A forum to address questions regarding enzyme-linked immunosorbent assay, most commonly known as ELISA.

• Tiffany Tan

  ELISA standard absorbances and result normalisation

  1) I obtained higher absorbances for my ELISA results than my college who performed the same analysis using the same kit. I wonder what would be the reasons for higher absorbances? 2) Also, I1) I obtained higher absorbances for my ELISA results than my college who performed the same analysis using the same kit. I wonder what would be the reasons for higher absorbances? 2) Also, I intended to normalise the result with alamarBlue absorbances(which correspond to cell metabolic activity/ viability); say for instance one of my ELISA absorbance is 2.34, and after converting it to protein concentration is 3400pg/mL, but after normalised with alamarBlue absorbance(0.32) then the amount would be so big. Or how should I normalise data? Hope someone would advice me on these =) Thanks

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    Tiffany Tan replied

    Thank you ! I really have an invaluable ELISA lesson here! I really appreciate all your helps and advices =D

    1 hour ago
• Kaija Strautins

  ELISA blocking agent: BSA or sodium caseinate?

  I'm making an in-house assay, where the ELISA plates are coated with streptavidin for the biotinylated peptides of interest. The manual suggests blocking with a PBS/Tween 20/sodium caseinateI'm making an in-house assay, where the ELISA plates are coated with streptavidin for the biotinylated peptides of interest. The manual suggests blocking with a PBS/Tween 20/sodium caseinate solution, but the lab doesn't have any. Is there any reason I should avoid using BSA (bovine serum albumin) instead as a blocking agent? It's used at other point in the assay (i.e. diluent for the secondary antibody) so I don't think it's an issue? Has anyone else had experience where one is superior/has limitations to the other?

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    Bodo Brocks replied

    BSA (1-3 %) should normally work well here. I rather would be reluctant using milk dreived proteins like casein on streptaviding plates, as these might more likely contain biotinylated proteinBSA (1-3 %) should normally work well here. I rather would be reluctant using milk dreived proteins like casein on streptaviding plates, as these might more likely contain biotinylated protein contamintations than BSA does. Bodo

    4 days ago
• Liubov Yurjevna Vergun

  Why modern ELISA use conjugate with polimer HRP?

  Last time I meet articles where for conjugation enzyme (HRP) - antibody is used polimer-HRP. What for usual-HRP is substituted polimer-HRP?
• Djafsia Boursou

  Is it advisable to use Human Serum Albumin for blocking the plate? In which cases?

  Does it exist any interference of BSA blocking when a sample of bovine origin is used for immune recognition.
• Marilina Piemontese

  ELISA assay detecting carbonylated protein in bone lysate sample

  Does anybody know a good Elisa kit to detect carbonylated protein? My sample is a bone protein lysate. Any suggestion about the use oF ANTI-REDUCING AGENT? If my lysate buffer contains Triton X-100,Does anybody know a good Elisa kit to detect carbonylated protein? My sample is a bone protein lysate. Any suggestion about the use oF ANTI-REDUCING AGENT? If my lysate buffer contains Triton X-100, this will affect the assay?
• Evelyn Lumngwena Ngwa

  HI Everyone, I have problems with my p24 ELISA. Backgrounds higher than some standard readings leading to negative values in some standards and some values completely out of range. Some advice

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    Liubov Yurjevna Vergun replied

    I would like to advice to use buffer with casein (milk without fat) and mertiolat. You can make own buffer or buy (Sigma or other company)? sometimes I used BSA but only V fraction.

    21 days ago
• John G Walsh

  Elisa for hiv p24

  Can someone recommend an ELISA (kit or protocol) for HIV p24 that is effective and of good value?

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    Liubov Yurjevna Vergun replied

    As for me good kits have company Abbott or BioRad

    21 days ago
• Fatima Yucel

  IgA-type monoclonal antibody purification method?

  Could anybody suggest a protocol for this Purification method?

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    Stephan T. Kiessig replied

    It's not necessary, you can run a cell culture supernatant in IEF, Blot the and stain with anti-IgA.

    24 days ago
• Jayharsh Panchal

  Acid Treatment for ELISA

  Can anyone let me know, when optimizing an assay, if a protein is too sticky and needs to undergo acid treatment, what type of acid should be used? What are the criteria for deciding the acid to beCan anyone let me know, when optimizing an assay, if a protein is too sticky and needs to undergo acid treatment, what type of acid should be used? What are the criteria for deciding the acid to be used? The protein I am using is too sticky - size 15 kD, forms dimer and multimer a lot.

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    David J Hurley replied

    We are trying to measure IGF-1 and IGF-2 in colostrum and neonatal serum most often when we use the acetic acid. We use an acetate buffer after treatment, as it seems to keep down re-association ofWe are trying to measure IGF-1 and IGF-2 in colostrum and neonatal serum most often when we use the acetic acid. We use an acetate buffer after treatment, as it seems to keep down re-association of the IFG and the binding protein so our IGF-1 and IGF-2 capture antibodies give more signal at a given level of peptide. We use HCl when we are trying to reduce protein aggrigates in viscous materials like snot or colostrum. This seems to work ok. We use 30 minutes stirring as it has been successful for us and it is our tradition. All the assays are in house.

    25 days ago
• Daniel Woldeyes

  ELISA blood source

  Is there any possibility to use capillary blood for ELISA? I have read an article that made use of blood from ear lobe; but I didn't see the detail of the protocol. Would anyone tell me about theIs there any possibility to use capillary blood for ELISA? I have read an article that made use of blood from ear lobe; but I didn't see the detail of the protocol. Would anyone tell me about the detail of this technique so that I can make use of it for my cystic echinococcosis study project? Thank You.

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    Daniel Woldeyes replied

    Thank you all

    Apr 14, 2012

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    Maria Zahid replied

    Yes, for surface protein of a virus-like particle..

    Mar 31, 2012
• Yuliya Perfilyeva

  Has anyone performed 48h or 72h ELISPOT assay?

  Recently we attempted an experiment in which we analyzed NK cells for IL-10 production in ELISPOT assay. We found spots in 18h and in 72h, but there were no spots in 48h, even though it was the sameRecently we attempted an experiment in which we analyzed NK cells for IL-10 production in ELISPOT assay. We found spots in 18h and in 72h, but there were no spots in 48h, even though it was the same cell suspension. Has anyone performed 48h or 72h ELISPOT assay?

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    Yuliya Perfilyeva replied

    Well it really explains everything

    Mar 22, 2012
• Lya Sepúlveda

  Quantification of pmsg by elisa

  I need to quantify the purified hormone PMSG (pregnant mare serum gonadotropin) by ELISA. The commercial kits are to serum samples. Can I use these kits to quantify the purified hormone? What bufferI need to quantify the purified hormone PMSG (pregnant mare serum gonadotropin) by ELISA. The commercial kits are to serum samples. Can I use these kits to quantify the purified hormone? What buffer should I use? What dilution should I use? Thanks.
• Andrew Lariviere

  Protein extraction for ELISA

  Hi, I am performing an ELISA with Arabidopsis tissue. After extracting the protein I add 6X volume of ice-cold acetone and place the samples in the freezer for 3 hours. I then vortex the samples, butHi, I am performing an ELISA with Arabidopsis tissue. After extracting the protein I add 6X volume of ice-cold acetone and place the samples in the freezer for 3 hours. I then vortex the samples, but I get 3 phases of material. A top, liquid acetone phase that I discard, a thin middle phase that looks like it might be the protein precipitate, and a 50-100 μL gel-like bottom phase. Another person in the lab who previously performed ELISA with this protocol said he did not get the bottom phase and was able to dry the precipitate with no problem. If I include the bottom phase in the rest of the assay I do not get good results. It cannot be easily separated from the thin middle phase which I believe is the precipitated protein. Does anyone have any suggestions on how to separate the middle and bottom phases? And what the bottom phase may be? My extraction buffer contains 0.5 M tris (pH 8), 1% SDS, 30% sucrose 2.4 g/L aminocaprioc acid, and 0.72 g/L benzamidine. Thank you for you suggestions, AL

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    Andrew Lariviere replied

    Thank you for all the helpful feedback. Someone in a neighboring lab suggested I try TCA followed by acetone, instead of acetone alone, thinking the gel-like phase was sucrose. I didn't get aThank you for all the helpful feedback. Someone in a neighboring lab suggested I try TCA followed by acetone, instead of acetone alone, thinking the gel-like phase was sucrose. I didn't get a pellet with TCA. So I went ahead and finished the procedure and included the gel phase in the assay. I got a signal and it looks like it may work I'm am going to perform another round and see if I can repeat it. If not I'll try something else that may help to exclude the bottom phase, as suggested here, such as a non-ionic detergent or piercing the tube. Thanks again for your help!

    Mar 21, 2012
• Michelle Tegg

  My antibodies work in an indirect ELISA but together in a sandwich I get high background and low signal. Does anyone know what could be happening?

  In my indirect (antigen coat) I get ODs for my blank of around 0.02 as expected for both antibodies, and signal for my highest standard concentration of 500ng/mL is around 2.2. However in a sandwichIn my indirect (antigen coat) I get ODs for my blank of around 0.02 as expected for both antibodies, and signal for my highest standard concentration of 500ng/mL is around 2.2. However in a sandwich these antibodies produce background of around 0.3 or higher and a signal of only 0.5 for a concentration of 5000ng/mL. Other than that it does work and I'm seeing a gradient for the standard curve and plasma dilution series. Does anyone have any idea what could be happening here to suppress my signal and increase the background? Also has anyone ever heard of using 1-3% polyethyleneglycol in the incubation buffer to increase the reaction speed of the immune reaction, and increase the signal? I read it somewhere and wondered if anyone had tried it. Any help would be gratefully appreciated (and thanks to all who have helped me previously).

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    Stephan T. Kiessig replied

    Hi Wolfgang, you are not right. BSA has a a neutral pH much less binding than nonionic detergents. These molecules are smaller an can replace the solid phase protein.

    Porstmann_Kiessig_Enzyme_1992.pdf

    Mar 21, 2012