Human SEMA3A (Semaphorin 3A) CLIA Kit (HUES00730)

SKU:
HUES00730
€599

Description

ELISA Kit Technical ManualMSDS

Human SEMA3A (Semaphorin 3A) CLIA Kit

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 SEMA3A . 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 SEMA3A 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 SEMA3A, 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 SEMA3A. The concentration of Human SEMA3A in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human SEMA3A in samples. No significant cross-reactivity or interference between Human SEMA3A and analogues was observed.

Typical Data

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
(pg/mL)
RLU Average Corrected
200054503
56939
5572155693
100022905
25043
2397423946
50011274
10790
1103211004
2504879
5709
52945266
1252740
2476
26082580
62.51412
1210
13111283
31.25671
677
674646
027
29
28--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human SEMA3A were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human SEMA3A were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)100.04315.47856.96107.94314.58772.72
Standard deviation10.8723.4481.6712.7334.3555.33
C V (%)10.877.439.5311.7910.927.16

Recovery

The recovery of Human SEMA3A 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)88-10193
EDTA plasma (n=5)89-10294
Cell culture media (n=5)94-108101

Linearity

Samples were spiked with high concentrations of Human SEMA3A 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 (%)94-10989-10288-100
Average (%)1019493
1:4Range (%)95-11198-11394-109
Average (%)102103100
1:8Range (%)93-10789-9992-105
Average (%)999499
1:16Range (%)92-108102-11794-110
Average (%)100110102

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 CLIA 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 Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human SEMA3A (Semaphorin 3A) CLIA Kit (HUES00730) 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 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.
    11. Determine the RLU value of each well immediately.

Human SEMA3A (Semaphorin 3A) CLIA Kit (HUES00730) Protein Information

UniProt Protein Function:SEMA3A: Induces the collapse and paralysis of neuronal growth cones. Could serve as a ligand that guides specific growth cones by a motility-inhibiting mechanism. Binds to the complex neuropilin-1/plexin-1. Belongs to the semaphorin family.
UniProt Protein Details:

Protein type:Secreted; Motility/polarity/chemotaxis; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 7p12.1

Cellular Component: axon; extracellular region; extracellular space

Molecular Function:chemorepellent activity; semaphorin receptor binding

Biological Process: axon extension involved in axon guidance; axon guidance; negative regulation of axon extension involved in axon guidance; nerve development; neural crest cell migration; neuron migration; olfactory bulb development; regulation of axon extension involved in axon guidance; sensory system development; sympathetic nervous system development

Disease: Hypogonadotropic Hypogonadism 16 With Or Without Anosmia

NCBI Summary:This gene is a member of the semaphorin family and encodes a protein with an Ig-like C2-type (immunoglobulin-like) domain, a PSI domain and a Sema domain. This secreted protein can function as either a chemorepulsive agent, inhibiting axonal outgrowth, or as a chemoattractive agent, stimulating the growth of apical dendrites. In both cases, the protein is vital for normal neuronal pattern development. Increased expression of this protein is associated with schizophrenia and is seen in a variety of human tumor cell lines. Also, aberrant release of this protein is associated with the progression of Alzheimer's disease. [provided by RefSeq, Jul 2008]
UniProt Code:Q14563
NCBI GenInfo Identifier:8134674
NCBI Gene ID:10371
NCBI Accession:Q14563.1
UniProt Related Accession:Q14563
Molecular Weight:
NCBI Full Name:Semaphorin-3A
NCBI Synonym Full Names:semaphorin 3A
NCBI Official Symbol:SEMA3A  
NCBI Official Synonym Symbols:HH16; SemD; COLL1; SEMA1; SEMAD; SEMAL; coll-1; Hsema-I; SEMAIII; Hsema-III  
NCBI Protein Information:semaphorin-3A
UniProt Protein Name:Semaphorin-3A
UniProt Synonym Protein Names:Semaphorin III; Sema III
Protein Family:Semaphorin
UniProt Gene Name:SEMA3A  
UniProt Entry Name:SEM3A_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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