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Soluble Semaphorin 4D ELISA Assay Kit

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$730.00
SKU:
BI-20405

 Product Description

Package insert view pdf
 

 

 

Soluble Semaphorin ELISA Assay Kit:
For Research Use Only
Size:  1x96 wells
Sensitivity:  LOD: (0 pmol/l + 3 SD): 12 pmol/l; LLOQ: 31 pmol/l
Dynamic Range:  0 to 2000 pmol/l
Incubation Time:  > 4 hours
Sample Type:  EDTA plasma, heparin plasma, and citrate plasma 
Sample Size: 10 µL
Controls Included
Product Developed and Manufactured by Biomedica

Unit conversion: 1 pg/ml = 0.0127 pmol/l; 1 pmol/l=78.9 pg/ml (MW: 78.9 kDa)

 

 


Assay Background

Semaphorin 4D (SEMA4D or CD100) is a member of a family of transmembrane and secreted proteins that regulates key cellular functions and is involved in cell-cell communication (1-3). Most of the effects of SEMA4D is mediated by plexins (4, 5). SEMA4D participates in numerous physiological processes such as axon guidance, immune regulation, angiogenesis, tumor progression, and bone metabolism (6-9). Cleavage of SEMA4D near the cell membrane through matrix metalloproteinases leads to the biologically active soluble SEMA4D with a molecular weight of 120 kD consisting of 713 amino acids (3, 5, 10). SEMA4D has emerged to a novel therapeutic target in cancer and in bone diseases (11, 12). Semaphorin 4D is widely studied for its role in neural connectivity, vascularization, cell migration, the immune response, tumor progression, and bone remodeling.

This sSEMA4D ELISA utilizes two monoclonal anti-human Semaphorin 4D antibodies, both recognizingconformational epitopes on Semaphorin 4D. The epitopes have been mapped by overlapping cyclic peptides and
shown to involve amino acids AA30-AA34 and amino acids AA238-AA241, respectively.

Assay Principle:

This kit is a sandwich enzyme immunoassay for the direct determination of soluble Semaphorin 4D in human plasma samples. In a first step, STD/sample/CTRL are pipetted into the wells of the microtiter strips, which are precoated with anti Semaphorin 4D antibody. Semaphorin 4D present in the STD/sample/CTRL binds to the pre-coated antibody in the well. In the washing step all non-specific unbound material is removed. In a next step, the conjugate (bivalent Fab bacterial alkaline phosphatase fusion antibody-HRP) is pipetted into the wells and reacts with the soluble Semaphorin 4D forming a sandwich. After another washing step, the substrate (TMB Tetramethylbenzidine) is pipetted into the wells. The enzyme catalysed colour change of the substrate is directly proportional to the amount of Semaphorin 4D. This colour change is detectable with a standard microtiter plate ELISA reader. A dose response curve of the absorbance (optical density, OD at 450 nm) versus standard concentration is generated, using the values obtained from the standards. The concentration of Semaphorin 4D in the sample is determined directly from the dose response curve.

Assay Procedure:

  1. Pipette 100 μl ASYBUF (Assay buffer, red cap) into each well.
  2. Add 20 μl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective well. Swirl gently
  3. Cover tightly and incubate for 2 hours at room temperature (18-26°C).
  4. Aspirate and wash wells 5x with 300 μl diluted WASHBUF (Wash buffer). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel.
  5. Add 100 μl CONJ (Conjugate, amber cap) into each well. Swirl gently.
  6. Cover tightly and incubate for 1 hour at room temperature (18-26°C) in the dark.
  7. Aspirate and wash wells 5x with 300 μl diluted WASHBUF (Wash buffer). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel.
  8. Add 100 μl SUB (Substrate, blue cap) into each well. Swirl gently.
  9. Incubate for 30 min at room temperature (18-26°C) in the dark.
  10. Add 50 μl STOP (Stop solution, white cap) into each well. Swirl gently.
  11. Measure absorbance immediately at 450 nm with reference 630 nm, if available.

 

 

 Typical Standard Curve:

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Literature

 

1. Semaphorins command cells to move. Kruger RP et al., Nature Rev Mol Cell Biol, 2005; 6:789-800.
2. The semaphorins. Yazdani U and Terman JR, Genome Biol, 2006; 7(3): 211.
3. Biology and function of neuroimmune semaphorins 4A and 4D. Nkyimbeng-Takwi EH and Chapoval SP, Immunol Res, 2011; 50 (1):10-21.
4. Structural basis of semaphorin-plexin signalling B.J.C. Janssen BJC et al., Nature, 2010; 467:1118-1122.
5. Semaphorins and their receptors in immune cell interactions. Suzuki K et al., Nature Immunology, 2007;
9:17-23.
6. Sema4D induces angiogenesis through Met recruitment by Plexin B1. Conrotto P et al., Blood, 2005;
105:4321-4329.
7. Diverse roles for semaphorin-plexin signaling in the immune system. Takamatsu H et al., Trends Immunol, 2012; 33(3):127-135.
8. Bone cell communication factors and Semaphorins. Negishi-Koga T and Takayanagi H, Bonekey Rep, 2012; 1:183.
9. Suppression of bone formation by osteoclastic expression of semaphorin 4D. Negishi-Koga T et al., Nat Med, 2011; 17(11):1473-1480.
10. Soluble SEMA4D/CD100: A novel immunoregulator in infectious and inflammatory diseases. Maleki KT et al., Clinical Immunology, 2016; 163:52-59.
11. Anabolic bone formation via a site specific bone targeting delivery system by interfering with semaphorin 4D expression. Zhang Y et al., J Bone Miner Res, 2015; 30(2): 286-296.
12. Generation and preclinical characterization of an antibody specific for SEMA4D. Fisher TL et al., Mabs, 2016; 8(1):150-162.
13. Coagulation-induced elevated sSEMA 4D concentrations in human serum versus plasma measured by sandwich ELISA. Laber et al., 2018; submitted.

 

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