Questions related to Antigen
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.
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!
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.
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?
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.
- 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.
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!!
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.
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?
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?
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
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
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
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?
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?
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.
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.
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
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?
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?
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?
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.
Im looking for the Amino acid sequence of the exact peptide presented to the T CD4+
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?
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!
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?
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!
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?
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.
Can anyone suggest any reference for this statement?
There are approximately 1 x 104 physical particles of lentivirus for every pg of p24 antigen
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?
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?
- 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?
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
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.
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).
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?
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.
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.
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!
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?
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 ?
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!
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?
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.
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.
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?
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?
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.
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
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 ?
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.
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.
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!
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?
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 ?
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?
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,
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
Do Cancer survivor, (particularly solid tumor survivor) treated by Radiation and (or) Chemotherapy have high immunological memory against cancer antigen compared with Normal population ?
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 ?
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...
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?
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?
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.
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?
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.
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.