Interleukin 35 ELISA Kit

Catalog No. ABIN415132

Product Details Interleukin 35 ELISA Kit

Target: Interleukin 35 (IL35)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 15.6 pg/mL - 1000 pg/mL

Minimum Detection Limit: 15.6 pg/mL

Application: ELISA

Purpose: The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of IL35 in Serum,Plasma,Tissue Homogenate,Cell Lysate,Cell Culture Supernatant,Biological Fluids

Sample Type: Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate

Analytical Method: Quantitative

Specificity:

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

Cross-Reactivity (Details): No significant cross-reactivity or interference between Interleukin 35 (IL35) and analogues was observed.

Sensitivity: 5.8 pg/mL

Components:

• Pre-coated, ready to use 96-well strip plate, flat buttom
• Plate sealer for 96 wells
• Reference Standard
• Standard Diluent
• Detection Reagent A
• Detection Reagent B
• Assay Diluent A
• Assay Diluent B
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

Application Details

Application Notes:

• Limited by the current condition and scientific technology, we cannot completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.
• The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
• Kits from different batches may be a little different in detection range, sensitivity and color developing time.
• Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
• Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism.
• There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
• Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
• Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
• Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
• Kits from different manufacturers for the same item might produce different results, since we have not compared our products with other manufacturers.

Comment:

Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.

Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.

Information on antibodies:
The provided antibodies and their host vary in different kits.

Sample Volume: 100 μL

Assay Time: 3 h

Plate: Pre-coated

Protocol: The microplate provided in this kit has been pre-coated with a monoclonal antibody specific to IL12A. Standards or samples are then added to the appropriate microplate wells with a biotin-conjugated monoclonal antibody specific to IL27B. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain IL35, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of IL35 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reagent Preparation:

1. Bring all kit components and samples to room temperature (18-25 °C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.
2. Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 1,000pg/mL. Prepare 7 tubes containing 0.5 mL Standard Diluent and produce a double dilution series. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 1,000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.2pg/mL, 15.6pg/mL, and the last microcentrifuge tube with Standard Diluent is the blank as 0pg/mL.
3. Detection Reagent A and Detection Reagent B - If lyophilized reconstitute the Detection Reagent A with 150μL of Reagent Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B, respectively.
4. Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:

1. Making serial dilution in the wells directly is not permitted.
2. Prepare standards within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for one pipetting.
4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
5. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
6. Contaminated water or container for reagent preparation will influence the detection result.

Assay Precision:

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level 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 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%

Restrictions: For Research Use only

Handling

Precaution of Use: The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

Handling Advice: 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.

Storage: 4 °C

Storage Comment:

• For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4°C.
• For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
  Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
• For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.

Expiry Date: 6 months

References for Interleukin 35 ELISA Kit (ABIN415132)

Yayla, Torgutalp, Okatan, Yurteri, Küçükşahin, Dinçer, Gülöksüz, Sezer, Us, Turgay, Kinikli, Ateş: "Serum Interleukin 35 Levels in Systemic Sclerosis and Relationship With Clinical Features." in: Journal of clinical rheumatology : practical reports on rheumatic & musculoskeletal diseases, Vol. 26, Issue 3, pp. 83-86, (2020) (PubMed).

Massalska, Radzikowska, Kuca-Warnawin, Plebanczyk, Prochorec-Sobieszek, Skalska, Kurowska, Maldyk, Kontny, Gober, Maslinski: "CD4+FOXP3+ T Cells in Rheumatoid Arthritis Bone Marrow Are Partially Impaired." in: Cells, Vol. 9, Issue 3, (2020) (PubMed).

Donninelli, Saraf-Sinik, Mazziotti, Capone, Grasso, Battistini, Reynolds, Magliozzi, Volpe: "Interleukin-9 regulates macrophage activation in the progressive multiple sclerosis brain." in: Journal of neuroinflammation, Vol. 17, Issue 1, pp. 149, (2020) (PubMed).

Zhang, Peng, Ban, Zhang: "Upregulation of Interleukin 35 in Patients With Endometriosis Stimulates Cell Proliferation." in: Reproductive sciences (Thousand Oaks, Calif.), Vol. 25, Issue 3, pp. 443-451, (2018) (PubMed).

Lopez-Abente, Prieto-Sanchez, Muñoz-Fernandez, Correa-Rocha, Pion: "Human immunodeficiency virus type-1 induces a regulatory B cell-like phenotype in vitro." in: Cellular & molecular immunology, Vol. 15, Issue 10, pp. 917-933, (2018) (PubMed).

Kam, Liu, Cai, Mak, Wong, Chiu, Wong, Tsang, Tam: "Synoviocytes-derived Interleukin 35 Potentiates B Cell Response in Patients with Osteoarthritis and Rheumatoid Arthritis." in: The Journal of rheumatology, Vol. 45, Issue 4, pp. 563-573, (2018) (PubMed).

Li, Wang, Liu, Ding, Hao, Shao, Wang, Chen, Huang, Shao, Fu: "Lower level of IL‑35 and its reduced inhibition in Th17 cells in patients with bone marrow mononuclear cells Coombs test‑positive hemocytopenia." in: Molecular medicine reports, Vol. 17, Issue 2, pp. 2973-2981, (2018) (PubMed).

He, Liu, Liu: "Interleukin-35 as a New Biomarker of Renal Involvement in Lupus Nephritis Patients." in: The Tohoku journal of experimental medicine, Vol. 244, Issue 4, pp. 263-270, (2018) (PubMed).

Khoshkhui, Alyasin, Sarvestani, Amin, Ariaee: "Evaluation of serum interleukin- 35 level in children with persistent asthma." in: Asian Pacific journal of allergy and immunology, Vol. 35, Issue 2, pp. 91-95, (2017) (PubMed).

Zhou, Zhang, Chen, Wang, Hou: "Interleukin-35 as a predictor of prostate cancer in patients undergoing initial prostate biopsy." in: OncoTargets and therapy, Vol. 10, pp. 3485-3491, (2017) (PubMed).

Jin, Liu, Lin: "IL-35 may maintain homeostasis of the immune microenvironment in periodontitis." in: Experimental and therapeutic medicine, Vol. 14, Issue 6, pp. 5605-5610, (2017) (PubMed).

Roccatello, Sciascia, Di Simone, Solfietti, Naretto, Fenoglio, Baldovino, Menegatti: "New insights into immune mechanisms underlying response to Rituximab in patients with membranous nephropathy: A prospective study and a review of the literature." in: Autoimmunity reviews, Vol. 15, Issue 6, pp. 529-38, (2016) (PubMed).

Ringkowski, Loke, Huang, Ahmadzai, Herth, Thomas, Herbert: "Enhanced LPS-induced activation of IL-27 signalling in sarcoidosis." in: Respiratory medicine, Vol. 117, pp. 243-53, (2016) (PubMed).

Yin, Ge, Yang, Peng, Lu, Zhang, Wang: "The clinical utility of serum IL-35 in patients with polymyositis and dermatomyositis." in: Clinical rheumatology, Vol. 35, Issue 11, pp. 2715-2721, (2016) (PubMed).

Xiao, Wu, Yin, Han, Ding, Qiao, Lu, Deng, Bo, Gong: "Wogonin Inhibits Tumor-derived Regulatory Molecules by Suppressing STAT3 Signaling to Promote Tumor Immunity." in: Journal of immunotherapy (Hagerstown, Md. : 1997), Vol. 38, Issue 5, pp. 167-84, (2015) (PubMed).

Šenolt, Šumová, Jandová, Hulejová, Mann, Pavelka, Vencovský, Filková: "Interleukin 35 Synovial Fluid Levels Are Associated with Disease Activity of Rheumatoid Arthritis." in: PLoS ONE, Vol. 10, Issue 7, pp. e0132674, (2015) (PubMed).

Tomcik, Zerr, Palumbo-Zerr, Storkanova, Hulejova, Spiritovic, Kodet, Stork, Becvar, Vencovsky, Pavelka, Filkova, Distler, Senolt: "Interleukin-35 is upregulated in systemic sclerosis and its serum levels are associated with early disease." in: Rheumatology (Oxford, England), Vol. 54, Issue 12, pp. 2273-82, (2015) (PubMed).

Yu, Ge, Lu, Shi, Li, Zhang, Wang, Huang, Shao, Huang, Zhang, Nie, Zheng: "Anti-inflammatory effects of interleukin-35 in acquired aplastic anemia." in: Cytokine, Vol. 76, Issue 2, pp. 409-16, (2015) (PubMed).

Sun, Zhang, Yang, Zhang, Lv, Fu, Lv, Liu, Chen, Liu, Huang, Xue, Liu, Zhang, Li, Yang: "Interleukin 35 may contribute to the loss of immunological self-tolerance in patients with primary immune thrombocytopenia." in: British journal of haematology, Vol. 169, Issue 2, pp. 278-85, (2015) (PubMed).

Sun, Kong, Liu, Yu, Tian, Ma, Ji: "The imbalanced profile and clinical significance of T helper associated cytokines in bone marrow microenvironment of the patients with acute myeloid leukemia." in: Human immunology, Vol. 75, Issue 2, pp. 113-8, (2014) (PubMed).

Target Details for Interleukin 35

Target: Interleukin 35 (IL35)

Alternative Name: IL35 (IL35 Products)