Human SAA (Serum Amyloid A) ELISA Kit (HUES03080)

SKU:
HUES03080
€299

Description

ELISA Kit Technical ManualMSDS

Human SAA (Serum Amyloid A) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human SAA . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human SAA 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 SAA, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human SAA. The concentration of Human SAA in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range1.25-80 ng/mL
Sensitivity0.75 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human SAA in samples. No significant cross-reactivity or interference between Human SAA and analogues was observed.

Typical Data

As the OD 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)
O.D Average Corrected
802.402
2.436
2.4192.365
401.505
1.523
1.5141.46
200.911
0.905
0.9080.854
100.425
0.433
0.4290.375
50.24
0.222
0.2310.177
2.50.152
0.144
0.1480.094
1.250.094
0.112
0.1030.049
00.045
0.063
0.054--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)3.708.0735.344.028.8436.42
Standard deviation0.230.461.550.210.431.65
C V (%)6.225.704.395.224.864.53

Recovery

The recovery of Human SAA 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)84-9590
EDTA plasma (n=5)89-10094
Cell culture media (n=5)92-10397

Linearity

Samples were spiked with high concentrations of Human SAA 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)
1:2Range (%)89-10293-10891-105
Average (%)9610098
1:4Range (%)87-9981-9489-100
Average (%)938794
1:8Range (%)90-10287-9785-97
Average (%)969290
1:16Range (%)90-10383-9684-97
Average (%)959090

Kit Components & Storage

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 ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human SAA (Serum Amyloid A) ELISA Kit (HUES03080) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human SAA (Serum Amyloid A) ELISA Kit (HUES03080) Protein Information

UniProt Protein Function:SAA1: Major acute phase reactant. Apolipoprotein of the HDL complex. Reactive, secondary amyloidosis is characterized by the extracellular accumulation in various tissues of the SAA1 protein. These deposits are highly insoluble and resistant to proteolysis; they disrupt tissue structure and compromise function. Elevated serum SAA1 protein levels may be associated with lung cancer. Belongs to the SAA family.
UniProt Protein Details:

Protein type:Secreted, signal peptide; Secreted

Chromosomal Location of Human Ortholog: 11p15.1

Cellular Component: extracellular region

Molecular Function:heparin binding; G-protein-coupled receptor binding

Biological Process: positive regulation of interleukin-1 secretion; receptor-mediated endocytosis; platelet activation; neutrophil chemotaxis; elevation of cytosolic calcium ion concentration; positive regulation of cell adhesion; regulation of protein secretion; negative regulation of inflammatory response; acute-phase response; innate immune response; lymphocyte chemotaxis; macrophage chemotaxis; positive regulation of cytokine secretion

NCBI Summary:This gene encodes a member of the serum amyloid A family of apolipoproteins. The encoded protein is a major acute phase protein that is highly expressed in response to inflammation and tissue injury. This protein also plays an important role in HDL metabolism and cholesterol homeostasis. High levels of this protein are associated with chronic inflammatory diseases including atherosclerosis, rheumatoid arthritis, Alzheimer's disease and Crohn's disease. This protein may also be a potential biomarker for certain tumors. Alternate splicing results in multiple transcript variants that encode the same protein. A pseudogene of this gene is found on chromosome 11.[provided by RefSeq, Jun 2012]
UniProt Code:P0DJI8
NCBI GenInfo Identifier:395406826
NCBI Gene ID:6288
NCBI Accession:P0DJI8.1
UniProt Secondary Accession:P0DJI8,P02735, P02736, P02737, Q16730, Q16834, Q16835 Q16879, Q3KRB3, Q6FG67, Q96QN0, Q9UCK9,
UniProt Related Accession:P0DJI8
Molecular Weight:122
NCBI Full Name:Serum amyloid A-1 protein
NCBI Synonym Full Names:serum amyloid A1
NCBI Official Symbol:SAA1
NCBI Official Synonym Symbols:SAA; PIG4; SAA2; TP53I4
NCBI Protein Information:serum amyloid A-1 protein; serum amyloid A protein; tumor protein p53 inducible protein 4
UniProt Protein Name:Serum amyloid A-1 protein
UniProt Synonym Protein Names:Amyloid fibril protein AA
UniProt Gene Name:SAA1
UniProt Entry Name:SAA1_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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