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 BioCheck中國(guó)代理, BioCheck上海代理， BioCheck北京代理，BioCheck廣東代理， BioCheck江蘇代理BioCheck湖北代理,BioCheck天津,BioCheck黑龍江代理,BioCheck內(nèi)蒙古代理,BioCheck吉林代理,BioCheck福建代理, BioCheck江蘇代理, BioCheck浙江代理, BioCheck四川代理

BIOCHECK公司由創(chuàng)始人 Dr. John Chen, Medix創(chuàng)立, 是一家抓也提供腫瘤標(biāo)志物，心肌標(biāo)志物，激素類zui高性價(jià)比的抗體的公司。
  

簡(jiǎn)要原理

利用競(jìng)爭(zhēng)酶聯(lián)免疫方法，預(yù)先在微孔中包被羊抗兔抗體，實(shí)驗(yàn)時(shí)先后加入氯霉素標(biāo)準(zhǔn)品或待測(cè)樣本，氯霉素酶標(biāo)抗原和兔抗氯霉素抗體。經(jīng)過(guò)室溫溫育，反應(yīng)液中的兔抗氯霉素抗體與微孔板上的羊抗兔抗體結(jié)合，待測(cè)樣品中的抗原與氯霉素酶標(biāo)抗原競(jìng)爭(zhēng)微孔板上的兔抗氯霉素抗體。洗滌后，沒(méi)有與抗

體結(jié)合的待測(cè)樣品中的抗原或酶標(biāo)抗原被洗去，再加入反應(yīng)底物，結(jié)合的酶標(biāo)抗原的酶將底物轉(zhuǎn)化為藍(lán)色產(chǎn)物，加入終止液后顏色由藍(lán)色變?yōu)辄S色。反應(yīng)完成后，樣品中氯霉素含量越多，反應(yīng)呈色就越淺；反之，樣品中氯霉素含量越少，則呈色越深。利用標(biāo)準(zhǔn)曲線可計(jì)算出樣品中氯霉素含量。

技術(shù)參數(shù)

   檢測(cè)蜂蜜、蛋類、牛奶、奶粉、水產(chǎn)品、動(dòng)物組織(肌肉、肝臟等)、飼料、血清、血漿及尿液樣本中存在的氯霉素，定量限可達(dá)0.025 ppb。

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PD-1 ENZYME IMMUNOASSAY TEST KIT

Catalog Number: BC-1301

Enzyme Immunoassay for the Quantitative Determination of PD- 1 Concentration in Human Serum and Plasma

FOR RESEARCH USE ONLY

Not for use in diagnostic procedures

INTRODUCTION

Programmed cell death protein 1, (PD-1, CD279, PDCD1), is a cell surface receptor that is critical for the regulation of T cell inflammatory activity and maintains peripheral tolerance in the immune system (1). With an extracellular IgV domain, a transmembrane domain, and an intracellular tail with two phosphorylation sites (SHP-1 and SHP-2), PD- 1 is able to interact with ligands PD-L1 and PD-L2 to down-regulate the immune system and suppress T cell activity (2). Upon antigen recognition, an activated T cell expresses PD-1 on its surface and produces interferons, which induces expression of PD-L1, thereby promoting apoptosis or cell death. (3) However when hijacked by tumor cells, aberrant expression of PD-1 ligands inhibits immune-modulatory T cell activation, leading to disease progression (4).

Elevated levels of PD-1 in serum and plasma have been associated with rheumatoid arthritis and skin sclerosis (5,6). Also, PD-1 is predominantly expressed by tumor infiltrating T cells(7). Further research implies that using monoclonal antibodies to target the PD-1 immunologic checkpoint has contributed to breakthrough progress in understanding and treating cancers such as melanoma and other various non-small cell lung cancer (4).

PRINCIPLE OF THE ASSAY

The PD-1 ELISA is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes a unique monoclonal antibody pair directed against a distinct antigenic determinant on the PD-1 molecule. One mouse monoclonal anti- PD- 1 antibody is used for solid phase immobilization (on the microtiter wells). Another mouse monoclonal anti-PD-1 antibody conjugated to horseradish peroxidase (HRP) is in the enzyme conjugate solution. The test samples are allowed to react sequentially with the two antibodies, resulting in the PD-1 molecules to be sandwiched between the solid phase and enzyme-linked antibodies. After two separate 60- minute incubation steps at room temperature, the wells are rinsed with Wash Buffer to remove unbound labeled antibodies. TMB Reagent is added and incubated

for 30 minutes under dark conditions, resulting in the development of a blue color. The color development is stopped with the addition of Stop Solution, changing the color to yellow. The concentration of PD-1 is directly proportional to the color intensity of the test samples. Absorbance is measured spectrophotometrically at 450 nm.

REAGENTS AND MATERIALS PROVIDED

• Antibody-Coated Wells (1 plate, 96 wells)

Microtiter wells coated with mouse monoclonal anti-PD-1

• 50 ng/ml PD-1 Standard (0.5 mL /vial)

50 ng/mL PD-1 in phosphate buffer-BSA solution with preservatives

3. Standard and Sample Diluent (30 mL/bottle, 1 bottle)

Contains phosphate buffer-BSA solution with preservatives

• Enzyme Conjugate Reagent (12 mL/vial, 1 vial)

Contains mouse monoclonal anti-PD-1 conjugated to horseradish peroxidase

• 20X Wash Buffer (50 mL/bottle, 1 bottle) Phosphate buffer with detergents
• TMB Reagent (11 mL/bottle, 1 bottle)

Contains one-step TMB solution

• Stop Solution (11 mL/bottle, 1 bottle)

Contains diluted hydrochloric acid (1N HCl)

STORAGE CONDITIONS

• Store the unopened kit at 2-8°C upon receipt and when it is not in use, until the expiration shown on the kit label. Refer to the package label for the expiration date.
• Keep microtiter plate in a sealed bag with desiccant to minimize exposure to damp air.

REAGENT PREPARATION

• All reagents should be allowed to reach room temperature (18-25°C) before use.
• For each test run, prepare a fresh standard set.
• Dilute 50 ng/mL to 10 ng/mL with Standard/Sample diluent. Prepare two-fold serial dilutions of the 10 ng/mL Standard with Standard/Sample Diluent:
• 10 ng/mL: 0.12 mL of 50 ng/mL + 0.48 mL of Standard/Sample Diluent
• 5 ng/mL: 0.25 mL of 10 ng/mL+ 0.25 mL of Standard/Sample Diluent
• 2.5 ng/mL: 0.25 mL of 5 ng/mL+ 0.25 mL of Standard /Sample Diluent
• 1.25 ng/mL: 0.25 mL of 2.5 ng/ml + 0.25 mL of Standard/Sample Diluent
• 0.625 ng/mL: 0.25 mL of 1.25 ng/mL + 0.25 mL of Standard/Sample Diluent
• 0.313 ng/mL: 0.25 mL of 0.625 ng/mL + 0.25 mL of Standard/Sample Diluent
• 0.156 ng/mL: 0.25 mL of 0.313 ng/mL + 0.25 mL of Standard Diluent
• 0 ng/mL: 0.25 mL of Standard Diluent
• Patient samples need to be diluted 4-fold prior to use. Prepare a series of small tubes (i.e., 1.5 mL microcentrifuge tubes) and mix 60 µL of serum with 180 µLStandard/Sample Diluent.
• Working Wash Buffer: Preparation of 1X Wash Buffer from 20X Stock. Add 50 mL of 20X Wash Buffer Stock to 950 mL of DI H2O. The Working Wash Buffer is stable at 2-8°C for 30 days. NOTE: Any crystals that may be present due to high salt concentration must be redissolved at room temperature before making the dilution.

ASSAY PROCEDURE

• Prepare Standards. See Reagent Preparation.
• Dilute samples 1:4 dilution. See Reagent Preparation.
• Secure the desired number of coated wells in the holder.
• Dispense 100 mL of PD-1 standards, and DILUTED specimens into the appropriate wells.
• Incubate for 60 minutes at room temperature (18-25 °C).
• Remove incubation mixture by flicking plate contents into a waste container. Rinse and flick the microtiter wells 5 times with 300 m L Working Wash Buffer. Strike the wells onto absorbent paper or paper towels to remove all residual water droplets.
• Dispense 100 mL of PD-1 Working Enzyme Conjugate Reagent into each well.
• Incubate for 60 minutes at room temperature (18-25 °C).
• Remove incubation mixture by flicking plate contents into a waste container. Rinse and flick the microtiter wells 5 times with 300 uL Working Wash Buffer. Strike the wells onto absorbent paper or paper towels to remove all residual water droplets.
• Dispense 100 mL TMB solution into each well.
• Incubate for 30 minutes at room temperature (18-25 °C).
• Stop the reaction by adding 100 mL of Stop Solution into each well.
• Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color compley.
• Read absorbance at 450 nm with a microtiter well reader within 15 minutes.

CALCULATION OF RESULTS

• Calculate the mean absorbance value (OD₄₅₀) for each set of reference standards, controls and samples.
• Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in ng/ml on graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis.
• The corresponding concentration of PD-1 (ng/mL) can be determined from the standard curve using the mean absorbance value for each sample. Depending on experience and/or the availability of computer capability, other methods of

data reduction may be employed.

• The obtained values of the samples should be multiplied by the dilution factor of 4 to obtain PD-1 results in ng/ml.

EXAMPLE OF STANDARD CURVE

Results of a typical standard run with absorbency readings at 450 nm shown on the Y axis against PD -1 concentrations shown on the X axis. NOTE: This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each laboratory must generate its own data and standard curve in each experiment.

PERFORMANCE CHARACTERISTICS

• Sensitivity

The minimum detectable concentration of the PD-1 ELISA assay as measured by 2SD from the mean of a zero standard is estimated to be 0.15 ng/ml.

• Precision

• Intra-Assay Precision

Within-run precision was determined by replicate determinations of three different samples in one assay. Within-assay variability is shown below:

2

• Inter-Assay Precision

Between-run precision was determined by replicate measurements of three different samples over a series of individually calibrated assays. Between-assay variability is shown below:

• Recovery and Linearity Studies

• Recovery

Samples were spiked with known PD-1 levels and assayed in duplicate. The mean recovery was 105.3%.

• Linearity

Three samples were serially diluted to determine linearity. The mean recovery was 99.6%.

Mean = 97.4%

REFERENCES

• Fife, B. T., & Pauken, K. E. (2011). The role of the PD-1 pathway in autoimmunity and peripheral tolerance. Annals of the New York Academy of Sciences, 1217(1), 45-59.
• Riella, L. V., Paterson, A. M., Sharpe, A. H., & Chandraker, A. (2012). Role of the PD-1 Pathway in the Immune Response. American Journal of Transplantation?: Official Journal of the American Society of

Transplantation and the American Society of Transplant

Surgeons, 12(10), 2575–2587.

• Zak, Krzysztof & Ki, Rados?aw & Przetocka, Sara & Golik, Przemys?aw & Guzik, Katarzyna & Musielak, Bogdan & Dömling, Alexander & Dubin, Grzegorz & Holak, Tad. (2015). Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1. Structure. 23. . 10.1016/j.str.2015.09.010.
• Zoran Gatalica, Carrie Snyder, Todd Maney, Anatole Ghazalpour, Daniel Holterman, Nianqing Xiao, Peggy Overberg, Inga Rose, Gargi D. Basu, Semir Vranic, Henry T. Lynch, Daniel D. Von Hoff and Omid Hamid. Programmed Cell Death 1 (PD-1) and Its Ligand (PD-L1) in Common Cancers and Their Correlation with Molecular Cancer Type.

Cancer Epidemiol Biomarkers Prev. December 1 2014 (23) (12) 2965-2970.

• Greisen, S., Rasmussen, T., Stengaard-Pedersen, K., Hetland, M., Hørslev-Petersen, K., Hvid, M., & Deleuran, B. (2013). Increased

soluble programmed death-1 (sPD-1) is associated with disease activity and radiographic progression in early rheumatoid arthritis. Scandinavian Journal of Rheumatology,43(2), 101-108.

• Yanaba, K., Hayashi, M., Yoshihara, Y., & Nakagawa, H. (2016). Serum levels of soluble programmed death-1 and programmed death ligand-1 in systemic sclerosis: Association with extent of skin sclerosis. The Journal of Dermatology,43(8), 954-957.
• Ahmadzadeh, M., Johnson, L. A., Heemskerk, B., Wunderlich, J. R., Dudley, M. E., White, D. E., & Rosenberg, S. A. (2009). Tumor antigen–specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood, 114(8), 1537–1544.

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