Human Anti-cyclic citrullinated peptide antibody (CCP-Ab) ELISA Kit BHE11406852

Mfr.No.

AE62292HU

Alternative Names

CCP-Ab

Background

This assay employs the competitive enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antibody specific to CCP-Ab. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated CCP-Ab and incubated. The competitive inhibition reaction is launched between with HRP labeled CCP-Ab and unlabeled CCP-Ab with the antibody. A substrate solution is added to the wells and the color develops in opposite to the amount of CCP-Ab in the sample. The color development is stopped and the intensity of the color is measured.

Categories

ELISA

Description

The most prominent clinical syndrome associated with a disordered hypersecretion of Calcitonin is medullary carcinoma of the thyroid (MTC). MTC is a tumor of the Calcitonin producing C-cells of the thyroid gland. Although MTC is rare, comprising 5 - 10% of all thyroid cancer, it is often fatal. It may occur sporadically or in a familial form that is transmitted as an autosomal dominant trait. MTC has great clinical importance because of its familial distribution. Further, it leant itself to be diagnosed early by serum Calcitonin and total cure for early sub-clinical disease is possible1. This is frequently associated with other clinical features and it has good potential for cure with surgery. Although a rare tumor, it can occur in a familial pattern1 as a Type II multiple endocrine neoplasia.

Host

Human

Reactivity

Human

Specificity

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

Description

ELISA

Analytical Method

Qualitative

Assay Time

4.5H

Components

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

Detection Method

Competitive ELISA

Detection Range

12.35-1000 pg/mL

Detection Wavelength

450 nm

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of CCP-Ab 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%

Method Type

Serum, Plasma

Precision

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%

Recovery

Matrices listed below were spiked with certain level of recombinant CCP-Ab and the recovery rates were calculated by comparing the measured value to the expected amount of CCP-Ab 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

Sample Volume

1-200 μL

Sensitivity

4.48 pg/mL

Uniprot	N/A

Description	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).
Storage	Store at 2-8°C. Please refer to Instruction Manual.
Expiry Date	Half Year

Applications	ELISA

Protocols Description	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.
Other Protocol	Bring all reagents and samples to room temperature before use. It is recommended that all samples, controls, and standards be assayed in duplicate. 1.Prepare all reagents, working standards, and samples as directed in the previous sections. 2.Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 2 - 8°C. 3.Set a Blank well without any solution. Add 50 µL of Standard or Sample to per well. Add 50 µL of HRP-Conjugate to each well (Note: Do not to Blank!). Mix well, Cover with the adhesive films provided. Incubate for 30 minutes at 37°C. 4.Aspirate each well and wash, repeating the process for a total of five washes. Wash by filling each well with Wash Buffer (250 µL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 5.Add 50 μL of Substrate A and 50 μL of Substrate B to each well, mix well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 6.Add 50 μL of Stop Solution to each well when the first four wells containing the highest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 7.Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate. 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.

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SKU	Supplier No.	Size	Your price	Quantity
BHE11406852	AE62292HU	48T	$371.94		Add to Cart
BHE11406852	AE62292HU	96T	$608.50		Add to Cart

Human Anti-cyclic citrullinated peptide antibody (CCP-Ab) ELISA Kit BHE11406852

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