Human Adiponectin ELISA Kit

SKU:
HUFI02974
€499

Description

ELISA Kit Technical ManualMSDS

Human ADP (Adiponectin) ELISA Kit - Information

The Assay Genie Human ADP (Adiponectin) ELISA Kit can assay for Human ADP in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our Human ADP (Adiponectin) ELISA Kits Work?

The Assay 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, Assay 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 Assay 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 Human ADP 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 ADP (Adiponectin) ELISA Kit Data

Product Code

HUFI02974

UniprotQ15848
Alias

Adiponectin, AdipoQ, ADP, Acrp30, GBP28, ACDC, ADPN

Detection method

Sandwich ELISA, Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Human ADP concentrations in serum plasma and other biological fluids.

Size

96T

Range

1.56-100ng/ml

Sensitivity

0.938ng/ml

Storage

4'C for 6 months

Recovery

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

MatrixRecovery range(%)Average(%)
serum(n=5)85-10294
EDTA plasma(n=5)90-10494
UFH plasma(n=5)87-10293
Linearity

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

Sample1:21:41:8
serum(n=5)88-104%90-99%86-103%
EDTA plasma(n=5)82-96%83-95%85-99%
UFH plasma(n=5)81-91%88-100%88-99%
CV(%)

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

Note

For Research Use Only

Human ADP (Adiponectin) ELISA Kit Protocol

The below protocol is a sample protocol for Human ADP (Adiponectin) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISA Kits 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 ADP Antibody 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.

Assay 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 ADP (Adiponectin) 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 Assay Genie Human ADP (Adiponectin) 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 ADP Protein Information

UniProt Protein Function:adiponectin: Important adipokine involved in the control of fat metabolism and insulin sensitivity, with direct anti-diabetic, anti-atherogenic and anti-inflammatory activities. Stimulates AMPK phosphorylation and activation in the liver and the skeletal muscle, enhancing glucose utilization and fatty-acid combustion. Antagonizes TNF-alpha by negatively regulating its expression in various tissues such as liver and macrophages, and also by counteracting its effects. Inhibits endothelial NF-kappa-B signaling through a cAMP-dependent pathway. May play a role in cell growth, angiogenesis and tissue remodeling by binding and sequestering various growth factors with distinct binding affinities, depending on the type of complex, LMW, MMW or HMW. Homomultimer. Forms trimers, hexamers and 12- to 18-mers. The trimers (low molecular weight complexes / LMW) are assembled via non-covalent interactions of the collagen-like domains in a triple helix and hydrophobic interactions within the globular C1q domain. Several trimers can associate to form disulfide-linked hexamers (middle molecular weight complexes / MMW) and larger complexes (higher molecular weight / HMW). The HMW-complex assembly may rely aditionally on lysine hydroxylation and glycosylation. LMW, MMW and HMW complexes bind to HBEGF, MMW and HMW complexes bind to PDGFB, and HMW complex binds to FGF2. Interacts with CTRP9A via the C1q domain (heterotrimeric complex). Synthesized exclusively by adipocytes and secreted into plasma.
UniProt Protein Details:

Protein type:Secreted, signal peptide; Endoplasmic reticulum; Secreted; Hormone

Chromosomal Location of Human Ortholog: 3q27

Cellular Component: extracellular space; collagen; cell surface; endoplasmic reticulum; extracellular region

Molecular Function:identical protein binding; protein binding; protein homodimerization activity; hormone activity; cytokine activity; sialic acid binding; receptor binding

Biological Process: circadian rhythm; negative regulation of phagocytosis; negative regulation of MAP kinase activity; negative regulation of smooth muscle cell proliferation; negative regulation of hormone secretion; positive regulation of cellular protein metabolic process; response to glucocorticoid stimulus; membrane hyperpolarization; negative regulation of smooth muscle cell migration; glucose homeostasis; negative regulation of granulocyte differentiation; positive regulation of interleukin-8 production; positive regulation of glucose import; negative regulation of gluconeogenesis; response to glucose stimulus; adiponectin-mediated signaling pathway; negative regulation of protein amino acid autophosphorylation; negative regulation of blood pressure; negative regulation of cell migration; protein homooligomerization; response to nutrient; generation of precursor metabolites and energy; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of signal transduction; negative regulation of heterotypic cell-cell adhesion; glucose metabolic process; negative regulation of tumor necrosis factor production; negative regulation of I-kappaB kinase/NF-kappaB cascade; negative regulation of fat cell differentiation; negative regulation of synaptic transmission; response to sucrose stimulus; membrane depolarization; positive regulation of peptidyl-tyrosine phosphorylation; response to ethanol; fatty acid beta-oxidation; positive regulation of fatty acid metabolic process; cellular response to insulin stimulus; negative regulation of macrophage differentiation; negative regulation of low-density lipoprotein receptor biosynthetic process; negative regulation of inflammatory response; brown fat cell differentiation; response to hypoxia; fatty acid oxidation; positive regulation of protein amino acid phosphorylation; response to activity; negative regulation of transcription, DNA-dependent; positive regulation of myeloid cell apoptosis; positive regulation of blood pressure

Disease: Adiponectin, Serum Level Of, Quantitative Trait Locus 1

NCBI Summary:This gene is expressed in adipose tissue exclusively. It encodes a protein with similarity to collagens X and VIII and complement factor C1q. The encoded protein circulates in the plasma and is involved with metabolic and hormonal processes. Mutations in this gene are associated with adiponectin deficiency. Multiple alternatively spliced variants, encoding the same protein, have been identified. [provided by RefSeq, Apr 2010]
UniProt Code:Q15848
NCBI GenInfo Identifier:2493789
NCBI Gene ID:9370
NCBI Accession:Q15848.1
UniProt Secondary Accession:Q15848,Q58EX9,
UniProt Related Accession:Q15848
Molecular Weight:25.1 kDa (231 aa)
NCBI Full Name:Adiponectin
NCBI Synonym Full Names:adiponectin, C1Q and collagen domain containing
NCBI Official Symbol:ADIPOQ  
NCBI Official Synonym Symbols:ACDC; ADPN; APM1; APM-1; GBP28; ACRP30; ADIPQTL1  
NCBI Protein Information:adiponectin
UniProt Protein Name:Adiponectin
UniProt Synonym Protein Names:30 kDa adipocyte complement-related protein; Adipocyte complement-related 30 kDa protein; ACRP30; Adipocyte, C1q and collagen domain-containing protein; Adipose most abundant gene transcript 1 protein; apM-1; Gelatin-binding protein
Protein Family:Adiponectin
UniProt Gene Name:ADIPOQ  
UniProt Entry Name:ADIPO_HUMAN
View AllClose

Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
View AllClose