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antigen not binding to the plates

antigen not binding to the plates - ELISA Assay Forum

antigen not binding to the plates - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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  #1  
Old 05-02-2011, 10:38 PM
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Default antigen not binding to the plates



Hey there, the molecular weight of the peptide that we are using is 8812.0924.
The first time we did this experiment we took 2ug/mL of the peptide in binding buffer and added 100ul/well using multichannel pipette. But we saw that the antigen/peptide got washed away. So my supervisor told me to make a more concentrated solution so I made 10ug/mL in PBS this time. Please tell me why the peptide got washed away. I dont know how to proceed and not like I am a phd student. I am doing my masters and new to ELISA. I am so worried cuz I have to submit my thesis soon and I havent figured out why this is happening. So what we did this is we made 5ug/mL in PBS, 10ug/mL in PBS, 15ug/mL in PBS and 20ug/mL in PBS and my supervisor told me to check the fluorescence of all these solutions. The more the concentration the more the fluorescence obviously, so my supervisor told since the more concentrated once have more fluorescence, we use the more concentrated ones in the plate. So when we wash the plates we dont flick off the washing buffer but instead save it and check for fluorescence. I dont know if this the right way to proceed. Please give me ideas
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Old 05-03-2011, 03:11 PM
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Default Re: antigen not binding to the plates

Hi,

I work on a protein that is a dimer, each monomer being 12.7 kDa, and it does not bind well to polystyrene plates. I have no idea why, it just doesn't. So when we do ELISAs, either I bind a GFP-protein fusion to the plate (which works well), or I bind our protein's target receptor to the plate, then block, then add our protein. Some proteins just don't bind well to polystyrene.

You might want to opt for using western blotting with desitometric analysis instead of ELISA instead for quantification. It's not the best way, but it might work better.

Good luck!
Manders
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Old 05-03-2011, 06:08 PM
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Default Re: antigen not binding to the plates

OK, first you can optimize for binding with buffer pH.

You have two very popular options for binding buffer Carbonate/Bicarbonate buffer pH 9.0 & PBS pH 8.4.
Carb/Bicarb pH 9.0 is generally better.

Second you can upgrade your polystyrene plates to just higher quality like Pierce EIA (no. 15041)--just ELISA quality, so other brands will do. Or change to amine-binding maleic anhydryde plates for ELISA, they will chemically link to the amine in proteins (also from Pierce no. 15100); Block plates with SuperBlock or StartingBlock from Pierce, that way you won't have to troubleshoot the blocking step with your typical blockers.

Thirdly, ignoring the two recommendations above, you can optimize for a) binding or b) blocking. For a) you can extend binding to 1 hour at 37 C or ON at 4C. For b) if background is relatively high, time to start lowering try out the suggested Pierce blocker, typical manuf. instructions work great.
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Old 05-04-2011, 03:16 AM
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Default Re: antigen not binding to the plates

In my experience immulon 2HB and polysorp can offer advantages for antigen down assays over maxisorp and immulon 4HBX which are optimized for antibody binding. Active binding plates may also work. You may be in what I call a 'compost heap' situation, where the peptide does get bound to the plastic, but in no useful orientation for detection (a little like a squashed fly on the windshield). It may be useful to work with a biotinylated peptide, and capture the peptide via streptavidin, you can use one of the n-hydroxysuccinimide kits for labeling. The hope is that the peptide still retains binding activity after the biotinylation. Instead of using precoated streptavidin plates, preform the streptavidin and peptide (10 ug/mL streptavidin and 10 ug/mL biotinylated peptide in PBS) and dilute 10 fold in PBS before coating the complex on a maxisorp plate. Alternatively, with a relatively large peptide, it may be possible to find a capture antibody specific for an epitope independent from the detection reagent. It would be helpful if we knew more of the details and goals of the assay you are trying to develop. Good luck!
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Old 06-23-2011, 03:57 PM
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Default Re: antigen not binding to the plates

Passive adsorption has many advantage but also several drawbacks, that include desorption, binding capacity, and nonspecific binding. Because of the non covalent nature of the plastic/protein interactions, desorption may take place during the stages of the assay. But if the conditions of assay are standardized, leaching does not affect the viability of the majority of tests. For standardization is need having wide knowledge on coating target, not limited to molecular weight but also on other greater chemical characteristics such as, pI value, carbohydrate content, et cetera. From a theoretical point of view, the isoelectric point (pI) value of the used protein will influence buffer and pH choice. The best is to use a buffer with a pH value 1 to 2 units higher than the isoelectric point (pI) value of the protein being attached. This is, of course, not easy to determine in complex mixtures of proteins. The capacity for proteins to attach to microplate wells is influenced by the exact nature of the protein adsorbed to the specific plate used. Protein with high carbohydrate content is associated to bind poorly to plastic and, to clear hurdle, is need chemical activate plate, to realize antigen being covalently bound to prevent desorption (Elisadeveloper). On the last, I've realized easy chemical protocol, that can be furnished on request. The same attention, go observed to : a) ionic strength () of the coating buffer solution in combination with an optimal pH, give better results for the attachment of various peptides; b) lower pH coating when to use acidic protein to neutralize repulsive forces between protein and the solid phase; c) capacity of the plastic surfaces for adsorption molecule is finite, and influenced by the exact nature of the protein adsorbed to the specific plate used. Saturation levels of between 50 and 500 ng per well have been found valid for a variety of proteins when added as 50 ul volumes. This capacity is improved a lot when to use plate at high bound capacity (Danfive); d) appropriate post coating wash for ionic strength, pH, surface tension and washing force prevent desorpition of the coated molecule.
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