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Everything posted by Lena@mabtech.com


    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

    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

    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
  16. Specificity Mouse apoA-I ELISA

    Hi Dr. R, Thank you for your question! Yes, the mouse apoA1 ELISA assay is specific for mouse apoA1 and does not detect human apoA1. You should be aware that the buffer included in the kit is necessary for an accurate measurement of mouse apoA1, however this buffer does not contain any HAMA-blocking proteins so if you run human plasma in the mouse apoA1 ELISA a HAMA-effect might influence the results. This is of course not an issue if you will analyze mouse samples expressing human apoA1. The human apoA1 ELISA does not detect mouse apoA1, but it seems as mouse plasma is rather sticky so during low dilutions (1:10) it might give an unspecific signal. This is a link to our mouse apoA1 ELISA kit: https://www.mabtech.com/products/3750-1hp-2_mouse-apoa1-elisapro-kit This is a link to our human apoA1 ELISA kit: https://www.mabtech.com/products/3710-1hp-2_human-apoa1-elisapro-kit Best regards, Lena Beckman
  17. High levels in APO-A1 ELISA

    Dear C, Thank you for your reply! So you get nice looking standard curves and blanks, and you are following the protocol. This means that the assay is working and you are doing everything right. I think that the high levels you see are due to the storage and handling of the samples. We have done several experiments looking at plasma samples stored in different temperatures for different times. And the conclusion is that apoA1 starts to form these aggregates (leading to false high levels) after a couple of weeks in -20°C and warmer. This will not happen if the samples are fresh, freeze dried or stored in -80°C. The aggregation problem is exaggerated in samples with several freeze-thaw cycles. I would advise you to change the procedure with the samples so that they are stored in -80°C and minimize the number of freeze-thaw cycles. Best regards, Lena
  18. High levels in APO-A1 ELISA

    Dear C, Thank you for your question! When plasma/serum samples are stored in -20°C things happens to the lipoproteins in the samples that will affect the apolipoprotein A1 measurements. We have seen this phenomenon several times, and this is why we recommend not to store samples in -20°C (written in the data sheets). I have detected up to 300% (!) higher apoA1 levels in old and badly stored plasma samples. We cannot be totally sure what is really happening in the tube but it is known that apoA1 can form aggregates (look like coin rolls). The avidity of the antibodies to the aggregated apoA1 will be higher, leading to false high levels. We have tried to dissolve the aggregates but we have not managed to do this without affecting the measurements. So I really want to emphasize the importance of not storing samples in -20°C. Even though apoA1 is extreme in this sense, many different types of proteins are affected by handling and storage. This is not due to the methods, it is due to the analytes. So yes, your problem is most likely due to the fact that you have stored the samples in -20°C. To rule out some other possibilities, I have a couple of comments and questions for you. When analyzing apoA1 in plasma you need to do extensive dilutions, I hope you have seen the dilution guidelines at page 10 in the data sheet, the guidelines are also available here: https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/apolipoprotein-dilution. It is very important to be precise during the pipetting and to change pipet tips between the steps. To really sort this out it would be great if you can answer the following questions: How are your standard curves looking? How are your blanks looking? What kind of samples do you have and for how long do you store them in -20°C? How do you wash the plates? Thank you! Lena Beckman
  19. Technical questions for ApoB ELISA Pro kit

    Hi S! I’m glad I was able to help you with the questions, please do not hesitate to contact me again if you need any further assistance! Best regards, Lena
  20. Technical questions for ApoB ELISA Pro kit

    Dear S, Thank you for your questions! Yes you have absolutely right, we have two different type of ELISA kits: the development kit and ELISAPRO kit. The development kit includes the antibody-pair, standard and the SA-conjugate (either ALP or HRP) and is best suited for researchers that are used to coat ELISA plates, and wish to adjust and customize their own ELISA. The ELISAPRO kit, on the other hand, is a complete kit with pre-coated plates, Apo ELISA buffer, standard, detection antibody, SA-HRP, HRP-diluent, wash buffer, substrate and stop solution. The ELISAPRO kit is a more convenient and straightforward kit which saves time and reduces both inter- and intra-assay variation. Read more about the two different formats on the following link: https://www.mabtech.com/knowledge-center/product-guide/elisa-kits The detailed dilution guidelines for human serum/plasma samples are based on serum samples from fasting healthy subjects. The levels of apoB in cell line supernatants are influenced by several parameters such as type of cell line, cell confluency etc. why it is harder to give detailed dilution guidelines for such samples. The reason why we give detailed descriptions on how to handle human serum/plasma samples is also due to two other important factors: 1) To prevent interference by different LDL-particle sizes, all serum/plasma derived samples need to be diluted 2x in Triton X-100, followed by vortex for 5 seconds (this procedure is specific for when analyzing apoB). This is not necessary for cell line supernatants. 2) Serum /plasma samples may contain heterophilic antibodies that can, by cross-linking the assay antibodies used, result in false positive signals. The Apo ELISA buffer prevents the heterophilic antibodies for cross-linking. Heterophilic antibodies are not present in cell line supernatants. To answer your specific questions: 1) It is important to use a detergent in the buffers, I cannot say that one detergent is the best, in the development kit we leave this decision to the researcher. But as written in the protocol it is 0.05% Tween-20 in the “incubation buffer”. The “incubation buffer” can be used to dilute cell supernatants, standard, detection antibody and SA-HRP. 2) I incubate for approximately 15 min. 3) Due to the factors written above we always recommend to dilute serum/plasma samples in Apo ELISA buffer. When diluting cell supernatants it is also possible to use “incubation buffer”. However, it is important to use Tween-20 and BSA of high quality, and all our buffers are tested, validated and quality controlled. It is absolutely possible to use the development kit for assaying your cell supernatants, and to dilute the samples using an “incubation buffer”. But if you want to spare time (the time with preparing the buffers and coating the plates) and reduce inter- and intra-assay variability, I would recommend you to use the ELISAPRO kit. This is a link to our apoB ELISAPRO kits: http://www.mabtech.com/products?field_category_tid=66&field_analyte_tid[]=71&field_kit_plate_format_tid[]=1345&items_per_page=20&page=0 I really hope this answer can help you! Please let me know if I can be of any further assistance! Best regards, Lena Beckman