ELISA Kit for Semaphorin 4D (SEMA4D)

CD100; A8; BB18; GR3; M-Sema G; M-Sema-G; SEMAJ; Coll-4; Sema Domain,Immunoglobulin Domain(Ig),Transmembrane Domain(TM)and Short Cytoplasmic Domain 4D

Specificity

This assay has high sensitivity and excellent specificity for detection of Semaphorin 4D (SEMA4D).
No significant cross-reactivity or interference between Semaphorin 4D (SEMA4D) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Semaphorin 4D (SEMA4D) and the recovery rates were calculated by comparing the measured value to the expected amount of Semaphorin 4D (SEMA4D) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 80-98 88
EDTA plasma(n=5) 98-105 102
heparin plasma(n=5) 88-102 91

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Semaphorin 4D (SEMA4D) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Semaphorin 4D (SEMA4D) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Semaphorin 4D (SEMA4D) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 99-105% 78-90% 87-94% 99-105%
EDTA plasma(n=5) 94-105% 83-98% 92-101% 88-96%
heparin plasma(n=5) 80-91% 94-103% 82-90% 89-102%

Stability

The stability of 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.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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Magazine Citations
Expert Opin Ther Targets. Circulating semaphorin-4D and plexin-B1 levels in postmenopausal women with low bone mass: the 3-month effect of zoledronic acid, denosumab or teriparatide treatment Pubmed:25395071
Obesity (Silver Spring). Genome-wide analysis identifies an african-specific variant in SEMA4D associated with body mass index. pubmed:28296344
Clinical Lymphoma, Myeloma & Leukemia A Revised Staging System for Waldenström's Macroglobulinemia 10.1016/j.clml.2017.07.146
Clinical Lymphoma, Myeloma & Leukemia Extramedullary Multiple Myeloma: Clinical Features and Prognosis in Argentina 10.1016/j.clml.2017.07.144
Blood Cancer Journal Semaphorin 4D correlates with increased bone resorption, hypercalcemia, and disease stage in newly diagnosed patients with multiple myeloma Pubmed:29748532
Analytical Biochemistry A high-sensitivity enzyme immunoassay for the quantification of soluble human semaphorin 4D in plasma Pubmed: 30879960
Journal of Allergy and Clinical Immunology-In Practice Pathological and therapeutic implications of eosinophil-derived semaphorin 4D in eosinophilic chronic rhinosinusitis Pubmed: 32035658
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