Human SPC (Pulmonary Surfactant Associated Protein C) ELISA Kit (HUES02326)

SKU:
HUES02326
Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
€539

Description

ELISA Kit Technical ManualMSDS

Human SPC (Pulmonary Surfactant Associated Protein C) 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 SPC . 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 SPC 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 SPC, 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 SPC. The concentration of Human SPC 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 Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human SPC in samples. No significant cross-reactivity or interference between Human SPC 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
202.229
2.251
2.242.17
101.475
1.485
1.481.41
50.895
0.863
0.8790.809
2.50.433
0.465
0.4490.379
1.250.233
0.209
0.2210.151
0.630.166
0.16
0.1630.093
0.310.11
0.126
0.1180.048
00.062
0.078
0.07--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)1.022.676.951.002.866.72
Standard deviation0.070.150.300.070.130.36
C V (%)6.865.624.327.004.555.36

Recovery

The recovery of Human SPC 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)95-105100
EDTA plasma (n=5)88-9994
Cell culture media (n=5)88-9994

Linearity

Samples were spiked with high concentrations of Human SPC 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 (%)95-10988-9996-109
Average (%)10294102
1:4Range (%)91-10286-9784-94
Average (%)979189
1:8Range (%)87-10380-9384-94
Average (%)948789
1:16Range (%)91-10581-9087-102
Average (%)998694

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 SPC (Pulmonary Surfactant Associated Protein C) ELISA Kit (HUES02326) 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 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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 SPC (Pulmonary Surfactant Associated Protein C) ELISA Kit (HUES02326) Protein Information

UniProt Protein Function:SFTPC: Pulmonary surfactant associated proteins promote alveolar stability by lowering the surface tension at the air- liquid interface in the peripheral air spaces. Defects in SFTPC are the cause of pulmonary surfactant metabolism dysfunction type 2 (SMDP2); also called pulmonary alveolar proteinosis due to surfactant protein C deficiency. A rare disease associated with progressive respiratory insufficiency and lung disease with a variable clinical course, due to impaired surfactant homeostasis. It is characterized by alveolar filling with floccular material that stains positive using the periodic acid-Schiff method and is derived from surfactant phospholipids and protein components. Excessive lipoproteins accumulation in the alveoli results in severe respiratory distress. Genetic variations in SFTPC are a cause of susceptibility to respiratory distress syndrome in premature infants (RDS); also known as RDS in prematurity. RDS is a lung disease affecting usually premature newborn infants. It is characterized by deficient gas exchange, diffuse atelectasis, high-permeability lung edema and fibrin-rich alveolar deposits called 'hyaline membranes'. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Lipid-binding

Chromosomal Location of Human Ortholog: 8p21

Cellular Component: multivesicular body; extracellular space

Molecular Function:protein binding; protein homodimerization activity

Biological Process: circadian rhythm; response to cAMP; response to hyperoxia; response to retinoic acid; response to glucocorticoid stimulus; response to glucose stimulus; response to lipopolysaccharide; respiratory gaseous exchange; protein homooligomerization; response to vitamin A

Disease: Pulmonary Fibrosis, Idiopathic; Surfactant Metabolism Dysfunction, Pulmonary, 2

NCBI Summary:This gene encodes the pulmonary-associated surfactant protein C (SPC), an extremely hydrophobic surfactant protein essential for lung function and homeostasis after birth. Pulmonary surfactant is a surface-active lipoprotein complex composed of 90% lipids and 10% proteins which include plasma proteins and apolipoproteins SPA, SPB, SPC and SPD. The surfactant is secreted by the alveolar cells of the lung and maintains the stability of pulmonary tissue by reducing the surface tension of fluids that coat the lung. Multiple mutations in this gene have been identified, which cause pulmonary surfactant metabolism dysfunction type 2, also called pulmonary alveolar proteinosis due to surfactant protein C deficiency, and are associated with interstitial lung disease in older infants, children, and adults. Alternatively spliced transcript variants encoding different protein isoforms have been identified.[provided by RefSeq, Feb 2010]
UniProt Code:P11686
NCBI GenInfo Identifier:131425
NCBI Gene ID:6440
NCBI Accession:P11686.2
UniProt Related Accession:P11686
Molecular Weight:
NCBI Full Name:Pulmonary surfactant-associated protein C
NCBI Synonym Full Names:surfactant protein C
NCBI Official Symbol:SFTPC
NCBI Official Synonym Symbols:SP-C; PSP-C; SFTP2; SMDP2; BRICD6
NCBI Protein Information:pulmonary surfactant-associated protein C
UniProt Protein Name:Pulmonary surfactant-associated protein C
UniProt Synonym Protein Names:Pulmonary surfactant-associated proteolipid SPL(Val); SP5
UniProt Gene Name:SFTPC
UniProt Entry Name:PSPC_HUMAN
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