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1600/48T; 2600/9-Human IL-6 elisa試劑盒
  • 1600/48T; 2600/9-Human IL-6 elisa試劑盒
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地: 北京市通州區馬駒橋聯東U谷中區

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Human IL-6 elisa試劑盒
索萊寶ELISA試劑盒的優勢:
1,包被的酶標板單板可拆(能拆分成12個8孔的酶標條)
2,提供免費代測代檢服務,代出實驗數據
3,發文章高獎勵
4,磁鐵可吸附式包裝盒,包裝精美耐用

For research use only. Not for use in diagnostic procedures.

Human IL-6 elisa試劑盒

                                  MANUFACTURED AND DISTRIBUTED BY:

CountryCompany: ChinaBeijing Solarbio Science & Technology Co., Ltd

Address: NO.8, Liandong U Valley, Tongzhou District, Beijing, P.R.China.

     86-10-56371241    86-10-56371282    : service@solarbio.com

 

TABLE OF CONTENTS

 

SECTION                                          PAGE                                                                   

BACKGROUND...........................................................................................3

PRINCIPLE OF THE ASSAY......................................................................3

TECHNICAL HINTS AND LIMITATIONS...............................................4

PRECAUTIONS............................................................................................4

KIT COMPONENTS& STORAGE CONDITIONS.....................................5

OTHER SUPPLIES REQUIRED BUT NOT SUPPLIED............................6

SPECIMEN COLLECTION & STORAGE..................................................6

REAGENTS PREPARATION......................................................................6

ASSAY PROCEDURE .................................................................................8

CALCULATION OF RESULTS...................................................................8

PERFORMANCE CHARACTERISTICS....................................................10

REFERENCES..............................................................................................12

BACKGROUND  Human IL-6 elisa試劑盒

IL-6 is an interleukin that acts as both a pro-inflammatory and anti-inflammatory cytokine and is produced by T cells, macrophages, fibroblasts, osteoblasts, endothelial and other cells. IL-6 induces proliferation and differentiation and acts on B cells, T cells, thymocytes, and others. IL-6 is one of the most important mediators of fever and of the acute phase response.IL-6 can be secreted by macrophages in response to specific microbial molecules, referred to as pathogen associated molecular patterns (PAMPS). IL-6 in concert with TGF β is important for developing Th17 responses. IL-6 is relevant to many disease processes such as diabetes, atherosclerosis, depression, Alzheimer's disease, systemic lupus erythematosus, prostate cancer, breast cancer, and rheumatoid arthritis.

 

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-6 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-6 present is captured by the coated antibody after incubation. Following extensive washing, a biotin-conjugate antibody specific for IL-6 is added to detect the captured IL-6 protein in sample. For signal development, horseradish peroxidase (HRP)-conjugated Streptavidin is added, followed by Tetramethyl-benzidine (TMB) reagent. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Solution containing sulfuric acid is used to stop color development and the color intensity which is proportional to the quantity of bound protein is measurable at 450nm.

TECHNICAL HINTS AND LIMITATIONS

Human IL-6 elisa試劑盒

1.        This Solarbio ELISA should not be used beyond the expiration data on the kit label.

2.        To avoid cross-contamination, use a fresh reagent reservoir and pipette tips for each step.

3.        To ensure accurate results, some details, such as technique, plasticware and water sources should be emphasized.

4.        A thorough and consistent wash technique is essential for proper assay performance.

5.        A standard curve should be generated for each set of samples assayed.

6.        It is recommended that all standards and samples be assayed in duplicate.

7.        Avoid microbial contamination of reagents and buffers. Buffers containing protein should be made under aseptic conditions and be prepared fresh daily.

8.        In order to ensure the accuracy of the results, the standard curve should be made every time.

PRECAUTIONS

The Stop Solution suggested for use with this kit is an acid solution. Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling.

KIT COMPONENTS& STORAGE CONDITIONS

 

PART

SIZE

STORAGE OF OPENED/ RECONSTITUTED MATERIAL

Microwell Plate - antibody coated 96-well Microplate (8 wells ×12 strips)

1 plate

Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 1 month at 2 – 8℃**

Standard - lyophilized,1000 pg/ml upon reconstitution

2 vials

Aliquot and Store at -20°C** for six months

Concentrated Biotin-Conjugated antibody(100X) - 120 ul/vial

1 vial

Store at 2-8°C **for six months

Concentrated Streptavidin-HRP solution(100X) - 120 ul/vial

1 vial

Store at 2-8°C** for six months

Standard /sample Diluent - 16 ml/vial

1 bottle

Store at 2-8°C** for six months

Biotin-Conjugate antibody Diluent - 16 ml/vial

1 bottle

Store at 2-8°C** for six months

Streptavidin-HRP Diluent - 16 ml/vial

1 bottle

Store at 2-8°C** for six months

Wash Buffer Concentrate (20x) - 30 ml/vial

1 bottle

Store at 2-8°C** for six months

Substrate Solution - 12 ml/vial

1 bottle

Store at 2-8°C** for six months

Stop Solution - 12 ml/vial

1 bottle

Store at 2-8°C** for six months

Plate Cover Seals

 4 pieces

 

 

**Provided this is within the expiration date of the kit.

OTHER SUPPLIES REQUIRED BUT NOT SUPPLIED

1.        Microplate reader capable of measuring absorbance at 450 nm.

2.        Pipettes and pipette tips.

3.        Deionized or distilled water.

4.        Squirt bottle, manifold dispenser, or automated microplate washer.

5.        500 mL graduated cylinder.

6.        Human IL-6 controls (optional; available from Solarbio).

SPECIMEN COLLECTION & STORAGE

Cell Culture Supernates - Centrifuge cell culture media at 1000×g to remove debris. Assay immediay or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Serum - Use a serum separator tube (SST) and allow samples to clot for 2 hours at room temperature or overnight at 2-8℃. Centrifuge at approximay for 15 minutes at 1000×g. Assay immediay or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Plasma - Collect plasma using EDTA, heparin, or citrate as an anticoagulant. Centrifuge for 15 minutes at 1000×g within 30 minutes of collection. Assay immediay or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Note: The normal human serum or plasma samples are suggested to make a 1:2 dilution.

 

REAGENTS PREPARATION

1.        Temperature returning - Bring all kit components and specimen to room temperature (20-25℃) before use.

2.        Wash Buffer - Dilute 30mL of Wash Buffer Concentrate with 570mL of deionized or distilled water to prepare 600mL of Wash Buffer. If crystals have formed in the concentrate Wash Buffer, warm to room temperature and mix gently until the crystals have compley dissolved.

3.        StandardSpecimen - Reconstitute the Standard with 1.0mL of deionized or distilled water. This reconstitution produces a stock solution of 1000pg/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Pipette 500mL of Standard/Specimen Diluent into 500pg/ml tube and the remaining tubes. Use the stock solution of 1000pg/mL to produce a 2-fold dilution series (below). Mix each tube thoroughly and change pipette tips between each transfer. The 500pg/mL standard serves as the high standard. The Standard/specimen Diluent serves as the zero standard (0pg/mL).

*If you do not run out of re-melting standard, store it at -20. Diluted standard shall not be reused.

4.        Working solution of Biotin-Conjugate anti-human IL-6 antibody: Make a 1:100 dilution of the concentrated Biotin-Conjugate solution with the Biotin-Conjugate antibody Diluent in a clean plastic tube.

*The working solution should be used within one day after dilution.

5.        Working solution of Streptavidin-HRP: Make a 1:100 dilution of the concentrated Streptavidin-HRP solution with the Streptavidin-HRP Diluent in a clean plastic tube.

*The working solution should be used within one day after dilution.

Preparation of IL-6 standard dilutions

CALCULATION OF RESULTS

1.        The standard curve is used to determine the amount of specimens.

2.        First, average the duplicate readings for each standard, control, and sample. All O.D. values are subtracted by the mean value of blank control before result interpretation.

3.        Construct a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph.

4.        The data may be linearized by plotting the log of the IL-6 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

5.        This standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed.

Performance Characteristics

SENSITIVITY: The minimum detectable dose was 4pg/mL.

SPECIFICITY: This assay recognizes both natural and recombinant human IL-6. The factors listed below were prepared at 100ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.

 

 Factors assayed for cross-reactivity

Recombinant human

Recombinant mouse

Recombinant porcine

IL-6 sR?

IL-6

CNTF

IL-6 sR/sgp130

IL-10

G-CSF

IL-9

IL-11

IL-6

IL-10

IL-12

OSM

REPEATABILITY: The coefficient of variation of both intra-assay and inter-assay were less than 10%.

RECOVERYThe recovery of IL-6 spiked to three different levels in four samples throughout the range of the assay in various matrices was evaluated.

 

 Recovery of IL-6 in two matrices 

Sample Type

Average % of Expected Range (%)

Range (%)

Citrate plasma

86

82-91

Cell culture supernatants

95

90-100

LINEARITY: To assess the linearity of the assay, three samples were spiked with high concentrations of IL-6 in various matrices and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The plasma samples were initially diluted 1:1)

 The linearity of the assay

Dilution ratio

Recovery (%)

Citrate plasma

Cell culture supernatants

1:2

Average% of Expected

91

94

Range (%)

85-97

86-103

1:4

Average% of Expected

98

96

Range (%)

89-108

90-103

1:8

Average% of Expected

103

101

Range (%)

97-110

93-110

1:16

Average% of Expected

106

107

Range (%)

100-113

99-115

REFERENCES

1.        Hirano, T. et al. (1984) J. Immunol. 133:798.

2.        Kikutani, H. et al. (1985) J. Immunol. 134:990.

3.        Hirano, T. et al. (1986) Nature 324:73.

4.        Haegeman, G. et al. (1986) Eur. J. Biochem. 159:625.

5.        Van Snick, J. et al. (1988) Eur. J. Immunol. 18:193.

6.        Lotz, M. et al. (1988) J. Exp. Med. 167:1253.

7.        Hirano, T. et al. (1990) Immunol. Today 11:443.

8.        Hirano, T. et al. (1990) in Peptide Growth Factors and their Receptors I, Sporn, M.B.and A.B.Roberts eds., Springer-Verlag, New York, p. 663.

9.        Kishimoto, T. et al. (1992) Science 258:5593.

10.   Hirano, T. (1992) Clin. Immunol. Immunopathol. 62:S60.

 

 

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