Human ELA3B (Elastase 3B) ELISA Kit (HUES02390)

SKU:
HUES02390
€539

Description

ELISA Kit Technical ManualMSDS

Human ELA3B (Elastase 3B) 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 ELA3B . 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 ELA3B 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 ELA3B, 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 ELA3B. The concentration of Human ELA3B 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 ELA3B in samples. No significant cross-reactivity or interference between Human ELA3B 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
202.259
2.275
2.2672.176
101.547
1.583
1.5651.474
50.902
0.886
0.8940.803
2.50.482
0.52
0.5010.41
1.250.285
0.273
0.2790.188
0.630.19
0.182
0.1860.095
0.310.134
0.146
0.140.049
00.089
0.093
0.091--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ELA3B were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ELA3B were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.982.248.411.042.188.88
Standard deviation0.060.090.400.060.120.29
C V (%)6.124.024.765.775.503.27

Recovery

The recovery of Human ELA3B 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)86-9891
EDTA plasma (n=5)95-108102
Cell culture media (n=5)86-10092

Linearity

Samples were spiked with high concentrations of Human ELA3B 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 (%)96-11184-9590-105
Average (%)1039097
1:4Range (%)89-10383-9385-98
Average (%)948892
1:8Range (%)91-10587-10188-102
Average (%)969295
1:16Range (%)93-10983-9485-98
Average (%)998892

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 ELA3B (Elastase 3B) ELISA Kit (HUES02390) 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 ELA3B (Elastase 3B) ELISA Kit (HUES02390) Protein Information

UniProt Protein Function:CELA3B: Efficient protease with alanine specificity but only little elastolytic activity. Belongs to the peptidase S1 family. Elastase subfamily.
UniProt Protein Details:

Protein type:EC 3.4.21.70; Protease

Chromosomal Location of Human Ortholog: 1p36.12

Molecular Function:peptidase activity; serine-type endopeptidase activity

Biological Process: cholesterol metabolic process; proteolysis

NCBI Summary:Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. Humans have six elastase genes which encode the structurally similar proteins elastase 1, 2, 2A, 2B, 3A, and 3B. Unlike other elastases, elastase 3B has little elastolytic activity. Like most of the human elastases, elastase 3B is secreted from the pancreas as a zymogen and, like other serine proteases such as trypsin, chymotrypsin and kallikrein, it has a digestive function in the intestine. Elastase 3B preferentially cleaves proteins after alanine residues. Elastase 3B may also function in the intestinal transport and metabolism of cholesterol. Both elastase 3A and elastase 3B have been referred to as protease E and as elastase 1, and excretion of this protein in fecal material is frequently used as a measure of pancreatic function in clinical assays. [provided by RefSeq, May 2009]
UniProt Code:P08861
NCBI GenInfo Identifier:317373457
NCBI Gene ID:23436
NCBI Accession:P08861.3
UniProt Secondary Accession:P08861,P11423, Q5VU28, Q5VU29, Q5VU30, B2RE44,
UniProt Related Accession:P08861
Molecular Weight:29,263 Da
NCBI Full Name:Chymotrypsin-like elastase family member 3B
NCBI Synonym Full Names:chymotrypsin-like elastase family, member 3B
NCBI Official Symbol:CELA3B
NCBI Official Synonym Symbols:E1; CBPP; EL-1; ELA3B
NCBI Protein Information:chymotrypsin-like elastase family member 3B; elastase 1; protease E; elastase-3B; proteinase E; elastase IIIB; fecal elastase 1; pancreatic elastase 1; elastase 3B, pancreatic; pancreatic endopeptidase E; cholesterol-binding pancreatic protease
UniProt Protein Name:Chymotrypsin-like elastase family member 3B
UniProt Synonym Protein Names:Elastase IIIB; Elastase-3B; Protease E
Protein Family:Chymotrypsin-like elastase family
UniProt Gene Name:CELA3B
UniProt Entry Name:CEL3B_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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