EASIA
 
 
Can I useTMB with PeproTech’s EDKs?

A: PeproTech’s EDKs are optimized using ABTS and are therefore best used in conjunction with this substrate. The kit can still be used in combination with TMB, but only after some adjustments have been made:
• The avidin-HRP provided in the kit can not be used with TMB: streptavidin must be purchased separately.
• Dilutions of streptavidin will need to be optimized
• A stop reaction is generally needed when using streptavidin+TMB. Refer to manufacturer’s data sheet.
• The TMB reaction time, prior to the addition of stop solution, will need to be optimized.
• The plate is to be read at 450 nm with a correction at 650 nm when using recommended plates.


 

Why is D-mannitol added to the EDK components?

A: D-mannitol is added to the EDK components in order to aid in protein/antibody visualization. It does not alter ELISA results.


 

Can I use the curve on the EDK data sheet at my standard curve?

A: A separate standard curve must be run on each ELISA plate. In other words, the curve from one plate cannot be used for different plate. The curve that PeproTech provides on the EDK data sheet is for demonstration purpose only, as achieved in PeproTech’s laboratory.


 

What is the stability of the avidin-HRP included in PeproTechs ELISA Development kit? Web

A: The avidin-HRP included in the kit is stable for up to 1 month at 2-8C and for up to 2 years at -20C


 

Is there any step of the ELISA protocol that can be left over the weekend?

A: The plate may be coated with the capture antibody on Friday, left at 4C over the weekend, and resumed on Monday. Please note: changing incubation times may replique rolex datejust cause results to vary between plates.


 

Are PeproTech’s EDKs suitable to use with all sample types?

A: Although PeproTech has not tested all of its kits in every matrix available, they should be suitable for use in, but not limited to: serum, plasma, cell culture supernatant, urine and saliva.


 

Is a stop solution necessary to stop the reaction?

A: A stop solution is not needed when using avidin-HRP+ ABTS. In general, reliable standard curves are obtained when either O.D. reading do not exceed 0.2 units for the zero standard concentrations or 1.2 units for the highest standard concentration. If a stop solution is desired, 1% sodium dodecyl sulfate (SDS) may be used to end the reaction. Stop solutions are not used in PeproTech’s laboratory, though literature has shown that SDS is the optimal stop solution for ABTS.


 

How do I reduce the background in ELISA methods?

A: In general, a high background can be a result of one or more of the following. (Note: Additional suggestions for several procedures are found at the bottom of this document.)
1. Blocking was insufficient. Increase concentration of blocking agent. Increase incubation time with blocking agent. Try alternate blocking agent.
2. Reagents were too concentrated. Dilute primary antibody, secondary antibody, and/or streptavidin or avidin conjugate. Use recommended substrate concentration.
ELISA-specific suggestions for reducing high background:
1. Debris or other component interfered with plate reading. Use care in pipetting into or out of the wells to avoid air bubbles in the wells (break any air bubbles remaining on plate). Increase wash volumes to more effectively remove residual reagents.
2. Blocking was insufficient. Increase the concentration of blocking agent. Try alternate blocking agent.


 

How many plates can I run with one of PeproTech’s ELISA Development Kits (EDKs)?

A: One EDK contains enough material to run 10 standard plates at 100 wells each. This corresponds to 1000 ELISA plate wells.


 

My background is high in my ELISA. How may I lower the background?

A: Our standard procedure usually produces an assay with a background <0.2 OD. If a background higher than 0.2 OD observed, we suggest the following steps:
• Shorten incubation times, especially the substrate incubation. This will result in lowering the over-all signal.
• Decrease the concentration of the streptavidin-HRP conjugate.
• Include a greater number of washing steps.
• Increase soaks time.
• Try a lower binding capacity ELISA plate.
• Increase blocking time or try a alternative blocking agent.
• Optimize the standard diluent buffer formulation by adding additional animal protein.


 

What can cause poor reproducibility of results from ELISAs? How can it be fixed?

A: Cross-contamination of the wells or interfering substances resulted in variable signals. Use care when pipetting into and out of the wells, being careful to avoid air bubbles in the wells (break any air bubbles remaining on plate). Increase wash volumes to more effectively remove residual reagents.


 

How do I develop a sandwich ELISA using Cytosets?

A: Intended Use: These reagents have been prescreened to allow construction of a sandwich enzyme immunoassay (EIA) for the specific and quantitative measurement of cytokines. The enclosed procedures provide general guidelines for EIA preparation. All investigators should be aware that antibody concentrations, sample type, sample matrix, EIA procedure (time, temperature, etc.) can all affect the performance characteristics of the assay and must be optimized. These reagents are intended for research use only. They are not to be used for diagnostic purposes.

PlateCoating:
1. Dilute coating antibody in Coating Buffer to the concentration recommended on the accompanying CytoSetsTM information sheet. Refer to vial label for concentration of coating antibody.
2. Add 100 microliters of diluted Coating Antibody per well to polystyrene microplates. (e.g. Dynex Immulon 2 HB, Catalog Number: 6506 or Nunc Maxisorp, Catalog Number: 468667).
3. Cover the plates and incubate overnight (12 to 18 hours) at 2 - 8°C.
4. Aspirate the coating antibody from the wells and tap on absorbent paper to remove excess liquid.
5. Add 300 microliters of Blocking Solution to each well. Cover the plates and incubate for at least 2 hours at room temperature. If not used immediately, plates may be stored sealed for up to 5 days at 4°C in Blocking Solution (see below).
6. Prior to use, aspirate the Blocking Solution from the wells and tap on absorbent paper to remove excess liquid.
7. Wash the microplate 3 to 6x with 400 microliters per well of Wash Solution (see below) and tap on absorbent paper to remove all excess liquid after the final wash. Do not allow wells to dry completely at any time.

ELISA Method:
1. Add Standards and Samples to Microplate. Dilute standards and samples in Standard Diluent/Assay Buffer (see below) or in the assay matrix most relevant to your samples (e.g.: cell culture medium containing 10% fetal calf serum). Add 100 microliters of standards, samples and controls to appropriate wells in duplicate. Controls can include a reagent blank (Zero Standard Control) and a substrate blank.
Important Note: Some assays may require separate incubation of standards, samples and controls prior to addition of the biotinylated detection antibody (Step #2). Refer to the accompanying CytoSetsTM information sheet for specific instructions under standard incubation recommendations.

2. Add biotinylated detection antibody Dilute biotinylated detection antibody in Standard Diluent/Assay Buffer (see below) to the concentration recommended on the accompanying CytoSetsTM information sheet. Add 50 or 100 microliters of diluted detection antibody (as recommended on CytoSetsTM information sheet) to each well except chromagen blank, cover the plate and incubate for the time and temperature recommended on the accompanying CytoSetsTM information sheet. Aspirate solution from wells. Wash the microplate 3 to 6x with 400 microliters per well of Wash Solution (see below) and tap on absorbent paper to remove all excess liquid after the final wash.

3.Add Streptavidin-Horseradish Peroxidase Conjugate Dilute Streptavidin-HRP conjugate according to the manufacturer’s instructions. Streptavidin-HRP may be diluted in Standard Diluent/Assay Buffer (see below) if not otherwise specified by the manufacturer. Add 100 microliters of diluted Streptavidin-HRP per well, cover the microplate and incubate at room temperature for 15 to 45 minutes. (Note: Dilution and incubation time will vary depending on manufacturer and lot). Aspirate solution from wells. Wash the microplate 3 to 6x with 400 microliters per well of Wash Solution (see below) and tap on absorbent paper to remove all excess liquid after the final wash.

4.Add Substrate Prepare TMB (tetramethyl benzidine) substrate according to manufacturer’s instructions. Add 100 microliters of TMB to each well and incubate in the dark at room temperature for 10 to 60 minutes (generally 30 minutes) according to manufacturer instructions. (Note: Incubation time will vary depending on manufacturer and lot). Stop the reaction by addition of 50 or 100 microliters of Stop Solution (see below) to each well according to manufacturer’s instructions.

5.Read microplate Read microplate at 450 nm within 30 minutes of adding Stop Solution (reference filter: 630 or 650 nm). Calculate the average optical density at 450 nm for all Standards, Controls and Samples. Construct a standard curve by plotting each Standard optical density (ordinate) vs. the Standard concentration (abscissa) on semi-log graph paper. For plate readers with automated standard curve calculation capability, a log-log or four parameter curve fit algorithm may provide the best curve fit. Determine the concentration of each unknown sample from the standard curve.

For lot specific information refer to CytoSetsTM Information Sheet Suggested Solution Formulations All solutions must be prepared immediately prior to use
Coating Buffer A:
8.0 g NaCl
1.42 g Na2HPO4•2H2O
0.2 g KH2PO4
0.2 g KCl q.s. to 1 liter with distilled H2O, pH 7.4
Coating Buffer B:
4.3 g NaHCO3
5.3 g Na2CO3 q.s. to 1 liter with distilled H2O, pH 9.4
Blocking Solution:
8.0 g NaCl
1.42 g Na2HPO4•2H2O
0.2 g KH2PO4
0.2 g KCl
5.0 g bovine serum albumin (fraction V) q.s. to 1 liter with distilled H2O, pH 7.4
Standard Diluent/Assay Buffer:
8.0 g NaCl
1.42 g Na2HPO4•2H2O
0.2 g KH2PO4
0.2 g KCl 5.0 g bovine serum albumin (fraction V)
1 mL Tween 20 q.s. to 1 liter with distilled H2O, pH 7.4
Wash Buffer:
9.0 g NaCl
1 mL Tween 20 q.s. to 1 liter with distilled H2O, pH 7.4
Stop Solution:
1.8 N H2SO4

This procedure contains only recommendations. Investigators are advised to determine optimal buffer formulations, concentrations and incubation times for individual applications.


 

What is the difference between the Cytoscreen ELISA format and the EASIA format?

BioSource offers two types of ready-to-use ELISA kits: EASIA and Cytoscreen. Very similar in concept, both the EASIA and Cytoscreen kits contain all reagents necessary for completing of ELISA (enzyme linked immunosorbant assays).

The descriptions for these two kit formats are as follows: 

Our EASIA (Enzyme Amplified-Sensitivity Immuno Assay) kits contain 96-well plates coated with one or more F(ab)2 monoclonal capture antibodies. The standards in these kits are provided as lyophilized powders containing analyte at discrete levels plus plasma proteins. Standards are reconstituted by adding water. EASIA kits also include lyophilized controls with known levels of analyte in plasma protein. In performing the EASIA, sample, standard or control is pipetted onto the plate. Next, horseradish peroxidase conjugated detection antibody is added to complete a three member solid phase sandwich. Following a washing step, the HRP activity is quantitated by incubation with substrate solution (TMB plus H2O2). At the end of the incubation step, the HRP reaction is terminated by the addition of an acidic stop solution and the optical density of the solution in the wells is measured with a spectrophotometric ELISA plate reader. EASIA of ELISA are validated for measuring human serum, plasma, culture supernatant, and other biological fluids. 

Our Cytoscreen ELISA kits also contain pre-coated 96-well plates. The standard curve is constructed by reconstitution and serial dilution of a single vial of standard using standard diluent buffer provided in the kit. After samples or standards are pipetted into the wells, a biotin-labeled detection antibody is added, followed by a wash step and addition of horseradish-peroxidase(HRP)-conjugated streptavidin to complete a four member solid phase sandwich. As with the EASIA kits, the HRP activity is quantitated by incubation with substrate solution (TMB plus H2O2) followed by an acidic stop solution. The optical density is measured with a spectrophotometric ELISA plate reader. Cytoscreen of ELISA are validated for measuring analytes in serum or plasma, culture supernatant, and other buffered solutions, and are available for quantitation of human, mouse, rat, monkey, and pig samples. Several Cytoscreen kits are available as UltraSensitive formats, which measure sub-picogram levels.


 

Copyrights © 2015 & Gibthai Co., Ltd. All Rights Reserved