High Sensitive ELISA Kit for Interleukin 35 (IL35)

Specificity

This assay has high sensitivity and excellent specificity for detection of High Sensitive Interleukin 35 (IL35).
No significant cross-reactivity or interference between High Sensitive Interleukin 35 (IL35) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant High Sensitive Interleukin 35 (IL35) and the recovery rates were calculated by comparing the measured value to the expected amount of High Sensitive Interleukin 35 (IL35) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 88-102 98
EDTA plasma(n=5) 98-105 101
heparin plasma(n=5) 78-88 81

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level High Sensitive Interleukin 35 (IL35) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level High Sensitive Interleukin 35 (IL35) 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 High Sensitive Interleukin 35 (IL35) 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) 98-105% 87-101% 78-105% 85-102%
EDTA plasma(n=5) 91-98% 79-90% 80-102% 89-97%
heparin plasma(n=5) 80-91% 89-103% 95-102% 79-103%

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 Reference
Oncology Letters Aberrant expression of Treg-associated cytokine IL-35 along with IL-10 and TGF-β in acute myeloid leukemia. PubMed: 22783403
Journal of Maternal-Fetal and Neonatal Medicine Plasma IL-17, IL-35, interferon-γ, SOCS3 and TGF-β levels in pregnant women with preeclampsia, and their relation with severity of disease Pubmed: 24175856
Journal of Reproductive Immunology What is the impact of Th1/Th2 ratio, SOCS3, IL17, and IL35 levels in unexplained infertility? Pubmed: 24368037
Human Immunology The imbalanced profile and clinical significance of T helper associated cytokines in bone marrow microenvironment of the patients with acute myeloid leukemia ScienceDirect: S0198885913005776
Folia Biologica (Praha) Gene Expression of Subunits of the IL-12 Family Cytokines in moDCs Derived In Vitro from the Cord Blood of Children of Healthy and Allergic Mothers Pubmed:24785110
Molecular Medicine Reports Function of interleukin?17 and ?35 in the blood of patients with hepatitis B?related liver cirrhosis Pubmed:25323532
Rom J Morphol Embryol. The regular distribution and expression pattern of immunosuppressive cytokine IL-35 in mouse uterus during early pregnancy Pubmed:25611266
Journal of Immunotherapy Wogonin Inhibits Tumor-derived Regulatory Molecules by Suppressing STAT3 Signaling to Promote Tumor Immunity PubMed: 25962106
PLOS ONE Interleukin 35 Synovial Fluid Levels Are Associated with Disease Activity of Rheumatoid Arthritis PubMed: 26204444
Rheumatology (Oxford) Interleukin-35 is upregulated in systemic sclerosis and its serum levels are associated with early disease PubMed: 26231346
Cytokine Anti-inflammatory effects of interleukin-35 in acquired aplastic anemia PubMed: 26282938
J Biol Chem Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway PubMed: 26085094
Br J Haematol Interleukin 35 may contribute to the loss of immunological self‐tolerance in patients with primary immune thrombocytopenia PubMed: 25640666
Autoimmunity Reviews New insights into immune mechanisms underlying response to Rituximab in patients with membranous nephropathy: A prospective study and a review of the literature Pubmed:26876383
Clinical research Increased plasma concentrations of interleukin 35 in patients with coronary artery disease Journal:-19
Clinical Rheumatology The clinical utility of serum IL-35 in patients with polymyositis and dermatomyositis pubmed:27502600
Respiratory Medicine Enhanced LPS-induced activation of IL-27 signalling in sarcoidosis pubmed:27492538
International Journal of Molecular Medicine Effects of an intravitreal injection of interleukin-35-expressing plasmid on pro-inflammatory andanti-inflammatory cytokines. pubmed:27460435
上海口腔医学 IL-35在牙周炎及口腔扁平苔藓中的表达及相关性分析 QK:98604X
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