Intended use

This Avastin ELISA kit is intended Laboratory for Research use only ａnd is not for use in diagnostic or therapeutic procedures.The S Solution changes the color from blue to yellow ａnd the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of Avastin in the sample, this Avastin ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples ａnd allow the operator to produce a standard curve of Optical Density versus Avastin concentration. The concentration of Avastin in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Sample collection ａnd storages

1. Can’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.

2.  Extract as soon as possible after Specimen collection, Extracted according to the relevant literature.

Cell culture supernates ａnd plant exact fluids - Remove particulates by centrifugation and assay immediately or aliquot ａnd store samples at -20°C or -80°C. Avoid repeated freeze-thaw.

Materials required but not supplied

1.  Standard microplate reader(450nm)

2.  Precision pipettes ａnd Disposable pipette tips.

3.  37 ℃ incubator

Precautions

1.  Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.  Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

3.  Mix all reagents before using.

Remove all kit reagents from refrigerator ａnd allow them to reach room temperature ( 20-25°C)

Materials supplied

Note: Standard (S0 → S5) concentration was followed by: 0,0.5,1,2,4,8 ppb

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.

2.  Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.

3.  Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing sample well; Blank well doesn’t add anyting.

4.  Add 50μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C. 

5.  Aspirate each well ａnd wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate ａnd blot it against clean paper towels.

6.  Add chromogen solution A 50μl ａnd chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

7.  Add 50μl S Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not

appear uniform, gently tap the plate to ensure thorough mixing.

8.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

Calculation of results

1.  Calculate the mean absorbance value A450 for each set of reference standards ａnd samples.

2.  Divide the average A450 value for each standard, control ａnd test sample by the average A450 of standard0 ａnd multiply by 100 to obtain %B/B0 for each sample.

3.  Prepare a standard curve by plotting the average absorbance of each standard versus the corresponding concentrations of the standards on linear-log graph paper or the %B/B0 value for each standard versus the corresponding concentration of the standard on linear-log or logit-log graph paper. logit=ln(B/ B0)/(1－B/ B0)

4.  Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.

5.  The standard density is a X, the B/ B0 is a Y, sitting to mark the paper in the logit- log up draw a standard curve. According to the B/ B0 that need to be measured the sample can from sit to mark the density value that the paper looks up the sample up.

6.  The sensitivity by this assay is 0.25ppb

7.  The standard curve presented here is an example of the data typically produced with this kit; however, your results will not be identical to these.

8.  Standard curve match

Storage：  2-8℃.

validity： six months.

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!

原创作者：上海隆诚生物科技有限公司