Human Cytokeratin Fragment 21-1 ELISA Kit (HUFI00301)

Product Name:	Human Cytokeratin Fragment 21-1 ELISA Kit
Product Code:	HUFI00301
Size:	96 Assays
Alias:	CYFRA21-1, CytokeRatin Fragment Antigen 21-1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CYFRA21-1 concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human CYFRA21-1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CYFRA21-1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-95	91
EDTA plasma(n=5)	89-103	98
UFH plasma(n=5)	85-102	90

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CYFRA21-1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	87-105%	89-104%	93-104%
EDTA plasma(n=5)	88-101%	88-100%	88-101%
UFH plasma(n=5)	84-98%	80-95%	85-99%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P08727

UniProt Protein Function:	Function: Involved in the organization of myofibers. Together with KRT8, helps to link the contractile apparatus to dystrophin at the costameres of striated muscle. Ref.14
UniProt Protein Details:	Subunit structure: Heterotetramer of two type I and two type II keratins. Interacts with PNN and the actin-binding domain of DMD. Interacts with HCV core protein. Ref.13 Ref.14 Tissue specificity: Expressed in a defined zone of basal keratinocytes in the deep outer root sheath of hair follicles. Also observed in sweat gland and mammary gland ductal and secretory cells, bile ducts, gastrointestinal tract, bladder urothelium, oral epithelia, esophagus, ectocervical epithelium (at protein level). Expressed in epidermal basal cells, in nipple epidermis and a defined region of the hair follicle. Also seen in a subset of vascular wall cells in both the veins and artery of human umbilical cord, and in umbilical cord vascular smooth muscle. Observed in muscle fibers accumulating in the costameres of myoplasm at the sarcolemma in structures that contain dystrophin and spectrin. Ref.2 Ref.4 Ref.14 Developmental stage: Present in hair follicles at all stages of development. Ref.4 Domain: This keratin differs from all other IF proteins in lacking the C-terminal tail domain. Miscellaneous: There are two types of cytoskeletal and microfibrillar keratin: I (acidic; 40-55 kDa) and II (neutral to basic; 56-70 kDa). Sequence similarities: Belongs to the intermediate filament family.
NCBI Summary:	The protein encoded by this gene is a member of the keratin family. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. The type I cytokeratins consist of acidic proteins which are arranged in pairs of heterotypic keratin chains. Unlike its related family members, this smallest known acidic cytokeratin is not paired with a basic cytokeratin in epithelial cells. It is specifically expressed in the periderm, the transiently superficial layer that envelopes the developing epidermis. The type I cytokeratins are clustered in a region of chromosome 17q12-q21. [provided by RefSeq, Jul 2008]
UniProt Code:	P08727
NCBI GenInfo Identifier:	311033484
NCBI Gene ID:	3880
NCBI Accession:	P08727.4
UniProt Secondary Accession:	P08727,Q5XG83, Q6NW33, Q7L5M9, Q96A53, Q96FV1, Q9BYF9 Q9P1Y4, B2R874,
UniProt Related Accession:	P08727
Molecular Weight:	44,106 Da
NCBI Full Name:	Keratin, type I cytoskeletal 19
NCBI Synonym Full Names:	keratin 19
NCBI Official Symbol:	KRT19
NCBI Official Synonym Symbols:	K19; CK19; K1CS
NCBI Protein Information:	keratin, type I cytoskeletal 19; CK-19; keratin-19; cytokeratin 19; cytokeratin-19; keratin, type I, 40-kd; 40-kDa keratin intermediate filament
UniProt Protein Name:	Keratin, type I cytoskeletal 19
UniProt Synonym Protein Names:	Cytokeratin-19; CK-19; Keratin-19
Protein Family:	Keratin
UniProt Gene Name:	KRT19
UniProt Entry Name:	K1C19_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.