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I am working on Elisa optimization to detect the antibodies raised against plant glycans in human serum samples. I use anti-xylose and anti fucose as standards.
The issue is if the wells are not coated with antigen and add PBS with human serum samples, the OD values are very high than the ones coated with antigen.
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If you get a higher signal with avidin coating and human serum, I would conclude that there are biotinylated antibodies in serum (unlikely, there is no evidence yet that they exist) or there are antibodies against avidin (indicating a possible immunoreactivity against avian egg proteins of which avidin is one). You could try streptavidin instead, or avidin from eggs. There might be impurities in the plant based prep.
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Hi. I have a question of the MHCII expression in mammal. What I know so far is that the expression of MHCII is codominant, alleles from both mom and dad will be expressed. However, I would like to if the genotype of MHCII affect the phenotype. For example, would the MHCII wild type animals express more MHCII protein than the MHCII heterozygous animals? Also, would the MHCII WT animals process more antigen and have a stronger immune response than the MHCII heterozygous animals? Assuming only one specific type of antigen is processed in this case.
It will be very helpful if you can post some related literature. Thanks for your time!
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I have been finding the proof that this epitope called NLVPMVATV does not induce cytokine storm for days, but in vain. Could anyone please help me? I really need a reference paper to support this or else my project will be in a great trouble.
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Hi,
Check the references below:
HCMV UL83/pp65 (NLVPMVATV)
CMV pp65 peptide NLVPMVATV (HLA-A*0201) is a single peptide for stimulation of T cells. The peptide from human cytomegalovirus (CMV) phosphoprotein (pp65) is synthesized as it is presented by the MHC class I HLA-A*0201 allele and can be used for targeted in vitro generation and expansion of antigen-specific CD8+ T cells.
Reference:
"Although the anti-pp65 antibodies and the pp65 antigens are detected in immune-depressed patients with active viral infection [16], the antibody response to the pp65 antigen in normal infected individuals is not always detectable by immunoblotting [17]. It has been reported that the pp65 antigen is targeted by a cell-mediated immune response and that its vaccination can induce a pp65-specific CTL response"
Reference:
Hope it helps,
Best regards
Tomasz
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Based on the topics listed in https://www.nature.com/articles/d41586-020-03564-y the diversity of major topics categories under which research publications on COVID are grouped are:
  • Modelling epidemic, controlling spread
  • Public health
  • Diagnostics, testing
  • Mental health
  • Hospital mortality
It is obvious that Lateral Flow testing belongs to the third category on the list - diagnostics/testing. The software interface aspect of things is not addressed. One aspect that would belong to the software side is the reporting/registration of results. Please read the latest preprint of my paper on this subject at:
What are your thoughts on the impact on our preparedness for serious pandemics if software engineers develop methods to tackle online cheating at home? Please share your thoughts, let me know if I can quote you as a reviewer for this paper in a journal. Thanks in advance for your collaboration.
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One newspaper in the United Kingdom is reporting that some travelers are doctoring emails from Covid-19 testing laboratories, printing them out and simply handing them to airline staff before boarding flights.
Some Air Passengers Are Faking Negative Covid-19 Test Results, Per U.K. Reports (forbes.com)
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Hi all,
I recently performed a antibody-antigen affinity test using Fortebio Octet.
The format is as follows:
Antigen was captured by SA sensor then interacted with VHHs. Double reference was applied. But there is negative signal when the reference sensor(no ligand on it) dipped into the well of analytes except the buffer well.
The blue curve is the buffer and the other two are analytes with two concentrations.
Can anyone give me some suggestions on it?
Many thanks!
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Hi Iam having same issue on both SA and SSA sensor , kindly suggest me if any one of you have solved this issue
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I saw many papers are introducing or immunizing mice with ovalbumin (OVA), will this induce a cytokine storm? Is there any paper suggesting that OVA doesn't induce cytokine storm? I've been searching this for days, please help.
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No, there are essentially 3 major types of immune response to infection: an antiviral response which illicits interferon, a pro inflammatory response that makes il1, il6, tnfalpha etc wich is normally directed against bacterial and fungal infections ( and can lead to cytokine storm) and then the allergic response directed against allergens like ova that generates il4, il10, il13.
In mouse models, balb/c mice are typically used to study allergic responses as thy are inherently more allergic and c57bl/6 are used to study proinflammatory responses and diseases because they are inherently more prone to this type of immune reaction.
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I have this paper I am writing about a software interface idea and code I need to publish about Covid. However, the paper would be over 40 pages because a large part of it is to show that there is no literature discussing the issue which the software is meant for. Therefore I am spitting them into two. Where can I publish just the first paper which only introduces the issue?
EXCERPT FROM ABSTRACT of first paper:
In the paper, I simply demonstrate that there may be a hugely lucrative area of online cheating in the registration of Unsupervised Rapid Antigen Test Results.
KEYWORDS: Online Cheating; Registration of Lateral Flow test Results; COVID software; COVID mis-information; unsupervised tests; anti-fraud COVID testing
EXCERPT FROM CONCLUSION of first paper:
5. Please state two take-home conclusions or findings from this research work.
Answer:
  • There is currently no software for the reliable registration of completely unsupervised Rapid Antigen Test results, and this problem has not yet been addressed in science journals. Literature on academic online cheating does not address any form of COVID testing.
  • Creating software programs to detect cheating in the online registration of completely unsupervised Rapid Antigen Test results might open up a new lucrative area for research and software development.
FULL TEXT OF THE ABOVE FIRST PAPER:
Deleted research item The research item mentioned here has been deleted
Without that first paper, I will not be able to publish the one about the software interface.
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Hello, again, Donald O. Besong
Please do not let that rejection from that journal you mentioned be a discouraging event for you. Did they give any suggestions on how to modify your work?
I would look for other journals which might be suitable, particularly like I say, if they permit "Commentary"/"Opinion" type articles. Sometimes reading an issue of the journal helps you better understand their attitudes, audience and the way they want to give out information (and that tells you if the "Commentary"/"Opinion" type will be accepted in that journal).
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Dear colleagues,
does anyone know a good protocol to extract vaccine antigen for quantification? The idea is to separate the adjuvant from the antigen without major losses. Can anyone help me with this question? Thank you!!
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It might be not necessary to isolate the antigen if you can use a mass-spectrometric method. In the first step you have to digest all proteins in the sample with trypsin, and then quantify a known tryptic peptide from the antigen relative to an isotopically labeled peptide used as an internal standard.
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1. After adding HRPO to TMB substrate mixture, I am observing blue/ green color - which instantly turns to yellow color with brown particles even before adding stop solution. What could be the problem and how to rectify this?
2. I have replicated with antigen/ antibody and observed same yellow color with brown/ orange particles. TMB A and TMB B are clear/ pale blue. Same TMB solutions have previously been used to obtained appropriate color development.
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Randall Cameron Thorough washing is obviously essential, as remains of any reagents will affect the results. If there are traces of the HRP conjugate left, the TMB solution will turn blue instantly, traces of other reagents will compete with the intended interactions on the MTP's surface.
What works best in my experience is to fill the wells with washing buffer (PBST), expel it manually into the sink and beat the plate against a stack of sucking paper. Repeat at least 3 times. This is much superior over using a plate washer.
If you have to use a washer (because of automation, scaling up or GLP requirements) you need to carefully control the washing cycles and subject them to rigorous QC.
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Hi, I am trying to use the OCTET machine to measure affinities of HIS tagged-scFv's, I have tried immobilising both the binding antigen and the scFvs to the sensor by different means (biotinilation, His tag, Fc) however we are not able to get reproducible kD values. In the last set of experiments we loaded Fc-antigen to a sensor and analysed the binding and dissociation of soluble scFv at different concentrations (12-100 nM). This yielded similar binding to the antigen but different dissociation, even though the expected kD should be somewhere within (12 and 100 nM). Has anyone encountered similar problems? Could it be that the scFv is complexing with each other?
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Hi Rocio Rueda,
Octet will give us beautiful results, but it is really tricky to get it all set!
I am also trying to test different scFvs using Octet. We have tried streptavidin (binding antigen, scFv in solution) and His-tagged (binding scFv, peptide in solution) sensors but with no success.
We fortunately have the monoclonal antibody our scFv is derived from, and the Octet seems to work with the monoclonal antibody molecule (which makes sense, since it is a more stable entity). Let me ask you a couple questions so we might both solve this riddle:
- Does your scFv have any tag (such as SUMO, GST...)?
- How is your reference sensor signal compared to sample sensors? Does your scFv have greater signal? By reference sensor, I mean biosensor without ligand (antigen), but with analyte in solution (scFv).
- How is the ligation curve of your experiment (in terms of value and shape)? It should not saturate in order to run the association/dissociation steps.
- Have you tried higher concentrations of scFv/antigen? In my opinion, the concentration will depend on how your molecule is behaving on the control sensors...
Regarding the complexing your mentioned, I believe it is a characteristic of scFv molecules to form aggregates. Depending on the buffer you use, they quickly precipitate. I would recommend keeping your scFv at higher concentration with your best buffer so you can dilute your molecule in the assay buffer (such as kinetics buffer).
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Hi, I did an overnight digestion using an His-tagged HRV-3C protease, my antigen of interest and cleavage buffer (containing 50 mM Tris-HCl pH 7.5, 150mM NaCl, 1 mM EDTA, 5mM DTT). The final concentration of DTT in my sample is now 0.5 mM. Since I can´t use this sample for 5 days, can I just freeze it at -20°C, or this concentration of DTT could still affect my antigen at this temperature?
Thanks
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5~10mM DTT is recommended, not to mention if it is lower than that. In a short-term storage, number of freeze/thaw cycle is what should be considered, more than the storage temperature, -20 degree Celsius is ok for a one week keep, if longer I would suggest in -80.
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Hi,
I'm trying to run some immunoprecipitation experiments but am not getting the expected results.
I'm following the IP protocol provided by the supplier. In a nutshell, I react 100-500ug of cell homogenate with 2ug of antibody overnight then precipitate that with 100ul of protein G agarose beads. I elute the proteins by boiling the beads in 2x SDS sample buffer with 0.1M DTT.
The antibodies are validated for IP experiments by the supplier. I've tested the antibodies against a purified form of the antigen and it works. I also tested the crude cell homogenate to ensure the antigen I'm trying to pulldown is there.
The only thing left is the protein G beads so I want to see if I can somehow test the beads to make sure they're working properly.
Thanks in advance
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In addition to the suggestions by Dominique Liger, for a functional test of protein G beads, you could make a small column with e.g. 0.5ml of beads, pass a little of bovine (or human) serum through them, wash with PBS end elute with an acidic buffer (e.g. 50mM glycine HCl, pH 2.5). You then should find the IgGs in the eluate, detectable as protein by photometry or of course by Western, if you have suitable anti species ABs.
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Hi there,
We need an antibody for a plant protein recognition (100 kDa). No commercially versions are available. Antigen is not purified, so our own rabbit immunization is rule out at now.
Could anybody please recommend a company to prepare antibody based on protein sequence?
Thanks in advance
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Hi Sandra,
They are good
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I am sorry if this is a very basic question, but I am new to ELISA.
I am synthesizing a polypeptide to use in ELISA against infected serum from patients. We will be using individual reagents and not a kit.
However, I am unsure about the amount of polypeptide to synthesize. I know this will depend on how much is needed in the ELISA-peptide assay but this seems to vary from manuscript to manuscript. Is there a way to determine how much antigen to use?
I have seen protocols state to cover the wells with 100ul of 1uM polypeptide. I have seen manuscripts just state they cover the well with 5ug/ml polypeptide
Thanks in advance
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What molecular size is your peptide? If the size of the peptide is small you can conjugate the peptide to a carrier molecule. Since the adsorption of the antigen to the polystyrene is through the negative charges of the polystyrene, a small peptide can lead to a low adsorption efficiency. I hope it works for you
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This is the result of immunohistochemical staining using paraffin-embedded mouse sections. Dewaxing, hydration, antigen activation (ph6.0 citrate, autoclave), blocking, and incubation of primary and secondary antibodies were experienced. DAB used for color development and counterstaining was also performed. The mounting medium was used with softmount.
The results showed artificial disturbances in the figure, like bubbles or crystals. Does anyone know what is causing this? What should be done to avoid it?
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before mounting sections you should wash your lam with passaging through very strong base like KOH and then put them in D-water and next strong acid like HCL and again DW in this way your lam will became very clean then instantly put them in fresh filtered gelatin solution and leave them dry for a night and make sure not became polluted by covering them.
after sectioning and dewaxing, passage sections in another bath and make sure sections perfectly dewaxed when mount on lam also surface of lam most not encounter with wax specially under of mounted section.
use sampler for Entellan dropping on lam and make sure on bubble remain under lamella by pushing lamella.
do not heat Entellan or gelatin coated lam because distribution became disrupted and making bubble.
good luck.
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Hello I did 100 ns simulation using GROMACS a protein protein complex ( chain A for antigen - chain B & C for antibody) now I want to calculate the number of hydrogen bonds. I select chain A and chain B, but it shows nothing. What i should select for selection 1 and selection 2?
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Magnus Bertelsen yes, I tried but it shows nothing.
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I want to use ELISA to detect inactivated SARS-CoV-2 propagated in Vero cells using aptamers, what solution will you recommend for me to use after centrifugation of the sample, is it the supernatant liquid or the pellets.
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In addition, for quantification, you might be better off with a sandwich system, using two antibodies, one for analyte capture and one for analyte detection. Direct coating of the plate with the analyte IMHO is ok for qualitative testing, but not for quantification, as the surface of the plate is not really homogenous, and you'll usually observe a wide variation when replicating samples at saturating conditions..
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I would like to learn my students how to perform a co-immunoprecipitation experiment, coupled to western blot of primary antigen and the co-precipitant.
Does anyone have a good suggestion for this? Failsave and not to expensive?
It's possible to do transfection for this purpose and precipitate using a tag antibody.
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You can consider the setup from this paper by my friend:
doi: 10.18632/oncotarget.18899
She performed mutant p53 - Hsp70 co-IP; this interaction is very strong. DO1 antibody for p53 IP is great. HSP70 is also easy to detect. You may overexpress proteins or not - depends on which cell line you work.
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Hello every one,
I wants to separate, purify and culture the mice uterus epithelial cells and there are various protocol available, kindly if any one worked in this area suggest me the standard protocol with highest purity and viability of epithelial cells and secondly in case if we used magnetics beads or any other linker or antigen for the separation of epithelial cells then how to detach them from the epithelial cells at the end after getting the epithelial cells?
Your quick and kind response will be highly appreciated
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Subhash C. Juneja thank you very much Sir for your detailed and well explained answer and as i am new in this field of uterus epithelial cells isolation and purification so i will be very thankful if you shared your published protocol if possible and secondly after getting the endometrial epithelial cells how we have to conform that there are only pure epithelial cells without any other cells type or contaminations? Once again thank you for your time.
With best regards
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Dear All,
I need to use this protein as an antigen to immunize mice and get antibodies (ideally). I need an advise what to do if protein precipitated after buffer exchange (purified in denaturing condition ProBond protocol - exchange to PBS) .
I immunized mice, developed a Western Blot and did not see any antibodies (after second immunization).
What do you think?
Thank you!
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"What do you mean by use a native PAGE gel - should I load serum samples into a a PAGE so see a hypothetical protein band (there are so many proteins in the mice serum..)"
Native PAGE means essentially leaving the SDS out, so you'll sort your proteins according to their net charge and size and/or isoelectric point. Run your antigen and probe it with diluted serum or extracted IgG. Searching the web for "native PAGE" will lead to various protocols. For the electrotransfer, you'll need membranes on both sides of the gel.
But if your antigen is apparently pure, a dot blot or ELISA like assay (coating the plate with the antigen obtained at various purification steps) is actually much more straightforward, so native PAGE IMHO only makes sense if you want to demonstrate that SDS is the culprit in your assay, as it's a quite tedious process over all.
Instead of a buffer exchange, have you tried to concentrate your protein by centrifugation with a membrane concentrator, "dialysis" against solid PEG or salt precipitation (ammonium or sodium sulphate, possibly also NaCl?).
Could it be that the precipitate you are seeing is not the protein you're after?
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Hello community.
I am conducting some initial ELISAs of potential diagnostic antigens from bovine parasites.
I will be using infected and non-infected sera to see which candidate antigen has the best accuracy and affinity etc. All pretty standard.
However I would like to find an antigen to act as a positive control for all cows (separate from parasite status). So this would be an antigen with naturally occurring Ab in all the serum samples. Not much is obvious from the literature on what this could be. Some E coli antigen or extract maybe? I will also be using the complete parasite extract as a control for positive cows, but I would like an antigen that all individuals will respond to.
Does anyone regularly use a particular antigen?
Thanks!
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Thomas, probably, you can get some clue from this review or similar https://www.frontiersin.org/articles/10.3389/fimmu.2020.02139/full
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Are ELISAs considered "semi-quantitative" due to the nature of antibodies being able to bind more than one antigen at a time? Since it is not a true 1:1 ratio, is it rational to assume that the concentration values reported in ELISAs are less than their actual/true values? I am talking about ELISAs in general, although I use sandwich ELISAs the most.
For example, Primary ab binds to two antigens - secondary binds to the one antigen - conjugate reaction strep/tmb produces the colorimetric change. This reaction does not measure whether or not the antibody sandwich has one or two antigens bound. What are the reasons ELISAs are considered semi-quantitative?
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The ELISA has been considered the gold standard of immunoassays. ELISA is very sensitive and is used to detect and quantify analytes, including antibodies, antigens, proteins, glycoproteins, hormones etc.
Normally four major types of ELISA are commonly used i.e. 1) Direct ELISA (antigen-coated plate; screening antibody); 2) Indirect ELISA (antigen-coated plate; screening antigen/antibody); 3) Sandwich ELISA (antibody-coated plate; screening antigen) and 4) Competitive ELISA (screening antibody).
The ELISA is quantitative, qualitative, or semiquantitative depending up on the experimental target you set. For instance, the quantitative concentration results are plotted and compared to a standard curve. The qualitative results confirm or deny the presence of a particular antigen/antibody in a sample, while semiquantitative results compare the intensity of the signals, which can compare relative antigen levels in a sample.
Its very important to note that now a days very sensitive ELISA have been developed that efficiently detect the analytes accurately in the scale of picograms. Hence, considering ELISA as "semi-quantitative" may not be appropriate.
Best
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I am coating the well with blank 1X PBS and then Block it with complete RPMI media and after blocking give these well PBS wash and seed the stimulated PBMCs which have Antigen Secreting cells.
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With serum
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Im looking for the Amino acid sequence of the exact peptide presented to the T CD4+
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Tal vez esto te puede ayudar
Suerte
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The antigen that we are trying to detect in pathogens using monoclonal antibody or polyclonal antibody studies, are these not detected when they the organism to be studied is dead?
In addition to this, the protein (antigen), is it always present in the pathogen or is it heavily biologically secreted in pathogens when the pathogen are about to cause disease or produce toxin?
Does the antigen production differ in a pathogen at different stages or is it consistently same?
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Please do a literature search on expression pattern of the protein in your pathogen. Furthermore, if the antibody is specific for the antigen of your pathogen. Also if any studies have used a particular Ab please try them. Generally monoclonal are more specific than polyclonal.
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I will try a new ELISA protocol in my lab, and have to prepare the coating antigen. However, we only have inactived Mhyo cultures for this purpose. Has anyone tried to sonicate inactivated antigen for protein obtention and have good results? Do you think the inactivation could limit the protein quantification or even inhibit some epitops that could jeopardize my analysis? I appreciate if you guys give some light!
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Usually, a coating antigen is a soluble compound. Any larger particles need to be removed by filtration and/or centrifugation. From my experience, particulate materials lead to strange and irreproducible results.
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I want to test 8 antigens using in vivo (mice immunization and challenge) and in vitro assays (Elisa, elispot, growth inhibition, various cytokine conc measurements). Based on the data obtained and analyzed, 2 of these antigens will be selected for nonhuman primates (NHP) testing. My question is, How do I go about selecting the 2 antigens for the NHP experiments Do I use the elisa OD values, elidpot SI values, parasitaemia and cytokine conc values? If so, what will be the cut-off value for each assay? What if the different antigens perform differently in the different assays?
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I totally agree with colleaque Balam Saidou.Strain of causative agent of Malaria a parasite variant that is genetically unique and induce specific immune response expressed antigens in lab.animals(mice) that yield high immunogenicity are selectef as vaccine.mor detailed in attached ref.
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Does anyone know a good way of predicting E. coli K-antigen identity from NGS data? It seems like there are lots of options for O and H antigens (e.g. the excellent ECTyper) but none for the K antigen. I'm hoping to type some strains to better understand the host ranges of a phage collection. Any help gratefully received!
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If you have assembled genomes, this should be easily done. The most simple way I could think of right now is similarity based analysis, for example, BLAST.
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I got two .pdb files for antigen and antibody. I need to dock them, but I don't have a file with antibody-antigen conjugate. Is it possible to compute binding?
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Hi Mykta,
As Martin says, you can use the free online server above. However, if you want a bit more control and accuracy, then you might want to use something to probe the protein for likely binding sites first using something such as DeepSite (https://www.playmolecule.com/). This will identify likely binding sites which you can then focus your search on to produce more accurate binding predictions. You can perform slightly more detailed predictions using something such as Autodock Vina. If you can get hold of Schrodinger's Glide, then I recommend that, but it can be expensive.
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Hi everyone,
I am working on a lateral flow rapid test using the sandwich method. I tried different parameters to get the result in the full test but no success. However, it works in the half test format.
When there is no sample and conjugate pad and I put the antigen and conjugate AuNPs in a well and run the half strip, it works and when I run the full strip it did not even show a faint line.
Any idea about what to do to solve the problem.
P.S. I want to use the strip for detecting the antigen in 100 ng/mL concentration.
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It look that most of the conjugate is stuck on the pad and doesn't continue to the membrane. Try to separate the Sample application pad & conjugate pad (use glass fiber for conjugate), sorther sample pad also better, try add serfactant like triton or tween to the conjugate.
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Can anyone suggest any reference for this statement?
There are approximately 1 x 104 physical particles of lentivirus for every pg of p24 antigen
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There are two references:
one from research paper: Briggs, J.A.G.; Simon, M.N.; Gross, I.; Kräusslich, H.G.; Fuller, S.D.; Vogt, V.M.; Johnson, M.C. The stoichiometry of Gag protein in HIV-1. Nat. Struct. Mol. Biol. 2004, 11, 672–675. [CrossRef] [PubMed]
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When someone is tested for antigen and it's negative, it doesn't automatically say it's really negative, swab tests must follow through. Right? What if, the test must be all swab, so redundancy will be out of question. Don't you think so? Can you please enlighten me?
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I agree with you. The swab test for covid-19 is an accurate and reliable test for diagnosing covid-19. A positive test means one is likely to have covid-19. A negative test means one probably does not have covid-19 at the time of the test. Nevertheless, antigen tests are still fairly accurate, particularly when someone is experiencing symptoms and their viral load is very high. However, they can be less accurate when someone has a lower viral load, like in someone without symptoms. This could lead to false negative test results.
In situation where test turnaround time is critical, there is value in providing immediate results with antigen tests, even though they may have lower sensitivity. This will help quickly identify people with COVID-19, thereby adopting infection prevention and control measures and thus preventing transmission.
Best.
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To avoid false negative results (or false positive, if any), how long after completion of H. pylori eradication treatment is the ideal time for conducting stool antigen test?
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One month after completion of antibiotics course
Two weeks after stoppage of ppi.
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  • As we known, the naive B cell activation depend on T cell. The antigen alone cannot trigger B cell differentiation. However, whether the memory B cell could be activated by antigen without Th cell signaling?
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Actually, with certain antigens - ones that have a repetitive structure - primary B cells CAN be activated without T cell help. These antigens are known as T-independent antigens.
But memory B cells are absolutely dependent on T cell help. I spent several years as a post-doc working with Norman Klinman at Scripps on this precise topic. Look for a review by Phyllis-Jean Linton and Norman Klinman; she published that back in the late 80's early '90s. I do not remember the name of the journal.
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When running a FLUOROSPOT assay you need to do the following (briefly)
1) incubate cells on a bed of capture antibodies
2) remove cells
3) add fluorescent-labeled detection antibodies
Why cant the fluorescent-labeled detection antibodies be added along with the cells to enable real-time detection of antigen. As long as the detection antibody binds to only one epitope on the antigen it should work i.e. sandwich pairs for homogeneous ELISA
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The FluoroSpot assay combines the sensitivity of ELISpot with the capacity to study secretion of several analytes simultaneously, secreted at the single-cell level. Each spot will represent the analyte signature of single cell. Presence of cells and the accompanying cellular debris (if any) will interfere with the final results as it may give rise to artefactual spots. Additionally, presence of dead cells may result in high background staining. So, it is necessary to wash out the cells after they are done.
The analysis of FluoroSpot assays is based on spot parameters such as spot size, spot intensity, spot gradient (fading of staining intensity from center to periphery of spot) and position. Therefore, as mentioned by John Hildyard , maintaining “neatness of spots” is an important factor.
Best.
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I'm not pretty sure I mix the NP-KLH and Alum correctly. I just vortex for about 30 minutes. Is that right?
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Adjuvant can be selected to elicit a tailar_made immune response to specific as well as biodegradable and nano _particles exhabit adjuvant be side homogenicity of the vaccine.
https://,www ncbi.nlm.nil.gov_>pmc
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Dear experts and colleagues,
I have an lateral flow assay that previously worked but this time the test line did not appear.
1) I have tried different vials of Antibody for test line and different vials of Antigen as the deterioration of protein may happen but nothing changed.
2) I check the reaction between Antigen and antibody in conjugation by dotting antigen on membrane and use conjugation as sample. It worked well.
3) I made another strip and dotted the Antibody on the membrane as test line (no control line). Then, I mixed the Antigen with conjugation and put the strip into this mixture. The dot appeared.
From above experiments, I assumed that the test line did not appear in full strip (having both test line and control line) experiment is due to the the Antigen did not react with the conjugation on the conjugation pad. So, after going conjugation pad, the sample contained Antigen and conjugation instead of Antigen+conjugation.
Have you experience the same situation? Could you please give me some advices and suggestion. Thank you for your help.
For your information: The conjugation pad was treated with sucrose, trehalose, tween 20%, BSA. The membrane was not treated.
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You said you are using the same monoclonal antibody for both capture and detection. Is your antigen divalent/multivalent? It is difficult to use the same monoclonal antibody for both capture and detection if your antigen is monovalent as both the detector and the capture antibody will be competing for the same epitope on the antigen molecule. Have you checked for non-specific signal when you did the spot test using the antibody spot by adding just the conjugate without the antigen and showed no signal?
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Antigen determines the specificity of the immune answer and the affinity of immune receptors (TCR and antibodies). A single vaccine with a single antigen will lead to a single CDR. Boosters aim to revive the dormant memory cells. Based on the logic of classical immunology, boosting with the same vaccine will not increase the affinity of antibodies/TCR.
If the given antigen varies from the vaccine, how boosting will increase the affinity of the antibody towards the antigen?
The only answer I think, is that by boosting we enhance the numbers of less specific antibodies to a given antigen, thus compensating the reduced affinity by increasing the avidity (quantity of less specific immune receptors).
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Dear Colleagues,
Thank you for your interest and answer to my question, but let me disagree with you and explain why.
Here I discuss theoretical two cases: when memory B cell enters into the Germinal center, and when Naive B-cells enter into the germinal center.
1) It's known that Somatic Hypermutation (SHM) and Class-switch recombination (CSR) occur upon Germinal Center reaction, but those processes are specific to a given antigen, in this case, a vaccine. Moreover, if IgM memory B cell enters into a germinal center reaction after booster injection, it only becomes more specific to a vaccine, and thus, less specific to an antigen that differs from the vaccine (a mutated form).
Please, also mark here, that IgG memory cells will not undergo a class-switch recombination.
2) A follicular B cell is a naive B cell, whose naive IgM receptor has some weak specificity towards the antigen (in this case - a vaccine). The repertoire of the immune receptor is fixed in naive cells, and it is defined by the probabilities of V-D-J recombination (3 x 10 in the power of 11 for immunoglobulin), which occurs in the Bone Marrow (for B cells) and in the Thymus (for T cells). The number of B cells with a given VDJ is equal to the number of total naive B cells pool divided by the probability of VDJ recombination. For example:
m = a/(3x10 in the power of 11); where:
m- number of B cells with the same VDJ recombination
a- total pool of naive B cells
3x10 in the power of 11 - a probability of VDJ
m is a naive B cell that has plasticity, meaning that it can undergo SHM and CSR when undergoing the germinal center reaction. However, when it comes out of the germinal center as a memory cell, it loses its ability to undergo major changes in the CDR (Complementarity-determining region). Thus, if a vaccine to a single protein, intended to protect against "a" antigen differs from the mutated
"c" variant with minimal changes, the antibody against "a" variant cannot be specific to "c". Moreover, boosting with "a" will decrease the number of "m" naive cells, that could otherwise undergo better specific changes when encountering with "c" mutated antigen. In other words, every time you boost with a vaccine that is different from a pathogen protein even minimally you decrease the number of cells that could be more specific to a mutant by stealing them from a naive pool and deviating towards the vaccine (n(c)=m-a; where n(c) - is a number of naive cells from m pool that could undergo germinal center reaction and give rise to BCR specific to mutated protein).
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Can we use AmberS99 for antibody (protein) and Charmm36 for antigen (drug like small non-protein) in md simulations? Or do we have to use only one forcefield for both?
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I have to simulate antibody (protein ) and antigen (carbohydrate) complex. Problem is glycam is a part of Amber software which is not available in my department. We only use Gromacs. Is there any other forcefield for carbohydrate that we can use?
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I have docked antibody Fab region with protein antigen using Zdock. Now I have to run MD simulation on Gromacs version 2021.3. Should I follow GROMACS protein in water tutorial or protein-ligand complex tutorial.
I need to study interactions between both proteins.
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Saleha Hafeez You can follow the Lysozyme in water tutorial by Dr. Justin. However, during the analysis, you will need to create two separate indexes for both the protein and then perform the analysis.
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I performed an indirect ELISA and I used BSA and skimmed milk with different concentration for blocking separately but the result didn‘t change and negative control change color. Also I test different dilution of the antigen, primary antibidy and secondary antibody. But there were no difference between the positive and negative control. I hope some experts can give me a solution.
Looking forward to your reply.
Zahra
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If either the positive or negative control does not produce the expected result, it indicates that the investigator should reconsider his or her experimental procedure. Zahra Rezaei
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Dear all, I have been working on antigen presenting experiments in cancer pathogenesis, recently, I am just wondering that is there any tumor-associated antigen that can be chosen to stimulate T cell proliferation? Thanks a lot!
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A regularly updated database of antigenic peptides effectively presented by tumor cells can be found in the link below.
You can also refer to the papers I have attached.
Best.
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I am investigating T cell responses to peptide-vaccine constructs in mice. The general scheme is 10 days after immunization (peptide X-CFA) at the base of the tail, I harvest the inguinal lymph nodes and isolate T cells. I co-culture the isolated T cells with irradiated feeder cells from the spleen of an unimmunized mouse together with no peptide (unstim) or peptide (stim). I also use a positive control of PPD (antigen in CFA). The ratio of irradiated feeders:T cells I use is 100k:400K (per 200 ul in a well).
I read out proliferation via CFSE on day 3 where I stain only the isolated T cells (not the feeders) prior to setting up the culture. The problem is, I seem to get quite a large amount of proliferation in the unstimulated condition in which there is no antigen/peptide. Further, I don't seem to be getting a response to the peptide I immunize with anymore.
I optimized my protocol over the past few months and I was getting quite good results in terms of proliferation. But ever since I switched to irradiating my feeder cells as opposed to treating them chemically with mitomycin C, it seems my assay is not working.
Is it a problem with my co-culture i.e. are my feeder cells not presenting the peptide? Why am I seeing such significant proliferation in my unstimulated condition?
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Chenglong Wang Thanks for you thoughtful response.
Let me know whether or not your mice colonies are infected. As for the foxp3 cells, did you find a specific source citing this? You are saying that in the absence of stimulation, these cells may induce low levels of proliferation among all T cells in culture? Please elaborate.
My proliferated cells show dye dilution as well as increased FSC. Please see my data attached below. This experiment compared using irradiated vs mitomycin treated feeders. I assayed both the LN and spleen of CFA-immunized animal and did a recall response with PPD. I compared an old batch of PPD we had (which works very well) and a new batch we recently got, which does not seem to work...Anyways, the point is to show you what my unstimulated and stimulated cultures look like in terms of the dye dilution and FSC. I am using cell-trace far-red (APC). These graphs simply come from initial gating on T cells followed by doublet discrimination. Note that you can see that irradiation seems to minimize feeder cell proliferation as there are less unlabeled (APC) cells in my gate compared to the mitomycin treatment.
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I want to release the antibody from an antigen-antibody complex formed in an immunoprecipitation assay, and then the antibody should be separated from the antigen. Can anyone help me in this regard ?
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If you are not bothered about denaturation, you could simply boil in SDS sample buffer and separate the proteins on gels. If your antigen is around 50 or 25 kDa, use non-reducing sample buffer to move the Ab to 150K on the gel. Otherwise I would conjugate the Ab to CNBr sepharose and elute with acid as noted by others.
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I want to test in vivo immune response with different model antigens, is there any well-characerized model antigen similar but irrelevant to OVA? It would be appreciated with relevant work paper or links attached. Many thanks!
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You're Welcome Celine Chen Please do look up.
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Hi, we are using IFNg-ELISpot assay on PBMC stimulated with Tet Tox and KLH. Unfortunately we are seeing higher spot counts in our media only wells and not sure why, could anyone please shed some ideas?
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Hi Vanessa,
I would believe your media got contaminated with bacterial components, such as LPS. Depending on the immunological status of your cells previous to the stimulus, you may detect more spots than expected with these type of contaminants, even in control conditions.
Another interesting point to pay attention is the spot size among different stimuli. Are the control well spots similar in size with those detected in the presence of your specific antigen? Sometimes, different stimulation may generate spots of different size and intensity.
Thus, I suggest you to add antibiotics to your media previous to its addition to the cells on the ELISPOT plate.
Best of luck,
Eduardo
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immunization with NP-OVA hapten antigen
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Usually, NP-OVA is 4-hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin protein through lysine. Commercial alum suspensions are often directly mixed with the antigen.
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Looking for a reliable source of Canine and Feline-specific pathogen antigen.
Thank you.
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In the absence of purified antigens, I could think of vaccines for dogs and cats, but that will depend on the nature of the use.
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If mRNA meet an organism what will be the mechanism of action and the barriers he must cross?
The higher body is used to seeing many types of antigens break in. The immune system is able to cope and then keep in memory an imprint in humoral or cellular form. This is the classic pattern. Is the introduction of mRNA in an unusual form, is not at issue. The stabilizers and other additives of the vaccine are well mastered and are then used perfectly by the manufacturers of vaccines.
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Adverse reactions after vaccination occur for a variety of reasons.
In classical vaccine manufacture, there are often contaminants from the host cells used to produce large amounts of the virus - this is the classic egg component issue found in flu vaccine production.
Alternately, when attenuated viruses are used, sometimes the vaccinated person fails to respond to the vaccine, and the attenuated virus cases the adverse reaction. This is the cause of the rare side effect of measles vaccination, sub-sclerosing pan-encephalitis.
Sometimes the vaccine triggers immune cross-reactions to other antigens; these can be self antigens or exogenous antigens. This is why hay fever is such a common allergen - there is a pathogenic fungus that triggers a cross reaction to the hay allergen in some people.
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I am running and experiment where I use cryopreserved PBMCs as the following:
1) thawing and incubation on RPMI-1640 in CO2 incubator overnight
2) activation with IFN-alpha for 15 min
3) fixation with BD fix/lyse buffer (10 min)
4) surface antigen staining
5) permeabilization with BD perm III buffer (30 min on ice)
6) intracellular antigen staining
When i run flow cytometry I get this image on FSC and SSC. I can't identify which population is lymphocytes as i see CD3+ in both population 2 and 3.
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It iw true you might need experience to read the flow cytometry result, bit maybe say it could depend on both. Either you make sure your standard ( which could subtract the errors from controls) or from the properties of the lymphocytes. Size, density, granules, nucleus. You might see, but maybe is it "determinated"?. Maybe sqy it is a low experimental result.
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is LC/MS MS appropriate for antigen detection?
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Hello,
You can refer to the research article given below. It will be helpful.
Best.
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I have a basic immunology question that I am not able to find a definitive answer to so far.
B cells require activation by a T helper cells in order to turn into a memory B cell.
But is it necessary for the T helper cell and the B cell it activated to be specific to the same antigen?
My colleague says that CD40-CD40L and CD28-CD80 co-stimulations are not enough and describes a different process;
B cells must internalized the BCR-antigen complex, process the antigen and present it on an MHC-II molecule to a T helper. Only when a T cell is activated this way can it can activate the presenting B cell. Meaning, they must necessarily recognize the same antigen for the B cell activation and maturation to occur (of course, different epitopes in that molecule, but still the same protein antigen).
I was not taught that this step is critical; but that any stimulated and licensed T helper can provide the required co-stimulation to any activated B cell. Antigen-specific activation occurs stochastically, because both APC, T helpers and B cells are present in the same area when an antigen is incountered.
So far I can't find a clear answer for which of the two option is true, either in the basic or more specific literature.
Can anybody shed light on this, preferably with reference to proper sources?
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That makes sense.
Thank you for the input!
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When you use RIA, with a control the unknown sample with antibodies, you bare in mind the binding sites of the antibodies. However you still need to measure labelled antigen (radioactive) and not labelled antibody; labelled antibody and not labelled antigen. What is the formula you need to use, what to do with the mix of antibody/antigen/labelled/
unlabelled and why would you need to do it?
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thank you!!!
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Recently, I want to verify the certain treatment enhancing on antigen presenting ability of macropohages.
I searched the literitures, using OT.II T lymphocytes and OVA antigen, OT.1 lymphocyte and OVA antigen are often chosen to verify the ability on CD4 and CD8 T lymphocyte differentiation and proliferation.
I want to konw whether there is any other method to do the experiment. Because we do not have OT.II or OT.I mice.
How about co-culture the Naive T cells and macrophages in the presence of certain antigen "X". If this method is acceptable, what antigen "X" could be?
ps: Infectious and malignant diseases like tuberculosis and lung cancer are our intersted field.
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A follow-up idea: one way to look at a larger portion of the T cell population and response would be to use a superantigen, such as staphylococcal enterotoxin B [SEB]. Superantigens bind class II MHC on antigen-presenting cells and to multiple Vbeta regions on the TCR. I cite SEB because it binds to I-Ab on C57Bl/6 mice. The problem here is that your readout may only be detectable in CD4 T cells and not CD8 T cells, but I do know that SEB on C57Bl/6 APC triggers a significant proliferation response by T cells.
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Hello
I have a problem with ELISA test
I try to do Ag detection ELISA for quantification my antigen
So i hyperimmunized rabbits and guinea pigs with the same antigen then designed the capture ELISA As bellow:
1- coating with Guinea pig Ab from hyperimmune serum // antigen // rabbit Ab from hyperimmune serum// antirabbit HRP conjugate// substrate then stope solution.
2- coating with Guinea pig Ab from normal serum serum // antigen // rabbit Ab from hyperimmune serum// antirabbit HRP conjugate// substrate then stope solution.
3- coating with Guinea pig Ab from hyperimmune serum // PBS // rabbit Ab from hyperimmune serum// antirabbit HRP conjugate// substrate then stope solution.
4- coating with Guinea pig Ab from normal serum serum //PBS // rabbit Ab from hyperimmune serum// antirabbit HRP conjugate// substrate then stope solution.
5- coating with Guinea pig Ab from hyperimmune serum // PBS // PBS// antirabbit HRP conjugate// substrate then stope solution.
6- coating with Guinea pig Ab from normal serum serum //PBS // PBS// antirabbit HRP conjugate// substrate then stope solution.
As a result in the cases 2. 3. 4 i have got OD less but very close to the OD of the case 1, while 5 and 6 were equal the blank's OD.
My question, is it possible to have such ant species reaction between rabbits and guinea pig sera / or Ig G. and how can i solve this reaction
Thanks in advance for your comments
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The other possibility is that the rabbit hyper-immune serum contains antibodies that cross reacting with guinea pig Ig. This is not an uncommon finding. One way to eliminate this is by adding purified guinea pig IgG to the rabbit hyper-immune serum and filtering it [I use Acrodisc 13mm syringe filters with 0.2 um HT Tuffyn membranes] prior to adding to the plate. The guinea pig IgG will bind to any anti-guinea pig antibodies in your rabbit hyper-immune serum and those immune complexes will be retained by the filter. I used to do this routinely to block anti-mouse Ig cross reactive antibodies in a commercial goat anti-rat Ig product for FACS.
You will have to do some test runs to determine that correct amount of guinea pig IgG to add.
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I have generated a complex of antigen and antibody using haddock and used that cluster for MD run using Gromacs. Now I need to find out the inter hydrogen bonds formed between antigen and antibody complex. what commands do I need to give ?
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ok, thank u, sir, actually these no. of hydrogen bonds are may be individual H - bonds present in each protein 200 and 1600, but I am not sure.
and one more thing is it possible to take one protein as a ligand and run MD as ligand and protein complex ?
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We want to place a large order, but we are not sure how reliable they are. Our local suppliers costs us a lot more, since they also source it from similar foreign merchants, and we wanted to cut down the expense. 
We want the product shipped to India, and would like to have some assurance before placing the order. Has anyone had any experience with them in terms of international shipping? Any suggestions or information in the regard will be appreciated. Also, if anyone has used their own products, please let us know of your experience.
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See this Nature Article about the "Independent Validation" Program set up by antibodies-online.com to fight the Reproducibility Crisis (https://www.nature.com/articles/521274a)... antibodies-online.com cleary strives to provide reliable products... other than that: antibodies-online has been serving the scientific community since 2006 with offices in Europe and the U.S.
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Hello everyone,
I am currently trying to understand CAR-T´s mechanisms of action, but got stuck at a certain aspect.
2nd gen. CAR´s upwards require co-stimulation of e.g. intracellular CD28 domain to support T-cell proliferation and persistence. What I dont understand: How is the CD28 domain stimulated?
The CD80 of the target cell cannot bind directly to it, since the CD28 costimulating domain is intracellular. Is it activated along with an antigen binding to the CAR?
Looking forward to your response.
Regards,
Matthias
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Dear Matthias,
All the ligand-receptor bindings are not just bindings, tow examples for the typical consequences of the binding, one is multimerization, like TCR engagement, the other one is to recruit other molecules for splicing, like Notch signaling.
The transmembrane domains are often ignored, but as in TCR signaling events, they are quite significant for multimerization. Sole TCR would never work, they need to be clustered together to deliver the signals.
So for your questions, the CAR structure always compose an extracellular domain, a TM domain, and an ICD. The ECD, like anti-CD19 scfv, or others, is responsible for binding, and then clustered, then the signaling of CD28, amplified, and delivered.
I can't put a reference here, but I would suggest a key term for your retrieval: TCR signaling and clustering.@Matthias Müller
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When the antigen-presenting cell process the pathogen, does it have any preferences in choosing which piece of fragments to present to T help cells?
Thanks in advance!
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The length alone is not sufficient, the sequence of the peptide determines the affinity of the peptide to the MHC (
Article Peptide binding by class I and class II MHC molecules
)
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I saw that my antigen of study can activate T cells once in co-culture with PBMC. How can I actually demonstrate that it is internalized by dendritic cells and presented?
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Demonstrating presentation by dendritic cells means having to show that a particular peptide made it into the dendritic cell and back to the surface on MHC.
This requires antigen specific T-cells to respond to the dendritic cell.
Internalization can be demonstrated by using a radioactive labeled antigen (or other label). Add to your PBMC, wait an appropriate amount of time (you will need to determine this). Then treat cells with a strong protease to strip off any antigen on the surface. Then spin the cells down, measure residual radioactivity. If you don't wish to label the antigen you can also do this with an antigen capture assay by lysing the cells and detecting antigen. Controls consist of cells at 4 degrees C during the antigen incubation. The antigen will stick, but will not be taken into the cells.
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I am trying to perform indirect ELISA for newly purified recombinant antibody. I coated the antigen and use BSA as my negative control but I am not sure what can I use as a positive control. would you possibly help me with that ?
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Lora Benoit Thanks for the help
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I have C6/36 cell lines infected with serum sample. Can i do NS1 antigen detection in supernatant culture fluid? Does it requires purification step?
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Hi, arigo has a Dengue virus NS1 ELISA Kit that can be used to detected Dengue virus NS1 from serum, plasma, and cell culture supernatants samples. Please refer to the detail at https://www.arigobio.com/Dengue-virus-NS1-ELISA-Kit-ARG81357.html.
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Hello
I'm growing EBV-LCL cells but they are not growing well. Any idea why this is happening? Can it be caused by the reactivation of EPV virus stressing LCL cells? Should I use EPV antigen test?
Any suggestions are appreciated.
Regards
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In that case you may find the attached file interesting Melika
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I would like to determine whether my vaccine candidate is able to elicit a cellular immune response in Balb/c mice. I was thinking of isolating the splenocytes from the inoculated mice, stimulating them with the peptide antigen and then doing FACS to determine the numbers of CD4+ and CD8+ cells that have proliferated. How long should I incubate the antigen with the splenocytes before harvesting the T cells for FACS?
I would also like to do a cell-mediated cytotoxicity LDH study to determine whether a cytotoxic T cell response has been elicited. In this case i would like to incubate the macrophages from the isolated splenocytes with antigen and then add the T cells from the splenocytes the following day to determine whether they are able to kill the macrophages. I was thinking of just incubating the T cells O/N in culture medium before adding them to the peptide-stimulated macrophages the next day. My questions are the following: Should the T-cells be stimulated in any manner prior to the addition to stimulated macrophages? Also, how long should i incubate the T cells with the macrophages before measuring the LDH released?
I am looking forward to your responses,
Daria
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Thank you very much Martin. Your help and advice is very much appreciated. I will let you know how things fare:-)
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we are developing a test to detect myoglobin in plasma, serum and whole blood samples. When using a recombinant antigen, the sensitivity of the test is satisfactory, the concentration of the antigen is appropriate. Clinically validated samples also work well, but negative samples have a strong false positive reaction. buffer solutions described, the membrane is dried in the desired humidity and temperature. How can the problem be solved? Thank you for your answers, colleagues!
for antibody dilution PBS 0.01 M + 2.5% BSA V = 100 ml Na2HPO4 - 0.116 g NaH2PO4x2H2O - 0.028 g NaCl - 0.877 g BSA - 2.5 g ph = 7.2 -7.4 for diluting the conjugate concentrate PBS No. 4 V = 100 ml NaCl - 0.877 g KH2PO4 - 0.136 g BSA - 0.1 g NaN3 10% - 100 μl ph = 7.5 sucrose - 10 g for sample PBS 0.01 M + TWIN V = 100 ml Na2HPO4 - 0.116 g NaH2PO4x2H2O - 0.028 g NaCl - 0.877 g TWIN-20 10% - 250 μl NaN3 10% - 200 μl
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Dear Yulia,
look for cross-reactivity. Have you checked the peptide sequence that you have been using as immunogen for homologies with other proteins (e.g. bz BLASTing it against GenBank) ?
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protocol for Fitc conjugation of fmdv antigen . Related any document
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Thank you
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Do Cancer survivor, (particularly solid tumor survivor) treated by Radiation and (or) Chemotherapy have high immunological memory against cancer antigen compared with Normal population ?
or
Do cancer survivor are highly resistance to "De Novo" cancer i.e cancer that is not associated with past treatment (or) previous cancer compared to Normal population ?
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Hi All ..
Please find relevant article addressing the aforementioned Question
Han, J., Zhao, Y., Shirai, K. et al. Resident and circulating memory T cells persist for years in melanoma patients with durable responses to immunotherapy. Nat Cancer 2, 300–311 (2021). https://doi.org/10.1038/s43018-021-00180-1
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Hello,
I am trying to stain glioblastoma cells with an anti-CD56 antibody in PE. For adherence of the cells, I am using PBS with magnesium and calcium.
Almost every cell seems to express CD56, but I only obtain very weak signals in the PE channel. Even when I am adjusting the exposure time.
Could this be because of the PBS that I am using? I've read somewhere that ions might hinder antigen bindung of the antibody...
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Not at the relatively low concentration of these ions in this buffer. You wold need several M to possible have any adverse affect. Worth doing the incubation at room temperature or 37c to be sure. Reserve 4c for overnight incubation.
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Dear colleagues I am looking for a freezing tissue protocol which can preserve good morphology and antigenicity for immunofluorescence. The scientific literature is scarce on this topic. I am not sure how to snap frozen, liquid nitrogen or isopentan? How to store samples, in -20, -80 or liquid nitrogen, which option is better? OCT cryopreservative should be used to store samples? Can it improve morphology quality?
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Do you mind post one publication you used with the acetone protocol. I got very interested in try it.
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Need to design antibody against antigen.
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Do you need to specifically discriminate Rosetta cells against other bacteria) e.g other E. coli)?
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For an adoptive transfer would like to isolate CD8 T cells that express granzyme K and EOMES from the spleen. Is it possible to first isolate CD8 T cells with an isolation kit, followed by the use of antibodies for granzyme K and EOMES?
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If you're worried about antibodies sticking to your cells after isolation, I can recommend the Fab-TACS® system. Instead of high affinity antibodies, low affinity Fab fragments are used to positively select cells. They completely dissociate from the surface after isolation, so you don't have to worry about antibodies remaining on your cells. You can check out the products here:
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I am trying to develop a uPAD that is similar to wax printed paper based ELISA techniques that have been developed.
However, I noticed that such devices are based on sandwich ELISA by immobilizing an antibody in the test zone as the analyte is then an antigen. I seem to understand antibodies adsorb well (are immobilized well) on paper.
In my case, I want to perform an indirect ELISA such that an antigen would be immobilized in the test zone. After which blocking / washing would be done before adding the sample (containing an antibody which would be the analyte in this case), washing, and then adding the substrate for the colorimetric reaction.
However, I am having difficulty finding information on whether antigens can be immobilized effectively on cellulose; will they be dislodged from the test zone when applying wash and different solutions when running the test?
I appreciate any help on this matter.
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Hey, I am not sure if I understood your goal. But as I have done already Nitrocellulose based ELISA (MBA-membrane based assay) I would recommended to take a look my paperz
Take a look the protacol.
If you need any extra help, DM me.
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How many antibodies can be used, or in other words, how many antigens/molecules can I detect with immunohistochemistry?
I read somewhere that only two antibodies can be used but is this really correct?
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Most immunohistochemistry (IHC) is performed with a single primary antibody, however there are protocols for performing multiplexed IHC. One common example is double staining with DAB (brown or black staining) and alkaline phosphatase (red). However, it is generally recommended to use immunofluorescence if you need to probe for multiple antigens on the same slide.
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As COVID-19 has shown the strain variations, various mutants, if we developed vaccination at one place, r we sure that perticular gene has not mutated take example of E-gene of coronavirus.
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I appreciated way in the paper Antigen-detection in the diagnosis of SARS-CoV-2 infection using rapid immunoassays: interim guidance, 11 September 2020 (WHO) by Muhammad Yousuf
And also the experiment like for finding the common and differences of each newly discovered strain of COVID within a short span of time.
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I have cryopreserved OCT mounted human nasal polyp tissue slides, which I want to study post-immuno-fluorescence staining. I have the following concerns-
Whether an antigen retrieval step is required? If so how to retrieve antigen?
Which anti-fade/mounting medium should I use?
I will appreciate it if someone could suggest a good protocol or at least addresses my concerns. Actually, the protocol, I saw, is not suggesting any antigen retrieval step. Further, most of the available anti-fade mounting media are xylene based and I guess, for OCT frozen tissue, staining does not require xylene. Therefore, I have a concern about the use of these anti-fade mounting media.
Thank you
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Hi Mohammad Asad , this is the protocol I use, for animal tissue in OCT at least. you can start by washing the tissue in phosphate buffer (physiological pH). You should also wash in this buffer between each step. Then block for 1hour in 1:100 dilution of bovine Serum albumin and normal goat serum. Wash and incubate in primary antibody for ~18 hours (overnight)(this step will need to be troubleshooted based on the antibody you are using), then incubate with secondary raised to the primary antibody, for one hour. (The secondary antibody should be biotinylated) Then incubate in avidin biotin complex. This complex needs to be pre incubated for at least 10-20 minutes before being applied to the tissue (1:500 avidin and 1:500 biotin), once the complex is formed, let it incubate on the tissue for 1h. Then, add your PE dazzle 594 conjugated to streptavidin and incubate again for an hour. Hope this helps!
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