Human Aβ42 (Amyloid Beta 42) ELISA Kit
Catalogue No: EH2685
Size: 48T/96T
Reactivity: Human
Detection Range: 15.625-1000pg/ml
Sensitivity:
Application: For quantitative detection of Aβ42 in serum, plasma, tissue homogenates and other biological fluids.
Storage: 4°C for 6 mo
NOTE: FOR RESEARCH USE ONLY.
Kit Compo
Item Specifications(48T/96T) Storage
ELISA Microplate(Dismountable) 8×6 /8×12 4°C/-20°C
Lyophilized Standard 1 vial/2 vial 4°C/-20°C
Sample / Standard Dilution Buffer 10ml/20ml 4°C
Biotin-labeled Antibody (Concentrated) 60ul/120ul 4°C
Antibody Dilution Buffer 5ml/10ml 4°C
HRP-Streptavidin Co
SABC Dilution Buffer 5ml/10ml 4°C
TMB Substrate 5ml/10ml 4°C(shading light)
Stop Solution 5ml/10ml 4°C
Wash Buffer (25X) 15ml/30ml 4°C
Plate Sealer 3/5pieces
Product Des
Principle of the Assay
This kit was ba
antibody were added to the wells subsequently, and washed with wash buffer. HRP- Streptavidin was added and unbound co
O.D. absorbance at 450nm in a microplate reader, and then theco
Precautions
1. To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using standards and a small number of samples is recommended.
2. After opening and before using, keep plate dry.
3. Before using the Kit, spin tubes and bring down all compo
4. Storage TMB reagents avoid light.
5. Washing process is very im
6. Duplicate well assay is recommended for both standard and sample testing.
7. Don’t let Micro plate dry at the assay, for dry plate will inactivate active compo
8. Don’t reuse tips and tubes to avoid cross contamination.
9. Avoid using the reagents from different batches together.
Material Required but Not Supplied
1. Microplate reader (wavelength: 450nm)
2. 37°C incubator
3. Automated plate washer
4. Precision single and multi-channel pipette and disposable tips
5. Clean tubes and Eppendorf tubes
6. Deio
Manual Washing
Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other absorbent material. Fill each well completely with 350ul wash buffer and soak for 1 to 2 minutes, then aspirate co
filter papers or other absorbent material. Repeat this procedure two more times for a total of THREE washes.3
Automatic Washing
Aspirate all wells, and then wash plate THREE times with 350ul wash buffer. After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer shall be set for soaking 1 minute.
Sample Collection and Storage
Isolate test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20°C for long term. Avoid multiple freeze-thaw cycles.
l Serum: Place whole blood sample at room temperature for 2 hours or put it at 4°C overnight and centrifugation for 20 minutes at approximately 1000×g, Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.
l Plasma: Collect plasma using EDTA-Na2 as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, high cholesterol samples.
l Tissue Homogenates: As hemolysis blood has relation to assay result, it is necessary to remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Mince tissue after weighing it and get it homogenized in PBS (the volume depends on the weight of the tissue. Generally speaking, 9mL PBS would be appropriate to 1 gram tissue
pieces. Some protease inhibitors are recommended to add into the PBS) with a glass homogenizer on ice. To further break the cells, you can so
l Cell Culture supernate: Centrifuge supernatant for 20 minutes at 1000×g at 2 - 8°C to
remove insoluble impurity and cell debris. Collect the clear supernate and carry out the assay immediately.
l Other Biological Fluids: Centrifuge samples for 20 minutes at 1000×g at 2-8°C. Collect supernatant and carry out the assay immediately.
l Sample Preparation: Samples shall be clear and transparent and remove suspended solids by centrifugation.
Note: Samples to be used within 5 days can be stored at 4°C, besides that, samples must be stored at -20°C (assay ≤1 month) or -80°C(assay≤2 months) to avoid loss of bioactivity and contamination. Hemolyzed samples are not suitable for this assay.
Sample Dilution Guideline
End user should estimate the co
solutions are for reference only:
l High co
l Medium co
l Low co
l Very low co
Reagent Preparation and Storage
Put the kit at room temperature for 20 minutes before use
1, Wash Buffer:
Dilute 30mL Co
until the crystals have completely been dissolved. The solution should be cooled to room temperature before use.
2, Standard:
1). 1000pg/ml of standard solution: Add 1 ml Sample / Standard dilution buffer into one Standard tube, keep the tube at room temperature for 10 minutes and mix them thoroughly.
2).500pg/ml→15.625pg/ml of standard solutions: Label 6 Eppendorf tubes with 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml, respectively. Add 0.3 ml of the Sample/Standard dilution buffer into each tube. Add 0.3 ml of the above 1000pg/ml standard solution into 1st tube and mix them thoroughly. Transfer 0.3 ml from 1st tube to 2nd tube and
mix them thoroughly. Transfer 0.3 ml from 2nd tube to 3rd tube and mix them thoroughly, and so on.
Note: It is best to use Standard Solutions within 2 hours. The Standard Solution shall be at 4°C up to 12 hours. Or store it at -20 °C up to 48 hours. Avoid repeated freeze-thaw cycles.5
3, Preparation of Biotin-labeled Antibody Working Solution
Prepare it within 1 hour before experiment.
1) Calculate required total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume)
2) Dilute the Biotin-detection antibody with Antibody Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl Biotin-labeled antibody into 99μl Antibody Dilution Buffer.)
4, Preparation of HRP-Streptavidin Co
Prepare it within 30 minutes before experiment.
1) Calculate required total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume)
2) Dilute the SABC with SABC Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl of SABC into 99μl of SABC Dilution Buffer.)
Assay Procedure
Before adding reagents into wells, equilibrate TMB Substrate for 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
1. Set standard, test sample and co
2. Aliquot 0.1ml of 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml, standard solutions into the standard wells.
3. Add 0.1 ml of Sample/Standard Dilution Buffer into the co
4. Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids) into test sample wells.
5. Seal the plate with a cover and incubate at 37 °C for 90 minutes.
6. Remove the cover and discard the plate content, and wash plate 2 times with Wash Buffer. Do NOT let the wells dry completely at any time.
7. Add 0.1 ml Biotin-labeled antibody working solution into above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the sidewall.
8. Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash Buffer, and let the wash buffer stay in the wells for 1 minute each time.
10. Add 0.1 ml of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.6
11. Remove the cover and wash plate 5 times with Wash Buffer, and let the wash buffer stay in the wells for 1-2 minute each time.
12. Add 90μl TMB Substrate into each well, cover the plate and incubate at 37°C in dark within 15-30 minutes. (Note: This incubation time is for reference only, end user shall determine the optimal time.) It will turn blue in the first 3-4 wells (with most co
13. Add 50μl Stop Solution into each well and mix them thoroughly. The color changes to yellow immediately.
14. Read the O.D. absorbance at 450 nm in Microplate Reader immediately after adding the stop solution. Regarding calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zero
well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective co
software curve expert to 1.3, please visit: http://www.givei.cn
Note: If the samples measured were diluted, multiply the dilution factor to the co
Summary
1. Wash plate 2 times before adding standard, sample and co
2. Add 100μL standard or sample to each well and incubate for 90 minutes at 37°C
3. Aspirate and wash plates 2 times
4. Add 100μL Biotin-labeled antibody working solution to each well and incubate for 60 minutes at 37°C
5. Aspirate and wash plates 3 times
6. Add 100μL SABC Working Solution into each well and incubate for 30 minutes at 37°C
7. Aspirate and wash plates 5 times
8. Add 90μL TMB Substrate. Incubate 15 -30 minutes at 37°C
9. Add 50μL Stop Solution. Read at 450nm immediately
10. Calculation
Typical Data & Standard Curve
Results of a typical standard operation of a Aβ42 ELISA Kit are listed below. This standard curve was generated at our lab for demo
X pg/ml 0 15.625 31.25 62.5 125 250 500 10007
Y OD450 0.046 0.097 0.178 0.28 0.542 0.944 1.578 2.125
Specificity
This assay has high sensitivity and excellent specificity for detection of Aβ42 . No significant cross-reactivity or interference between Aβ42 and analogues was observed.
Note: Limited by current skills and knowledge, it is difficult for us to complete the cross- reactivity detection between Aβ42 and all the analogues, therefore, cross reaction may still exist.
Recovery
Matrices listed below were spiked with certain level of Aβ42 and the recovery rates were calculated by comparing the measured value to the expected amount of Aβ42 in samples. Matrix Recovery Range (%) Average (%) Serum(n=5) 89-99 94
EDTA Plasma(n=5) 91-99 94
Heparin Plasma(n=5) 91-101 958
Linearity
The linearity of the kit was assayed by testing samples spiked with appropriate co
Sample 1:2 1:4 1:8 1:16
Serum(n=5) 87-104% 88-102% 85-104% 88-98%
EDTA Plasma(n=5) 83-99% 83-97% 88-101% 84-96%
Heparin Plasma(n=5) 87-97% 83- 84- 81-99%
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Aβ 42 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Aβ 42 were tested on 3 different plates, 8 replicates in each plate.
CV (%) = SD/meanX100
Intra-Assay: CV<8%
Inter-Assay: CV<10%
Stability
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 10% within the expiration date under appropriate storage condition. Standard(n=5) 37°C for 1 mo
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