VC(Vitamin C) ELISA Kit (UNLE00001)

Product Name:	VC (Vitamin C) ELISA Kit
SKU:	UNLE00001
Synonyms:	AA, L-Ascorbic Acid, Ascorbate
Reactivity:	General
Assay Type:	Competitive Inhibition
Sensitivity:	192.1 ng/mL
Standard:	40000 ng/mL
Range:	625-40000 ng/mL
Sample Type:	Serum, Plasma, and other biological fluids
Assay Length:	2h
Specificity:	This assay has high sensitivity and excellent specificity for detection of VC. Nosignificant cross-reactivity or interference between VC and analogues was observed.
Research Area:	Metabolic pathway, Infection immunity, Nutrition metabolism

Kit Components	Quantity (96T)	Storage Condition
Pre-Coated Microplate	12 strips × 8 wells	4°C/-20°C
Standard (Lyophilized)	2 vials	4°C/-20°C
Biotinylated-Conjugate (100×)	60 μL	4°C/-20°C
Streptavidin-HRP (100×)	120 μL	4°C/-20°C
Standard/Sample Diluent Buffer	20 mL	4°C/-20°C
Biotinylated-Conjugate Diluent	10 mL	4°C/-20°C
HRP Diluent	12 mL	4°C/-20°C
Wash Buffer (25×)	20 mL	4°C/-20°C
TMB Substrate Solution	10 mL	4°C/-20°C (store in dark)
Stop Reagent	6 mL	4°C/-20°C
Plate Covers	2 pieces	RT

Materials Required, Not Supplied:

Microplate reader capable of measuring absorbance at 450 ± 10 nm
High-speed centrifuge
Electro-heating standing-temperature cultivator
Absorbent paper
Double distilled water or deionized water
Single or multi-channel pipettes with high precision and disposable tips
Precision pipettes to deliver 2 μL to 1 mL volumes

This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with VC protein. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to VC. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of VC in the samples is then determined by comparing the OD of the samples to the standard curve.

Precision:

Intra-assay Precision (Precision within an assay): CV% < 8%
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.

Inter-assay Precision (Precision between assays): CV% < 10%
Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.

Recovery:

Matrices listed below were spiked with a certain level of recombinant VC, and the recovery rates were calculated by comparing the measured value to the expected amount of VC in the samples.

Matrix	Recovery range	Average
Serum (n = 5)	85-97%	91%
EDTA Plasma (n = 5)	91-105%	98%
Heparin Plasma (n = 5)	81-95%	101%

Linearity:

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

Matrix	1:2	1:4	1:8	1:16
Serum (n = 5)	93-101%	97-103%	95-107%	87-102%
EDTA Plasma (n = 5)	95-104%	93-102%	79-94%	87-98%
Heparin Plasma (n = 5)	86-93%	86-97%	85-103%	89-97%

Step 1:	After the kit is equilibrated at room temperature, add 50 μL of Standard Working Solution (gradual dilution refers to Reagent Preparation) or 50 μL of sample to each well. Immediately add 50 μL of 1× Biotinylated-Conjugate Working Solution to each well, mix well, and incubate at 37°C for 60 minutes.
Step 2:	Discard the liquid in the plate, add 200 μL 1× Wash Buffer to each well, and wash the plate 3 times. Pat dry against clean absorbent paper, add 100 μL 1× Streptavidin-HRP Working Solution to each well, and incubate at 37°C for 60 minutes.
Step 3:	Discard the liquid in the plate, add 200 μL 1× Wash Buffer to each well, and wash the plate 5 times. Pat dry against clean absorbent paper, add 90 μL TMB Substrate Solution to each well, and incubate at 37°C for 20 minutes in the dark.
Step 4:	Add 50 μL Stop Reagent to each well, shake the plate on a plate shaker for 1 minute to mix. Record the OD at 450 nm immediately and calculate the results.

Serum:	Samples should be collected into a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C. Centrifuge at 1000 × g for 20 minutes. Assay freshly prepared serum immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 1000 × g and 2-8°C for 15 minutes within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.
Tissue Homogenates:	Rinse tissues in pre-cooled PBS to completely remove excess blood and weigh them before homogenization. Mince tissues into small pieces and homogenize them in fresh lysis buffer (choose based on subcellular location of the target protein; PBS is suitable for most tissues) (w:v = 1:9, e.g., 900 µL lysis buffer per 100 mg tissue) using a glass homogenizer or micro tissue grinder on ice. Ultrasound the suspension with an ultrasonic cell disrupter until the solution is clear. Centrifuge homogenates for 5 minutes at 10000 × g. Collect the supernatant and assay immediately or store in aliquots at ≤ -20°C. *Note: Tissue homogenates are recommended to be tested for protein concentration simultaneously to obtain accurate concentration of the test substance per mg of protein. For protein detection, you can purchase our BCA Protein Concentration Determination Kit.
Cell Lysates:	Adherent cells: Wash gently with pre-cooled PBS, detach with trypsin, and collect by centrifugation at 1000 × g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash cells 3 times in pre-cooled PBS. Resuspend cells in fresh lysis buffer at a concentration of 10⁷ cells/mL. Ultrasonication can be applied if necessary until the solution is clear. Centrifuge at 1500 × g for 10 minutes at 2-8°C to remove cellular debris. Assay immediately or store in aliquots at ≤ -20°C.
Urine:	Collect the first urine of the day (mid-stream) into a sterile container. Centrifuge to remove particulate matter, assay immediately, or aliquot and store at ≤ -20°C. Avoid repeated freeze-thaw cycles.
Saliva:	Collect saliva using a collection device or equivalent. Centrifuge samples at 1000 × g at 2-8°C for 15 minutes. Remove particulates and assay immediately or store in aliquots at ≤ -20°C. Avoid repeated freeze-thaw cycles.
Cell Culture Supernatants and Other Biological Fluids:	Centrifuge samples at 1000 × g for 20 minutes. Collect the supernatant and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.