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Human Semaphorin-4D (SEMA4D) ELISA Kit


Species Reactivity

Human (Homo sapiens)

UniProt

Abbreviation

SEMA4D

Alternative Names

XXyac-YM68G7.1; C9orf164; CD100; FLJ33485; FLJ34282; FLJ39737; FLJ46484; M-sema-G; MGC169138; MGC169141; SEMAJ; coll-4; OTTHUMP00000021622|OTTHUMP00000208950|OTTHUMP00000208951|OTTHUMP00000208957|se

Range

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Sensitivity

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Sample Type

Serum, Plasma, Other biological fluids

Detection Method

Sandwich

Analysis Method

Quantitive

Assay Duration

1-4.5h

Sample Volume

1-200 μL

Detection Wavelengt

450 nm

Cat

AE64482HU

Size

96T 48T 96T*5 96T*50

Price

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Availability

3-5 working days

Manual

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Test principle:
This assay employs a two-site sandwich ELISA to quantitate SEMA4D in samples. An antibody specific for SEMA4D has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and anySEMA4D present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for SEMA4D is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of SEMA4D bound in the initial step. The color development is stopped and the intensity of the color is measured.
Product Overview:
CD100 was first identified as a cell surface protein of resting T cells; previous studies had shown that it was involved in lymphocyte activation. Hall et al. (1996) cloned CD100 from a cDNA library of phytohemagglutinin-activated human T cells. Sequence analysis showed that CD100 encodes an 863-amino acid polypeptide containing a secretory signal sequence, a single transmembrane domain, an immunoglobulin (Ig)-like domain, and a conserved 500-amino acid sema domain. Thus, Hall et al. (1996) stated that CD100 is a member of the semaphorin family and the first semaphorin believed to be involved in the immune system. See SEMA3F and SEMA3B. Northern blot analysis revealed that CD100 is expressed primarily as a 4.5-kb message in a variety of tissues, both hematopoietic and nonhematopoietic.
Components:

Reagents

Quantity

Reagents

Quantity

Assay plate (96 Wells)

1

Instruction manual

1

Standard (lyophilized)

2

Sample Diluent

1 x 20 mL

Biotin-Conjugate (concentrate 100 x)

1 x 120 μL

Biotin-Conjugate Diluent

1 x 20 mL

Streptavidin-HRP  (concentrate 100 x)

1 x 120 μL

Streptavidin-HRP Diluent   

1 x 20 mL

Wash Buffer (concentrate 25 x)

1 x 20 mL

Substrate Solution

1 x 12 mL

Stop Solution

1 x 10 mL

Adhesive Films

4

Specificity:
This assay has high sensitivity and excellent specificity for detection of Human SEMA4D. No significant cross-reactivity or interference between Human SEMA4D and analogues was observed.
Recovery:
Matrices listed below were spiked with certain level of recombinant Human SEMA4D and the recovery rates were calculated by comparing the measured value to the expected amount of Human SEMA4D in samples.

Sample Type

Number

Recovery range (%)

Average(%)

Serum

10

90-101

96

EDTA plasma

10

89-97

93

Heparin plasma

10

91-99

95

Precision:
Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%
Linearity:
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human SEMA4D and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample Type

1:2

1:4

1:8

1:16

Serum

78-89%

81-99%

92-103%

95-105%

EDTA plasma

91-101%

90-98%

93-101%

91-98%

Heparin plasma

92-103%

93-102%

92-99%

91-101%

Stability:
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
Sample collection and storage:
Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Kits storage instructions:
Store at 2-8°C. Please refer to Instruction Manual.