Immunology - Science topic

May be this article can help

I think it will be best if you read a Basic Immunology book that has a chapter on hematopoiesis (Basic Immunology by ABul K Abbas and others) to understand why and how you can make the discriminations of different cell populations. Company catalogs will provide you information on the antibodies one can purchase from them, basic knowledge is in the books.

With best wishes,

It's better if you provide more details about your questions. based on my experience, the flowjo software can show distinct populations based on their marker expression intensity. in a flowjo plot, one dot represent one cell, so you can also get the percentage of every population you have. these are basic functions that flowjo have, for more detailed, you can search on the official FLOWJO website or there are some useful youtube video that show how to analyse flow data by flowjo.

Hi, also you could give a try to ENCODE. Check out the following link, there is an overview of the immune cell lines profiled by different omics.

Not really. There's been a ton of effort going into developing tools to do this but most are in early stage development and not ready for routine usage. The main issue is a lack of training data that can be used to refine these algorithms.

There are new in vitro high-throughput functional TCR antigen profiling techniques that have come online in previous years, including the one we developed: https://www.researchgate.net/publication/336310777_Rapid_selection_and_identification_of_functional_CD8_T_cell_epitopes_from_large_peptide-coding_libraries

These approaches are powerful because they let you directly test large libraries of candidate sequences in an unbiased way. If you have any interest in trying out our method, send me a DM and we can talk further.

Progenitor cells in the tumor are usually Granzyme K⁺ and slowly proliferate. As the cells flip towards transitory/effector-like they become Granzyme B⁺ and stay Granzyme B⁺ as they further exhaust. So in essence there are Ki-67⁺ (with the capacity to proliferate) cytolytic cells and non-proliferating/terminally exhausted subsets.

It is stochastic, or random. The receptor repertoire based on vdj, alpha beta v delta gamma and n and p additions result in a nearly unlimited. Umber of potential receptor rearrangement. The naive t cells enter secondary lymphoid organs at the instruction of chemokines and 4andomly test against antigen presented in the context of correct mhc. In this scenario, it I'd the antigen that selects the correct tcr, resulting in clone proliferation. The t cell is the passive player.

Vipul Batra Thank you very much for your information!

Can I also get recommendations for universities offering masters in the same subject abroad as well. I am also looking for those options as well.

Hi André Cronemberger Andrade I read your comment and I would like to know if you have any protocol for what you mention about sensitizing the elisa plate to later detect surface proteins in exosomes. Do you use elisa plates of any particular brand?

I would appreciate it if you could help me, thanks in advance.

Do you already have reagents or a kit to measure total IgE and anti-dsDNA IgE? I would suspect the most straight forward way to measure total IgE is with a kit. Then for the anti-dsDNA IgE, you'll need to coat plates with dsDNA (followed by serum, then conjugated anti-IgE). Unfortunately, you'll probably just have to experiment to find the best dilution. But for anti-dsDNA IgE in an otherwise healthy mouse, I'd probably not dilute the sample at all and just report the result as O.D. Then test different dilutions depending on how you're setting up your standard.

Also these are good:

1- Writing Research Papers. Student's Book: From Essay to Research Paper

Dorothy E. Zemach, Daniel Broudy, Chris Valvona - 2013 - 128 pages

2- English for Writing Research Papers (English for Academic Research)

Part of: English for Academic Research (11 Books) | by Adrian Wallwork | Mar 3, 2016

3- https://www.scribbr.com/category/research-paper/

https://www.nature.com/articles/s41592-021-01380-4 Deep Learning based approaches for protein structure prediction have sent shock waves through the structural biology community. We anticipate far-reaching and long-lasting impact.

As far as I understand, your data points/conditions are nested within individuals (donors) creating non-independence. In other words, the same individuals provide data for each condition. If that is true, then a paired test such as repeated measures ANOVA would be most appropriate. One way to examine this is by looking at whether the observations across conditions are correlated. For paired observations, you will typically find a positive correlation of the DV scores across conditions reflecting the non-independence of the data points.

Hello Hasanthika C. Adhihetty

Immunology is a branch of biomedical science that deals with the study of immune system in health and disease. It has applications in various disciplines of medicine like oncology, virology, bacteriology, and organ transplant. With a Masters in immunology, you have various options open like Clinical Research Assistant in hospitals and clinics, technical support in clinical instrumentation like flow cytometry and ELISA-based techniques, and as a teaching faculty in universities.

Immunology is an interesting subject. If you wish and have a liking for immunology you can study further and complete your PhD in immunology and become an immunologist. This will be an added advantage as it will help you acquire a much higher position in academics, research or pharmaceutical industry.

All the Best.

Have a look at these useful links.

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Sorry it is outside my aera of expertise

Let's change it this way, Its possible to get serum from EDTA-K₃ plasma? the answer is yes.

Briefly, Centrifuge sample for remove of blood cells and keep buffy coat plasma at -20 °C. After thawing of the sample, dispense in another tube 1.0 ml of plasma add 26 μl of 1 M CaCl₂ and incubate overnight in +4 °C. A clott is present at the end of incubation that must be squeezed and removed, obtaining clotting factors free serum.

The difficulty is that if Antibodies bind nicely to your antigens it's usually that they are very affine to them and subsequently separating them is sometimes denaturing the antibodies themselves. They are some Mild elution buffer made up by Pierce you might give a try on that.

Other than that is to use maybe a higher salt content to protect antibodies but this is rarely working perfectly

Some more litterature you might want to read

China is the best choice, there are vast opportunities for the young

Hi,

Very important question. Moreover, the question is why (.... from my current knowledge please correct if I'm wrong) from more less 100 mAbs approved by FDA only 2 are bispecific ..... I think another trend is competitive for the "multispecificity" of mAbs. Its cocktails of mAbs (cocktails therapy). See some examples below:

1 https://www.fda.gov/news-events/press-announcements/fda-approves-first-treatment-ebola-virus

2 https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-monoclonal-antibodies-treatment-covid-19-0

3 https://www.fda.gov/media/106517/download

4 https://www.fda.gov/drugs/drug-safety-and-availability/fda-authorizes-bamlanivimab-and-etesevimab-monoclonal-antibody-therapy-post-exposure-prophylaxis

I think that cocktails therapy will be a more dynamic trend than "multispecificity" (because of higher and higher availability of different monospecific monoclonals)... and many other reasons,

Best regards

Tomasz

Fcs alternatives for HL 60 neutrophil like cell line is FPS (slightly different).

FoxP3 + should be CD4+CD25+ Tregs. of Couse,FoxP3 should overlap with CD3+ T cells. usually CD3 should be expressed on the cell surface, while FoxP3 should be expressed in the nuclear of cells. they should co-expression in one cells. if you see they are differently localized. it means it is a unspecific binding and it is not correct. I often see some published paper they are wrong! confocal will detect them more accurately!

Overall, PRNT has been reliable and a mainstay for virology and vaccine development. Here are the limitations I've found personally:

1) The throughput of the assay is somewhat limited due to the necessity of plaque counting as opposed to an automated read-out of reporter gene activity. I've really only done manual counts, and it looks to be somewhat difficult to automate the process. There are scanners with software for counting, but the visibility of the plaques and contrast with the uninfected monolayer can be limiting and the software may not give accurate counts. Related to this is the need to have the plaque size of the target virus being uniform so that counting is accurate.

One workaround is to examine the dilution series of each antibody and count only the two wells that bracket the endpoint, such as the 50% reduction in plaque counts. That can often be done in a hurry or to get preliminary data around the endpoint titer, but you lose a lot of information that way.

Another caveat for plaque counting either by human or machine is the necessity for cell monolayers to be uniform and complete enough in each well to get accurate plaque counts. If the cells do not form a 100% uniform monolayer by the end of the plaque growth against which the plaques can be counted, then it gets tricky, especially if the plaques/monolayers are stained with a dye rather than by immunochemical staining which may be better at showing the infected cells on a bad monolayer but is more time intensive.

2) The biosafety containment needs to be commensurate with the target virus biosafety. Often, researchers use the replicating virus of interest in the PRNT as opposed to a pseudovirus or chimeric virus which can be made to have lower biosafety requirements but will often have a reporter transgene if one is going to all the trouble of constructing these.

3) For some viruses that are more difficult to neutralize than others, it can be a little tricky to normalize the input virus between assays. This is more of a problem with the relationship between virus and neutralizing antibody concentrations, but practically speaking, inherent variability of the virus input can alter the results up or down depending on how much virus ended up in that assay. That can create more work in trying to put a large dataset together and have the assay data from each run match each other, say by the use of the results of a standard serum/antibody used in each assay.

4) One meta-problem that I've encountered is not the assay's fault, but rather the design of the assay with respect to the target cell. Viruses can enter the cells of various cell types in various tissues by different mechanisms, so the use of a non-physiological cell type for the neutralization assay can give nice, consistent results, but results that aren't useful going forward. For example, the human cytomegalovirus (HCMV) field for years employed primary human foreskin fibroblasts as the cells in the assay for which the neutralization of virus infection by antibodies was measured. This use is/was due to the very limited range of cell types that are available for permissivity of this virus for replication and plaque formation, so the cell type used is one of convenience (which itself is relative since these cells have limited passages). HCMV vaccine development experiments used these cells, and it wasn't for decades was it discovered that viral entry into endothelial cells is a more accurate assessment of neutralizing antibody responses compared to blocking entry/infection in a fibroblast. Thus, the fibroblast data wasn't particularly enlightening in the context of neutralization mediated protection. The cell type in the assay should be scrutinized when embarking on PRNT assays rather than treating them like another reagent in the assay.

I submitted a survey. Best of luck!

Live cell imaging with specific marker proteins

Have a look at this useful link.

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Dear Dr Larenas,

I invite you to read these articles to understand the relationship between SARS-CoV-2 and allergen immunotherapy:

Best regards,

Pr Hambaba

Ashraful Hoque sir, please share your opinion on this...

Hello, I find these interesting articles:

Best regards,

Pr. Hambaba

Dear Nicolas,

I invite you to see this article:

Best regards,

Pr. Hambaba

Polyvalent vaccine commonly used in vaccination policy from different serotypes for the same disease.The occurance of vaccine Anti_BRD clonality is a lot and more narrow but if antibody enhancement in vaccinated region ,no clonality elsewhere to compasate,you must consider these variant invaccinatin programm under scientfic base vaccine must be strain variant vaccine(Include the variant in the region.

Hi Samuel,

Even though this discussion is from 2016, I was wondering if you are able to comment on your observations of the Hela cell lysis? In particular, do you still see the membrane, just non-spherical? I am currently attempting a hypotonic lysis of HEK cells, but for the wash step am only adding water to a cell pellet and discarding the supernatant with out resuspension as I want to avoid premature lysis of cells before a final resuspension step in water and sample transfer. If, I can, I will attach an observation of HEK cells after 15 mins. The membrane is not circular and you can visibly see strands coming from the cell after staining with Propidium Iodide. I plan to run some proteomics experiments on the cells, so I need to be absolutely sure that they are lysed. Any help would be greatly appreciated!

Cl.tetani spores after infection to wounds germinate due to the action of tetanolysine which favour anaerobe enviroment for getmination and production of tetanospasmin which resposible for stiffness of muscles mainly massetric muscles thigh.

Immunoprophylaxis of tetanus is restricted to toxin neutrlization.Tetanus toxoid is an aluminum hydroxide suspension is given for routine propgylaxis and a single injection will induce protective immunity in to 10 to 14 days

@* My answer is no.

IsoFlow in Beckman coulter or sheath fluid in BD machine are the isotonic buffers solutions which may be replaced by Autoclaved and filter sterilized PBS.

You should be extra cautious while using in-house sheath buffer as any particle or growth may block your tubes in flowcytometer.

We have used Autoclaved and filter sterilized PBS in BD instrument and it never caused any issue.

Once you use any in-house buffer, the parent company will consider it void of warranty. So, never let the company people know that your are not using their isoflow.

Good luck,

Dear Abdelwahab Zemam

Please see my CFSE proliferation protocol attached and two papers for the results. The outline is for B cells, but it's easily adaptable to any other lymphocyte population.

Is there any public patient RNAseq data repository related to COVID? If there is, and if they have FASTQs of them, any research may be done with those data by you at home. It may depend on the amount of meta-data connected to them, but you can search some relationship between some factors and some gene/gene set expressions.

Huggins, Blocksom and Nooman (1936), reported that the more centrally located bones of the extremities, the cranial bones and the sternum of Rats, Rabbits and Pigeon have temperatures similar to that of the peritoneal cavity. Whereas the peripheral bone marrow of the extremities showed significantly lower temperatures. (https://journals.physiology.org/doi/abs/10.1152/ajplegacy.1936.115.2.395)

However, in 1952, Nicholas Petrakis, published a paper on the determination of temperature of the human bone marrow in the Journal of Applied Physiology. The authors actually reported the temperature of the human bone marrow but the article is closed access so you have to pay to get it. here is the link https://journals.physiology.org/doi/abs/10.1152/jappl.1952.4.7.549?journalCode=jappl

I think yes.

Thanks

I am looking forward your publish.@Maral Barzegar-Amini

Please see the link below.

this URL maybe hep you:

IgM is basically 5 IgGs glued together, which means that for the same affinity, you will nevertheless have substantially different avidity.

IgM is usually made first, and the individual Fab regions are usually pretty rubbish at binding antigens, but IgM has ten in total: the premise is that "rubbish x10" works out being quite good on aggregate. Getting ten crap binding sites in very close proximity can compensate for the fact that individually they're not very good. The body then takes whatever IgMs kinda work and hypermutates them to make (potentially much higher affinity) IgGs.

If you translate an IgM straight to an IgG context (without this secondary hypermutation), you lose the pentameric structure, and you're working on the base avidity of an IgG, which has only two binding sites. What works for IgM is not, consequently, guaranteed to work for an IgG.

So, short answer: affinity is likely unchanged. Avidity is markedly reduced.

In this case, it might be better to use another proliferation marker such as EDU because BrdU staining is mainly depending on denaturation by Hcl or you could try heating instead of Hcl treatment.

Good

Even though i don't know how to get a project in whatsoever field but i think you can contact via various sources in order to know more about the same .I suggest you to get a project on role of B and T cells in fighting against Corona virus with special reference on enhancement of helper t- cells in B cell activation.

Dear all,

I haven't found a paper yet that claims THP1 is a classical monocyte. Would you know of one? Thank you!

Dear Prof. Gheita, Md Abu Monsur Dinar, Hans-Joachim Kremer, thanks for your valued contribution. I appreciate your viewpoints.

think this idea is achieved at the biological level possible but very difficult and complex. But possible implementation by bioinformatics as well as via computer Algorithm. Good luck to do this .

Many thanks for this excellent questions. These advance immunological information needs to more and extra to describe and explain this type of stimulation of cell mediated immunity ( T cells Immunity.

Hi janmaris@Janmaris Marin

you showed that you got good results of destaining the first antibody, I wonder if the striping buffer that you used was glycine-SDS（pH 2.0）in the Daniel Pirici article which your mentioned above.

Best wishes,

Lixin

Designed as a two way analysis of variance (treatment x tank with three replicates per tank) and the analysis showed no significant tank effect, you are effectively getting the same experiment as 12 replicates/treatment in a one way AOV..

It is difficult to find out the LDL oxidation process at 234 nm. You will get nice conformation by Thiobarbituric Acid-Reactive Substances (TBARS) assay.

Being a clinician with an on going project on arthritis and HCQ200MG as primary drug of chronic symptomatic arthritis, Immune Modulator is more helpful than any other Immune inhibitors.

Most of morbidity or mortality are because of immune component of COVID which can affect any organ and system.

Commercial guidelines for Medicine is most important challenge for medical professionals to be encountered

Hi (copy from your question into discussion loop ...)

If your aim is study immunogenicity of human drugs in animal models then best way is analysis of guidances by FDA/EMA :) and total ADA (anti drug antibodies), fraction of Nab's (neutralising - high affinity antibodies) and seroconversion time. Such data could be colected after sigle dose of human protein. Sometimes pilot study is useful. Sometimes best way to analyse immunogenicity of human drug in animal model is simply analysis of drug pharmacokinetics. Then you just see what the difference is with Nab's because this fraction of antibodies increases the clearance of the drug that is administered. You can measure the most important cytokines and cells. The meaning of such work increases when your model is relevant in pharmacokinetics and pharmacodynamic meaning. Please remember that using the animal models for comparision test versus reference drug immunogenicity could be valuable (see guidelines below) but translation into humans impossible and off value. Please remember that different doses of the duman drug can induce different immune responce. For example higher dose of the drug may give lower immune responce if drug has immunosupresive action. Moreover in case of some drugs multiple dosing may decrease immune responce for example ustekinumab or infliximab ....

Some explanations from old FDA guidance could be usefull in your work (document is not available now)

„Animal immunogenicity assessments generally do not predict potential immunogenic responses to protein products in humans. However, when differences in manufacturing (e.g., impurities or excipients) between the proposed product and the reference product may result in differences in immunogenicity,

measurement of anti-protein antibody responses in animals may provide useful information relevant to patient safety. Additionally, significant differences in the immune response profile in inbred strains of mice, for example, may indicate that the proposed product and the reference product differ in one or more product attributes not captured by other analytical methods.”

GUIDANCE FOR INDUSTRY SCIENTIFIC CONSIDERATIONS IN DEMONSTRATING BIOSIMILARITY TO A REFERENCE PRODUCT FDA 2012

" A risk-based evaluation, focused on the mechanism of action of the therapeutic protein product as well as results of animal and in vitro evaluations should be performed to determine the need for collection of preand post-dose cytokine levels in the early phase of clinical development. In case of a clinical adverse event, such an evaluation may provide evidence to support the clinical diagnosis of cytokine release syndrome and help distinguish this entity from other acute drug reactions "

Guidance for Industry Immunogenicity Assessment for Therapeutic Protein Products

The immunogenicity of therapeutic proteins- what you don’t know can hurt YOU and the patient

" Therapeutic proteins show species differences in most cases. Thus, human proteins will be recognised as foreign proteins by animals. For this reason, the predictivity of non-clinical studies for evaluation of immunogenicity is considered low."

but .......

" the comparison of the antibody response to the reference product in an animal model may be part of the comparability exercise both for similar biological medicinal products "

" Qualitative or quantitative difference(s) of product-related variants (e.g. glycosylation patterns, charge variants) may affect biological functions of the biotechnology-derived protein and are expected to be evaluated by appropriate in vitro assays. These differences and impurities may have an effect on immunogenic potential and the potential to cause hypersensitivity. It is acknowledged that these effects are difficult to predict from animal studies and should be further assessed in clinical studies. "

Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues

Some nice papers about could be useful

Hope it's helpful

Best regards

Tomasz

Now, in a new, non-peer-reviewed study published in the journal bioRxiv, researchers show that this decrease is related to the disappearance of a family of antibodies called immunoglobulin M, or IgM, in blood plasma. In other words, IgM antibodies play a major role in neutralizing the virus and are part of the arsenal that the immune system uses to fight

I just spoke to a rheumatologist about the case and she considered off the bat

Hi Ben, here is an earlier paper from 2012 on the topic:

Have a look when convenient

Here is link

Hi Mari,

Relative to ROS production: working with fresh human whole blood (not PBMCs), we found considerable DHR fluorescence following PMA stimulation in monocyte and neutrophil populations by flow cytometry. Anything between 20 - 30mM DHR123 in DMSO for a final cell-stain dilution of (1:20) was ok.

Stimulation time matters as well. A quick look at literature reveals that ROS generation is a fast process; so you may get good signal with PMA in whole blood anywhere starting from 30mins at RT. We noticed that the fold change in neutrophil oxidative index with PMA began to drop significantly after 2h of stimulation. Hence you want to stay within that range if using PMA.

With respect to other stimulants: these affect the fluorescence intensity as well. In my hands, Mtb bacteria whole cell lysate produced a relatively higher signal than the unstimulated control under conditions stated above but significantly less than that induced with PMA.

NB: We didn't try fixing cells for later acquisition; we read immediately after the staining process.

Best wishes,

Caleb

Please take a look at this useful link.

Both excellent choices but can add

Molecular Biology of the Cell in your shelf for some amazing reading

Dear Chaimaa Benlabsir

check the following link

it my be helpful

I hope this

molecular virology

may be