Human MIP 3b / CCL19 ELISA Kit

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SKU:
HUFI00002
€299

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human MIP 3b / CCL19 ELISA Kit - Information

The ELISA Genie MIP 3b / CCL19 ELISA Kit can assay for MIP-3 beta / CCL19 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our MIP 3b / CCL19 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound MIP-3 beta / CCL19 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human MIP-3 beta / CCL19 ELISA Kit - Data

Description May play a role not only in inflammatory and immunological responses but also in normal lymphocyte recirculation and homing. May play an important role in trafficking of T-cells in thymus, and T-cell and B-cell migration to secondary lymphoid organs. Binds to chemokine receptor CCR7. Recombinant CCL19 shows potent chemotactic activity for T-cells and B-cells but not for granulocytes and monocytes. Binds to atypical chemokine receptor ACKR4 and mediates the recruitment of beta-arrestin (ARRB1/2) to ACKR4.  
Post-Translational Modification  
Uniprot ID Q99731
Alias MIP-3 beta/ELC/CCL19/beta chemokine exodus-3/Beta-chemokine exodus-3/CC chemokine ligand 19/C-C motif chemokine 19/chemokine(C-C motif) ligand 19/CKb11/EBI1-ligand chemokine/ELCMIP-3-beta/Epstein-Barr virus-induced molecule 1 ligand chemokine/exodus-3/Macrophage inflammatory protein 3 beta/macrophage inflammatory protein 3-beta/MGC34433/MIP-3b/MIP3BCK beta-11/SCYA19EBI1 ligand chemokine/small inducible cytokine subfamily A(Cys-Cys) member 19/Small-inducible cytokine A19
Detection method Sandwich ELISA Double Antibody
Application This immunoassay kit allows for the in vitro quantitative determination of MIP-3 beta concentrations in serum plasma and other biological fluids.
Size 96T
Range 15.625-1000pg/ml
Sensitivity < 9.375pg/ml
Storage 4'C for 6 months
Recovery Matrices listed below were spiked with certain level of MIP-3 beta and the recovery rates were calculated by comparing the measured value to the expected amount of MIP-3 beta in samples.
MatrixRecovery range(%)Average(%)
serum(n=5) 85-102 92
EDTA plasma(n=5) 85-100 93
UFH plasma(n=5) 92-105 99
Linearity The linearity of the kit was assayed by testing samples spiked with appropriate concentration of MIP-3 beta and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:81:16
serum(n=5) 89-102% 85-103% 87-102% 87-103%
EDTA plasma(n=5) 83-98% 91-100% 85-101% 87-99%
UFH plasma(n=5) 85-96% 83-100% 80-99% 83-100%
CV(%) Intra-Assay: CV<8%Inter-Assay: CV<10%
Note For Research Use Only

Human MIP 3b / CCL19 ELISA Kit Protocol

The below protocol is a sample protocol for Human MIP 3b / CCL19 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human MIP 3b / CCL19 present in their sample.

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.

Sandwich Protocol

Sandwich ELISA Protocol

Kit 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.

Human MIP 3b / CCL19 ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Human MIP 3b / CCL19 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Human MIP 3b / CCL19 ELISA Kit Protein Information

UniProt Protein Function:CCL19: May play a role not only in inflammatory and immunological responses but also in normal lymphocyte recirculation and homing. May play an important role in trafficking of T-cells in thymus, and T-cell and B-cell migration to secondary lymphoid organs. Specifically binds to chemokine receptor CCR7. Recombinant CCL19 shows potent chemotactic activity for T-cells and B-cells but not for granulocytes and monocytes. Belongs to the intercrine beta (chemokine CC) family.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 9p13

Cellular Component: extracellular space; extracellular region

Molecular Function:CCR chemokine receptor binding; chemokine activity; chemokine receptor binding; CCR10 chemokine receptor binding; CCR7 chemokine receptor binding

Biological Process: formation of immunological synapse; positive regulation of interleukin-12 production; positive regulation of dendritic cell antigen processing and presentation; cell maturation; positive regulation of JNK cascade; positive regulation of endocytosis; positive regulation of NF-kappaB import into nucleus; positive regulation of interleukin-1 beta secretion; activation of JNK activity; positive regulation of receptor-mediated endocytosis; dendritic cell chemotaxis; cell communication; establishment of T cell polarity; positive regulation of T cell proliferation; inflammatory response; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of chemotaxis; response to virus; positive regulation of phosphoinositide 3-kinase activity; positive regulation of tumor necrosis factor production; cellular calcium ion homeostasis; positive regulation of protein kinase B signaling cascade; myeloid dendritic cell chemotaxis; release of sequestered calcium ion into cytosol; positive regulation of protein kinase activity; T cell costimulation; immune response; positive regulation of T-helper 1 cell differentiation

NCBI Summary:This antimicrobial gene is one of several CC cytokine genes clustered on the p-arm of chromosome 9. Cytokines are a family of secreted proteins involved in immunoregulatory and inflammatory processes. The CC cytokines are proteins characterized by two adjacent cysteines. The cytokine encoded by this gene may play a role in normal lymphocyte recirculation and homing. It also plays an important role in trafficking of T cells in thymus, and in T cell and B cell migration to secondary lymphoid organs. It specifically binds to chemokine receptor CCR7. [provided by RefSeq, Sep 2014]
UniProt Code:Q99731
NCBI GenInfo Identifier:2842763
NCBI Gene ID:6363
NCBI Accession:Q99731.1
UniProt Secondary Accession:Q99731,O00697, O00736,
UniProt Related Accession:Q99731
Molecular Weight:10,993 Da
NCBI Full Name:C-C motif chemokine 19
NCBI Synonym Full Names:chemokine (C-C motif) ligand 19
NCBI Official Symbol:CCL19  
NCBI Official Synonym Symbols:ELC; CKb11; MIP3B; MIP-3b; SCYA19  
NCBI Protein Information:C-C motif chemokine 19; exodus-3; CK beta-11; MIP-3-beta; EBI1 ligand chemokine; EBI1-ligand chemokine; CC chemokine ligand 19; beta chemokine exodus-3; beta-chemokine exodus-3; small-inducible cytokine A19; macrophage inflammatory protein 3 beta; macrophage inflammatory protein 3-beta; epstein-Barr virus-induced molecule 1 ligand chemokine; small inducible cytokine subfamily A (Cys-Cys), member 19
UniProt Protein Name:C-C motif chemokine 19
UniProt Synonym Protein Names:Beta-chemokine exodus-3; CK beta-11; Epstein-Barr virus-induced molecule 1 ligand chemokine; EBI1 ligand chemokine; ELC; Macrophage inflammatory protein 3 beta; MIP-3-beta; Small-inducible cytokine A19
UniProt Gene Name:CCL19  
UniProt Entry Name:CCL19_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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