Human TNF-β(Tumor Necrosis Factor Beta) ELISA Kit

I. Product Information：

This assay operates on the Sandwich ELISA principle. A microplate pre-coated with a Human TNF-β-specific capture antibody is provided. Following the addition of standards or samples, the target analyte binds to the immobilized antibody. The plate is then incubated sequentially with a biotinylated anti-Human TNF-β detection antibody and an Avidin-HRP conjugate. After washing, a substrate solution is added. A blue color develops exclusively in wells containing the complete complex (Human TNF-β, biotinylated antibody, and Avidin-HRP). The reaction is stopped with an acid solution, changing the color to yellow. Absorbance (OD) is measured at 450 nm ± 2 nm. The OD value correlates directly with Human TNF-β concentration, which is determined by interpolating sample OD values from a standard curve.

II.Sample Testing Protocol:

1. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in thosesamples and finally lead to wrong results.

2. We are only responsible for the kit itself, but not for the samples consumedduring the assay. The users should calculate the possible amount of thesamples used in the whole test.Please reserve sufficient samples in advance.

3. The detection range of the kit is not equivalent to the concentration range ofthe analyte in the sample.It is recommended to consult reference,conductpreliminary experiments,or seek technical support to estimate theconcentration of the analyte in your sample.If the analyte concentration in thesample is too high or too low,appropriate dilution or concentration of thesample should be performed.

4. If the sample type is not included in the manual,a preliminary experiment issuggested to verify the validity.

III. Reagent preparation：

1. Bring all reagents to room temperature(18-25℃) before If the kit will notbe used up in one assay, please only take out the necessary strips and reagentsfor present experiment,and store the remaining strips and reagents at requiredcondition.

2. Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer with 720 mL ofdeionized or distilled water to prepare 750 mL of Wash Buffer.Note:if crystals have formed in the concentrate, warm it in a 40℃ water bath and mixit gently until the crystals have completely dissolved.

3. Standard working solution:

① Centrifuge: Centrifuge the standard at 10,000×g for 1 min.

② Add 1mL of Reference Standard &Sample Diluent,let it stand for 10 minand invert it gently several times. After it dissolves fully, mix it thoroughlywith a pipette. This reconstitution produces a working solution of 1000pg/mL.
③ Serial dilution:Take 7 EP tubes,adding 250μL of reference standard&sampledilution buffer to each tube(The volume can be adjusted based on actual usage,e.g.,500μL/tube). Transfer 250μL of the 1000pg/mL standard working solution into thefirst tube and mix thoroughly to obtain the 1000pg/mL standard working solution.Continue the dilution step by step until the second-to-last tube. The last tube willserve as the blank, and no solution should be transferred from the second-to-lasttube. The standard working solution should be freshly prepared and usedimmediately.

4. Biotinylated Detection Ab working solution: Calculate the required amountbefore the experiment(100μL/well). In preparation,slightly more thancalculated should be prepared. Centrifuge the Concentrated Biotinylated Detection Ab at 800×g for 1 min, then dilute the 100×Concentrated Biotinylated Detection Ab to 1×working solution with Biotinylated Detection Ab Diluent (Concentrated Biotinylated Detection Ab: Biotinylated DetectionAb Diluent=1:99).

5. HRP Conjugate working solution: Calculate the required amount before theexperiment (100μL/well). In preparation,slightly more than calculated shouldbe prepared. Centrifuge the Concentrated HRP Conjugate at 800×g for 1 min, then dilute the 100×Concentrated HRP Conjugate to 1×working solution with HRP Conjugate Diluent (Concentrated HRP Conjugate:HRP Conjugate Diluent=1:99).

6. Experimental Operation Tips

① Solutions should be added to the bottom of the micro ELISA plate well,avoid touching the inside wall and causing foaming as much as possible.

② Make the tested strips in use immediately after the wash step.Do not allowwells to be dry.

③ After adding the Substrate Reagent.The reaction time can be shortened orextended according to the actual color change,but not more than 30 min.

④ Adding the stop solution should be done in the same order as the substratesolution.

IV. Assay Procedure:

V. Notice:
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