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Lena@mabtech.com

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Lena@mabtech.com last won the day on August 30 2019

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About Lena@mabtech.com

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  1. HOW TO SETUP ELISA KITS

    Dear Mr. Cao, Thank you! Please do so, and feel free to post more questions regarding immunoassays. We will try to answer, and if we cannot maybe some other forum guest knows the answer. Take care! Lena
  2. HOW TO SETUP ELISA KITS

    Dear Mr. Cao, Thank you for using our forum! I'm sorry that I haven't responded to you earlier, or that no one else has been able to give you an answer. I've no experience of detecting antibiotics or pesticides. I made a search on the internet and I could find several different suppliers, but I'm no able to recommend any special to you as I'm not familiar with them. I'll ask around in our organization and I hope someone knows, but since our focus is on cytokines, immunoglobulins and apolipoproteins, we are not experts on the detection of antibiotics. I really hope you get the ELISA to work! And please add a reply here on how you solved it, it might help some other researcher. Best regards, Lena
  3. HOW TO SETUP ELISA KITS

    Dear Mr. Cao, Welcome to our forum! You write that you want to detect pesticides and antibiotics using ELISA, we don’t have any ELISAs or antibodies that can be used for this. Our focus is cytokines, immunoglobulins and apolipoproteins. I can give you some general ELISA information. First I think you should visit our assay principle section on our website, it is very informative and can guide you in how an ELISA assay is working: https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-principle ELISA is an immunoassay that enables sensitive quantification of analytes in solution e.g. cell supernatants, plasma, serum, CSF, saliva etc. The assay relies on the antibodies that capture and detect the analyte of target, and the antibodies’ affinity, avidity and antigen interactions are essential for the quality of the assay. There are different types of ELISA’s, but we at Mabtech utilized Sandwich ELISA as this is the most sensitive ELISA assay. To set up a Sandwich ELISA you need: · ELISA plates, high protein binding plates, there are several different available, Corning and Nunc are examples of two brands that have this kind of plates. · An antibody pair with specificity against analyte you are interested in. Preferably, the antibody pair should have been validated for ELISA. The antibody pair should contain one capture antibody and one detection antibody. If the detection antibody is biotinylated you will also need an enzyme conjugate like Streptavidin-HRP. Streptavidin binds to the biotin part on the antibody. We have SA-HRP conjugates (link here). Some detection antibodies are directly conjugated with an enzyme e.g. HRP, then the SA-HRP conjugate is not needed. · A substrate that reacts with the enzyme. In the above examples, I have mentioned HRP, and a substrate that reacts with HRP is the TMB ELISA substrate (link here). · TMB substrate needs to be stopped with e.g. H2SO4 (0.2M). · An ELISA reader that can measure the absorbance. · Apart from this you also need several buffers, what buffers that should be used is dependent on the analyte and the antibodies, therefore I cannot recommend any specific. In our ELISAs these are the most common buffers: Coating buffer (PBS pH 7.4). Washing buffer (PBS pH 7.4 with 0.05% Tween), blocking/incubation buffer ( PBS pH 7.4 with 0.1% BSA and 0.05%Tween20) for blocking and dilution of antibodies and samples. I hope this can give you some advice! Good luck! Lena Beckman
  4. Student

    Hi Oliver, Welcome to our Forum! We have discussed your ELISA problem internally here at Mabtech. Generally, it is hard to analyze Protein A as it binds to several antibodies thereby increasing the risk for high background levels. But as you also can detect positive signals in the negative controls, something else must be wrong. We guess that what has happened in your ELISA is a linkage between the coating and the detection antibody. You use an anti-mouse antibody as coating, do you know the origin of the detection antibody? If it is made in mouse the linkage is obvious, but it is also possible that the polyclonal antibody binds the detection antibody due to cross-linkage. There are commercial available ELISAs for the detection of Protein A, unfortunately, we don’t provide such a kit. Let us know if you have any further comments or thoughts. Good Luck! /Lena
  5. LOW signal with plasma samples

    Hi Sanda, I think your standard curves look good, and the background levels are low. So the assays seem to have worked. The OD signal increases with development time, on both samples and standard, and I think 60 min is a good developing time for pNPP (30 min is a bit too short, why you shouldn't focus on those results). You have diluted the samples with a dilution factor of 2X and 3X, and 3X is often below the lowest standard point and you shouldn't use these measurements. Since you are analyzing plasma samples, you need to dilute the samples at least 2X in the ELISA diluent, so, therefore, you cannot dilute the samples less. The reason why you need to dilute the samples in ELISA diluent is that the diluent prevents false-positive read-outs which may be caused by interference of heterophilic antibodies found in serum and plasma. My guess is that your samples have these very low levels of cytokines. I'm not an expert on PTSD but several factors might affect the levels, like timepoint for sampling, or disease progress etc? Also very important to use a diluent that prevents false-positive read-outs, I hope this has been done previously as well. Best regards, Lena
  6. Antibody coating

    Hi Emma, Yes, I agree with Christian and it is possible to adjust the protocol a bit (but as Christian writes we have not done comparison studies). I sometimes coat in RT (for 3hrs) and block overnight instead, if that suits my experiment schedule better. If you are tight on time, you could instead go for the ELISAPRO kits, with precoated plates. That will save you both time and decrease variability. Link to ELISAPRO IL6: https://www.mabtech.com/products/human-il-6-elisa-pro-kit_3460-1hp-2 Link to ELISAPRO IFNalpha: https://www.mabtech.com/products/human-ifn-alpha-elisa-pro-kit_3425-1hp-2 Good luck! Lena
  7. Queries on Elisa for beginners

    Glad to help!
  8. Queries on Elisa for beginners

    Hi Priya! 1) Yes, the dilutions provided in the above image can be used for all your standard curves. 2) For almost all of our ELISA systems a blocking buffer containing 0.1% BSA is sufficient. But for some systems, e.g. Human IL4 ELISA, a higher BSA concentration is needed. Therefore, we recommend a blocking buffer with 1% BSA for human IL4 ELISA. The blocking buffer with 1%BSA can be used for all systems but this high concentration of 1%BSA is not necessary for IL1alpha nor IL1beta. 3) The standard should be reconstituted, aliquoted and frozen. Do not re-freeze thawed aliquots of the standard. There is no need of aliquoting the other components, they can all be kept in the fridge for 18months (date from delivery). Take out the antibody vial from the fridge, pipette what is needed and put the vial back in the fridge. Always use new and clean tips. Best regards, Lena
  9. Queries on Elisa for beginners

    Hi Priya! You are using the ELISA development kits, they are flexible kits that the researcher can adjust for its own needs. In the protocols for the development kits we leave several decisions to the researcher, this can feel a bit puzzling for beginners, and this is why ELISAPRO kits can be an option. But our development kits are very appreciated by our customers and when you have optimized it and get it to work it is a very price effective assay. Therefore, it is so wise that you ask these questions before starting the assay. 1) For every analyte ELISA-system we validate the standard and set a standard range. A standard range is the range in which determination of analyte concentration can be done with precision, accuracy and linearity. So different ELISAs can have different standard ranges. The turtorial (on our website) should only be used as an example. You will need to look in the datasheet for the specific ELISA to see the standard range for that specific analyte. If, for example, the standard range is between 1-1000 pg/ml, you need to prepare the standard points/dilutions so that they cover this range. The amount of standard in the standard vial is written in the datasheet. If (again only as example, you need to look in the specific datasheet), the standard vial contains 1ug, reconstitute the standard with for example 1ml of reconstitution buffer (what to reconstitute the standard with is also written in the datasheet/protocol). In this example you will have a standard stock solution of 1ug/ml. Then to a serial dilution of this standard. Exactly the length of the standard curve or the individual steps between the dilutions doesn’t really matter as long as you cover the standard range. It is preferably if the standard range consists of at least 5 points. I include a picture of how to do a serial dilution of a standard that covers the example of a standard range between 1-1000pg/ml. As you can see in the picture, the standard curve covers the standard range and actually go beyond the range. But if you only want to use the points that are within the standard range you can just exclude the 3160pg/ml (used in this example). The different standard point will get different OD values that are plotted in the standard curve. When you look at the OD values of your samples, they should have OD values that are within the OD values of the standard range. 2) Reconstitute the standard and aliquote the stock solution. If you are using 10ul for each assay I would add more than 10ul per aligoute, at least around 15-25ul (dependent on the tube) since it will be hard to get out every single drop from each tube. These aliqoutes can be stored in the freeze, but when you have thawed one tube it should not be refrozen, and preparations of the standard (further dilutions) should not be frozen and kept for later analysis. 3) No, it is not recommended to store the diluted antibodies/conjugates. Dilute what is needed in that step of the ELISA. (Example dilute detection antibody in 12ml ELISA diluent (enough for one plate) just before you wash away the samples.) I really hope this answer your question, and please continue asking! Best regards, Lena
  10. Queries on Elisa for beginners

    Hi Priya, Yes very wise to sort out the question marks before starting the assay! We are here (and on email) if you get more questions! Lena
  11. Queries on Elisa for beginners

    Hi Priya, Welcome to our forum and thanks for your interest in our products. I will try to answer your questions. 1) The ELISA plates need to be high-protein binding and there are several suppliers available. What plates that are preferred is up to the user. Strip plates are very convenient if you only use a couple of wells for each experiment, but these plates are generally a bit more expensive. Different plates could give different background readings but the difference is generally very minor. If possible (due to the ELISA reader), we recommend to read a reference wavelength (e.g. 650nm), which corresponds to the background from the plastic, subtract the reference OD values from the samples OD reading. 2) For best results, we recommend to coat the plates overnight at +4 degrees. It is possible to store the plates for some more hours but longer periods are not recommended, since this could affect the antibodies. I cannot give an exact time for how long it is possible to have the plates coated and stored before running the assay, this is dependent also on the specific antibodies. When I do freshly coated plates and do not have the time to do the analysis on them after the overnight incubation, I block the plates and put it back into the fridge. I have had plates in the fridge for about 72 hours (including coating and blocking time), and the assay still works. But please, be aware that this is nothing that we recommend. If you need to store plates you should consider pre-coated plates, they are very stable and can be stored for a very long time. 3) This is dependent upon what substrate you are using. If you are using SA-ALP you need a substrate that interacts with ALP, e.g. pNPP substrate (available in our webshop 3652-P10), this substrate requires around 45 to 60 min of developing time, it is possible to do as you write to test different time-points, again this is dependent on your study. pNPP substrate does not need to be stopped and should be read at 405 nm. If you instead are using SA-HRP you will need to have a substrate that interacts with HRP, e.g. TMB substrate (available in our webshop 3652-F10). The development time for TMB substrate is between 10-15 min and it should be stopped with e.g. sulfuric acid and directly read at 450 nm. 4) Concentration of stop solution is dependent upon stop solution used, we usually use sulfuric acid at a concentration of 0.2M. It seems like you are using our ELISA development kits, this is an adaptable and flexible kit that makes it possible for you to set up the assay in your own way. If you are new to ELISA I think you should consider to start with our ELISAPRO kits, these are complete kits with pre-coated plates and everything included for a straightforward assay. Read more here about our different ELISA formats: https://www.mabtech.com/knowledge-center/product-guide/elisa-kits This is another link where we describe the ELISA assay: https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-principle on the following pages you can also read about how to determine the sample concentration and other ELISA guidelines. Please let me know how things go and if you need any more assistance! Best regards, Lena Beckman
  12. What to expect from the mouse apoE ELISA?

    Hi M, We have tested plasma and serum pools from three different strains (NSA, C57Bl/6 and BALB/C) from two different companies using our Mouse apoE ELISA (link to product here: https://www.mabtech.com/products/mouse-apoe-elisa-pro-kit_3752-1hp-2). The mouse apoE values differed between 10-150 µg/ml dependent on mouse strain. The plasma/serum were run in triplicates (see table below). We have also tested C57Bl/6 and BALB/c plasma from a research group, and the apoE levels were in line with the results presented in the table. Therefore, we recommend a dilution factor between 5000x-10000x. I hope this is useful to you, let me know if you need any further information. Best regards, Lena
  13. apo-ELISAs and cerebrospinal fluid

    Hi! I just want to add two interesting articles to this discussion: Van Harten et al. are also using Mabtech's apoE ELISA to analyze apoE in CSF and plasma, they dilute the samples 1:2000 and 1:20000, respectively. Van Harten et al. (2017) CSF ApoE predicts clinical progression in nondemented APOEε4 carriers. Neurobiol Aging. Several apolipoproteins are present in cerebrospinal fluid, Slot et al. are using Mabtech's apoA1 ELISA du analyze apoA1 in CSF and plasma, and for apoA1 they dilute CSF and plasma 1:1000 and 1:100000, respectively. Slot et al. (2017) Apolipoprotein A1 in Cerebrospinal Fluid and Plasma and Progression to Alzheimer's Disease in Non-Demented Elderly. J Alzheimers Dis. Best regards, Lena
  14. High levels in APO-A1 ELISA

    Hi Neelika! Welcome to our forum! I recommend to store the samples in -80°C. We have not seen any problems with aggregation when the samples are fresh, freeze dried or stored in -80°C. Best regards, Lena
  15. High levels in APO-A1 ELISA

    Hi C, Thank you, very glad that you appreciate my support! Please let me know if I can assist you with anything further! Best regards, Lena Beckman
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