	10	90-101	96
EDTA plasma	10	89-97	93
Heparin plasma	10	91-99	95

 

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%
 

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Goat PDGF-BB and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample Type	1：2	1：4	1：8	1：16
Serum	78-89%	81-99%	92-103%	95-105%
EDTA plasma	91-101%	90-98%	93-101%	91-98%
Heparin plasma	92-103%	93-102%	92-99%	91-101%

 

The stability of ELISA 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. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
 

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
 

Store at 2-8°C. Please refer to Instruction Manual.