Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.78-50 ng/mL
Sensitivity:	0.47 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human ADP/Acrp30 in samples. No significant cross-reactivity or interference between Human ADP/Acrp30 and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human ADP/Acrp30. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human ADP/Acrp30 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human ADP/Acrp30, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human ADP/Acrp30. The concentration of Human ADP/Acrp30 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

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

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (ng/mL)	RLU	Average	Corrected
50	35981 37981	36981	36959
25	14778 15352	15065	15043
12.5	6820 6780	6800	6778
6.25	3187 3495	3341	3319
3.13	1876 1682	1779	1757
1.56	1083 999	1041	1019
0.78	644 720	682	660
0	21 23	22	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ADP/Acrp30 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ADP/Acrp30 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	2.35	4.95	24.81	2.50	5.30	25.21
Standard deviation	0.30	0.57	2.74	0.21	0.62	1.97
C V (%)	12.77	11.52	11.04	8.40	11.70	7.81

Recovery

The recovery of Human ADP/Acrp30 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	94-109	100
EDTA plasma (n=5)	100-112	106
Cell culture media (n=5)	96-111	104

Linearity

Samples were spiked with high concentrations of Human ADP/Acrp30 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	95-111	97-110	98-112
1:2	Average (%)	103	102	106
1:4	Range (%)	100-117	84-97	99-113
1:4	Average (%)	108	91	104
1:8	Range (%)	91-105	88-101	101-115
1:8	Average (%)	98	93	107
1:16	Range (%)	99-114	102-115	91-103
1:16	Average (%)	107	109	96

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.