Is there any difference between using an eppindorf plastic tube and using a glass tube for diluting the antibodies and the conjugate that are using in ELISA test? I have heard that if I use a plastic tube, that could bind proteins to the tube? Is there anyone has experienced such difference?
Thanks.
Polystyrole tubes will absorb small amounts of protein (about 100 ng/cm²). To avaoid even this, you have normally in your sample buffern an inert protein (like BSA) and a detergent. The last one will bind with his hydrophobic tail onto the syrole rings like in any ELISA plate.
Polyehylene or -propylen have a much lower protein binding capacity. So you can use them without any problems.
From my experience, i'd avoid using glass! Use disposable CLEAN plastic tubes and tips for every ELISA assay you do. I had many problems of my TMB substrate changing color in glass tubes.
Eppendorf, Corning and other manufacturers sell low-protein-binding plastic consumables as well as their regular line. I have never used glass, and I would be concerned about its cleanliness, compared to using brand new, disposable tools.
There is a technique, blank pipetting, by which you aspirate the fluid you are pipetting one or two times in a new tip before doing the transfer. This is in case proteins you are interested in are binding to the internal walls of the tip & reducing concentration in the expelled volume. Aspirating gently once or twice equilibrates the concentrations in the tip with those in your stock solution. I'm not sure this is a particularly big effect, but something to consider if you are worried about losing molecules.
The buffer you are using for diluting antibody and enzyme conjugate if it contains inert protein in the concentration of 0.1% to 0.3% or with 0.01% (v/v) of tween-20. Then you need not to worry about adherence of antibody and enzyme conjugate on the wall of either glass or plastic vials. If you are using plastic vials use low protein binding vials.
If you are worried a lot about protein binding to the tubes' walls, you can use low-binding protein eppendorf tubes. Do not use glass for this sensitive assay!
Tubes have a great impact on ELISA values if you are measuring at lower concentrations (5-100 ng/ml). An Eppendorf tube can non-specifically bind your protein and make it even disappear from detection. However, this only occurs if you have a buffer system like PBS without protein support. In general, you should use a protein-based diluent such as 1-5% BSA to make your dilutions and the tube effect will not apply. The diluent of course will depend on assay compatibility and should be chosen carefully. Glass is even worse than plastic in non-specific binding of proteins when using protein-free buffers. Low protein binding tubes definitively will help to reduce the issue.
yes, there are big differences. Though glas was used in the past and some might think that it is inert, it is not and can cause big differences. Glas is used for chemical applications and chemical analytes, but even then it is often recommended to use silanized glas or similar. For proteins (antigens, antibodies) it is highly recommended not to use gals, but PP tubes. Gals and PS tubes may bind high amounts on proteins depending on their polarity and so influence the concentration and test results. I have hands-on experience with a test, where the enzyme-conjugate (POD) binds to the surface and the test did not work at all.
There are special low bindung tubes available, especially for DNA-tests (primer, enzymes, controls). These or the PP tubes are generally used in the test kits for critical substances, PS only for non-critical like buffers.
In addition, tubes for sample preparation should always be PP tubes. Normal glas tubes may bind/adsorb the analytes; silanized glas could be an alternative, but is alwaysa higher hazard if shipped.
Thank you so much Oliver for sharing your own experience in such issue.
Polystyrole tubes will absorb small amounts of protein (about 100 ng/cm²). To avaoid even this, you have normally in your sample buffern an inert protein (like BSA) and a detergent. The last one will bind with his hydrophobic tail onto the syrole rings like in any ELISA plate.
Polyehylene or -propylen have a much lower protein binding capacity. So you can use them without any problems.
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