Immunization - Science topic

Hello,

I'm looking to isolate plasma cells and plasmablasts from primary B-cells obtained from mouse spleen (I plan to use CD138 to enrich for plasma cells and plasmablasts). My goal is to culture these cells and maintain their viability for 1-2 weeks. What type of media and cell activators would you recommend for this? Additionally, can I culture the cells in this media immediately after harvesting them?

Hello,

I am currently working on parental strategies for effective immunisation coverage. As part of the study, i will be assessing the impact of the intervention in reducing barriers to immuisation. Can i pleases get a recommendation of validated tool to use?

Thank you

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?

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?

Hello everyone,

I am planning to immunize my mice with OVA/Alum in order to study germinal center reaction. I'm planning to do for 6 days immunization. But I am struggling to find good and tried OVA/Alum immunization protocol for mice.

Would you kindly share your protocol on this topic?

Best

The deadly Coronavirus Disease 2019 (COVID-19) has claimed numerous lives and the number is increasing day by day. So, in this situation, a vaccine has been expected to decrease the mortality rate worldwide and save us from this disaster. But there have been some complications reported from the vaccination process, however rare or mild those are. Now that some vaccines have been approved for emergency use, we want to investigate whether these vaccines cause any after-effects. You are cordially invited to participate in this study by providing your valuable response if you have taken at least one dose of the covid vaccine.

Thanks in advance for your valuable contribution.

Whether someone is vaccinated or has previously had Covid-19, they are safely immune from getting the disease again. But does that mean they are also not contagious to others? Some research indicates they can still catch the virus and be contagious and should continue to wear a mask. What are your views based on reported research?

I have two peptides in lypholized form, one is basic and other is neutral, i have to use these peptides for an experiment for which i need to dissolve them into solution form, as per the literature there are different chemicals suggested for dissoluation of the acidic , basic and neutral peptides, but the problem is i can't decide which chemical is to choose as it is written in the literature that if the peptide fails to dissolve in a particular solution then try to dissolve it in another solution but i just wonder how could i do that if my peptides are already in the lypholized form, as i can't try to dissolve the peptide in different solution while they are in the lyphlized form in the same vial.

Thanks in advance.

RNA vaccines follow a different strategy, without using any "real" component of the virus at all. Instead, researchers aim to trick the human body into producing a specific virus component on its own. Since only this specific component is built, no complete virus can assemble itself. Nevertheless, the immune system learns to recognize the non-human components and trigger a defense reaction. So May I ask, What are your opinions about the safety and efficacy of the BNT162b2 mRNA Covid-19 Vaccine?

Has anyone prepared a vial of sigma adjuvant systems (Ribi's) when they either aren't using the whole vial in one go or need to 1 vial for different antigens? The product insert says you should warm the vial to 40-45 degrees before adding 2ml of an antigen-saline solution. If you are only reconstituting it 1ml saline as detailed in step 3, it's unclear if this heating step should still be carried out? I intend to use small volumes of the 1ml reconsituted adjuvant with 5 different antigens, and then save the rest for immunisations a couple of weeks later. Should the adjuvant be heated initially AND before combining with the relevant antigen? or should it occur just once, either before reconstitution or immediately prior to mixing - if so, which?

Thanks in advance,

Rebecca

I had a few questions regarding Convalescent Plasma Therapy

Why can't we use whole blood rather than convalescent plasma only? or Plasma + WBC (Adoptive cell transfer). This way we will transfer not only a humoral response but the cell-mediated immune response as well into the recipient. This should theoretically lead to a better outcome.

By giving plasma only, we transfer humoral immunity (in the form of immunoglobulins/antibodies) to the recipient and not the cell-mediated immunity.

The second question: Is there any difference if we use "serum" rather then plasma? I don't see if there is any need of adding "clotting factors" to the recipient's blood by using plasma rather then sera.

A study of the factors influencing the knowledge and attitude of mothers of under five children of a selected area of Kunderki, Moradabad U.P. regarding immunization and efficacy of a need based intervention strategy towards its improvement. In this experimental research study i need a conceptual framework .so please help me to design a conceptual model.

I have a 13 amino acids peptide and I want to inject it in mice to induce immunization. The injection will be in the tail vein. After 12 days I expect to have a peak in the production of antibodies, so in the 12th day after injection I will challenge these mice and matched controls with bacteria and perform a mortality curve. I believe this kind of immunization will be protective and prolong mice survival. How much peptide should I use to induce the immune response? One dose is enough? Do I need to inject it several times? When and how much?

If everything works well, I want to produce a monoclonal antibody against this peptide and inject it after the bacteria injections in non-immunized mice to see if this monoclonal antibody works as a new treatment for infections. How much antibody should I inject to try to treat wild-type mice with a monoclonal antibody? One dose is enough? Do I need to inject it several times? When and how much?

I want to perform splenocyte restimulation culture for the estimation of Th1 and Th2 cytokines by ELISA. As each groups consists of 5 mice immunized with a test vaccine preparation; can splenocyte isolated from intra group mice be mixed together?

I would like to know a good adjuvant to combine with some peptides of 20aa long to immunize rabbits and after recovery of antibodies.

Hi all. I am working with a novel orbivirus and wish to produce polyclonal antibody in ascites fluid for the purposes of IHC. Unfortunately our virus is only 10^5 or so, and we need 10^8 for immunization of mice to get ascites fluid for antibody.

I use PEG precipitation to concentrate virus for deep sequencing, but is there a way to concentrate virus another way? I feel that injecting polyethylene glycol into mice could be harmful. Any other less potentially injurious forms of virus precipitation? Thank you!

After some reading and checking for the Treg cells during helminthic infection, I found my self somewhat confused for the % populations measured for CD4 CD25 and FOXP3 for Control against Text groups (immunized, Immunized & infected and Immunized & infected+treated).

I've found in some results of one of my peers CNTRL is higher than other groups in some analysis but this is not the situation for some published articles.

Any clarification if it is a logic trend for CNTRL to be lower than test groups in Flow Cytometry analyzed cells or it may differs?

Any help will be highly appreciated!

Best,

Ehab

There are challenges facing parents in keeping paper immunisation card in maintaining proper vaccination records for children under 5 years. The parents are faced with multitude of problems and sometimes forget the paper card at the immunisation centre, or lose them altogether due to improper storage or just forget the date for the next immunisation schedule due to other competing priorities. If the immunisation cards was provided inform of ecard which could be loaded on a mobile phone and reminds the parents about the due dates for their children's next immunisation schedule. Would we possibly increase access to vaccines and improve completion of immunisation of children below 5 years?

I've been reading into the use of nanobodies in replacement of normal antibodies in different molecular techniques. I was wondering if someone could explain why nanobodies with IgG subclass-specificity has an advantage over normal monoclonal antibodies?

Also, why do you require immunisation prior to immune library production?

Thanks!

Our research group have developed a novel polysaccharide adjuvant we call Advax which has proved effective in multiple animal models and is also in late stage human development. Its major virtue is that it is non-inflammatory and thereby nonreactogenic.We are always looking for collaborators who might want to test it in their particular animal model or who want a GMP adjuvant for human trials. Data from each new model or application gives us additional insights into the action of this unique adjuvant.

What peptide sequences for immunisation will suitable? How to find out? 

which one is more appropriate for inducing AD (ICV injection of amyloid beta), immunization, Morris Water Maze test and hippocampus RNA extraction (RT-PCR)?

Hello, can someone explain what can cause a different antibody titer in immunized BALB/c strain mice ? I have immunized 3 BALB/c mice three times over 12 week period with the same amount of antigen, yet their immune response to antigen differs. Tested antisera of each mice with ELISA and got different titer and not sure what caused this. 

Thank you !

Hi

I'm going to administer an intramuscular injection of alum-absorbed diphtheria toxoid (twice with 3 weeks intervals) to Sprague-Dawley rats and I'm going to assay their effects two weeks after the second administration, but I can't find an appropriate dosage (limit of flocculation)...

I want to know if any of you has an idea about how much does the using of a DTT treated protein (as immunogen in mice) affects the mAb production process?

I will purify my r-protein (produced in E.coli) and cleave the MBP and His-tag off it and I have heard about the inclination of the r-protein to aggregate/precipitate when the tag cleavage takes place, my question is can I use the precipitated or aggregated protein for the mice immunization after oil immulsion?

I want to carry out a cost effectiveness study on a participatory action research aimed at improve immunisation coverage.

Hi all, 

Goin thru the online papers that describe immunisation and challenge experiments normally draws to a conclusion that the immunisation gives protection or even partial protection to the host. Are there any papers that showed no protection after the immunisation, even though the immunisation experiments are able to induce immune responses in the host? Thank you.

I also like to know if it's possible to use auto-antigens related to auto-immune diseases,  to detect auto antibodies that may be generated after immunization against worm candidate vaccine

thank you for your help

Hello everyone. I am doing immunization. after doing ELISA, i left serum collected from mice at room temperature for more than 10 hrs. Is there any chance that it is contaminated or my Antibody (anti-Agr2) is degraded?

Hi..

can anyone tell me what is the method for virus inactivation so that it can be used for immunization in animals, especially in mice.

There is low routine immunization coverage in non-compliance settlement in some rural areas. This is a block rejection of injected able vaccines(antigens) in the communities.

We all know that Zika has circulated in Africa in the past and possibly conferred immunity in the populations of these settings. Except to that, I will look for having additional insights from all of you.

I have tried extraction of RNA from various commercial rabies vaccine which currently used for pre exposure and post exposure for dogs. After that I have done RT- PCR, while running gel i got band for only one vaccine, i tried several times with several type of procedures, all the times i got band from only one vaccine. what may be reasons? is it due to some inhibitor substances which present in vaccines? simultaneously i have tested the serum samples of dog after the immunization for sero- protection assessment, dogs vaccinated with that vaccine showed band in RT PCR had better sero- protection compared to that of other vaccine. Is there any relation between this two?

In the specific case of antibodies and antibody fragments, why is hybridoma technology still standard practice over phage display affinity maturation?  what are the advantages and disadvantages of each?  Is there a way to eliminate immunization of mice all together?  I have found review articles from the 1990s, but I am having trouble finding recent information on the topic.

Dear All, I am working on development of hybridoma technology. I was used balb/C mice for immunization and SpO cell lines as myeloma cells. After 3rd booster spleen was removed  aseptically. fusion of mice cells with spo was carried out via PEG. The clone was screened after 14 days (HAT medium). The clon was pick from the well and performed serial dilution for single clone isolation. Now the clones are grow exponetially on HAT medium, But I am not getting antibodies in the medium. 

Can any one help me...

I will appreciate very much your opinion

I would appreciate if anyone can share information about HA1 immunizations in mice. Particularly Cal09 HA1 in C57BL/6 mice or BALB/c mice. Any information about the murine MHC class2 restriction of Cal09 HA1 will be appreciated.

Thank you.

In the late 1970s - beginning of the 1980s, we’d developed a pioneer approach to prepare antibodies against monooxygenase molecular isoforms without laborious purification of CYPs. To achieve the point, we’d conducted immunization with protein band from SDS-PAGE (bands were preliminary characterized by isozyme-specific peptide maps). Thereafter in the 1990s-2000s, I’ve been deeply involved in preparation of the recombinant CYPs, and therefore lost a little bit the track on current procedure with SDS-PAGE-CYPs. Namely, if one need to inject mice with a protein band excised from SDS-PAGE gel, is it necessary to use Freund's adjuvant while using this protocol, won't the acrylamide itself act as an immunogen (can it serve as a mild adjuvant and/or booster after 6 weeks)? Some people stain gel, fix with 2% glutaraldehyde and destain, then cut the band of interest, pulverize in an Eppendorf tube, freeze-dry (lyophilize), mix with PBS and adjuvant, emulsify and inject. Others rather fix first, wash to remove glutaraldehyde, stain with reversed staining (e.g. zinc, CuCl2) and then cut. And how much protein might be cumulated in several parallel bands run, in terms of would it be enough to inoculate depending whether one is injecting rabbits or mice.

I collect splenic cells from C57/BL6 mice at day 7  post immunization with OVA protein and CFA, then incubate the sorted CD8 T cells from splenocytes with OVA257-264 pulsed EL4 target cells for several hours. I wonder whether OVA immunization could give rise to CD8 T cell response and generation of CTLs? Thanks!

 I am unsure of the potenital cytokine profiles after the stimulation of splenic cells after immunization with different adjuvants (different mice). Since the adjuvant influences the cytokine production, would the immunizing agent alone with cultured spleen cells effect be effected by previous immunization with adjuvant. 

For example, a adjuvant influences IL-6 production during immunization but would the cultured splenic cells produce IL-6 when stimulated with the immunizing agent (no adjuvant and assuming the immunizing agent does not produce IL-6). 

I need to immunize mice subcutaneously with 500 ug OVA in Complete Freund's Adjuvant. How should I prepare the mix to do it correctly?

Hello,

I am going to work with a helminth crude antigen, so need a suitable adjuvant to appropriately elicit immune response in C57 black mice.

Thank you.

I am trying to determine the frequencies of antigen specific IFN-gamma-producing cells from the spleens of immunized mice by flow cytometric analysis. My understanding is that upon vaccination T-cells will be activated. So it is correct that for T-cell stimulation experiments I use antigen only? Some researchers use anti-CD3 and antiCD28 to activate T-cells. hence, should I use anti-CD3 and anti-CD28 as well? Thanks

I am doing my thesis on assessment of immunization status in children in District swat. I will be using multistage stratified sampling technique.This will be a house hold survey.

Can anybody suggest to me why I cannot get a suitably high titre after 4 immunizations in mice? I have conjugated toxin with cationic BSA and confirmed the conjugate by running on the gel that give significant difference from standard protein. After four immunizations in mice the titre is very low. What should I do? Please comment

I have a question

how many ug of protein from inactivated influenza virus simply to immunize (1 swine) help me please, I don’t know what to do I look everywhere and I do not find the information

With my protocol for purification virus I have only 70ug/ml (total protein SIV), IM NOT SURE if 70ug/ml is sufficient to immune 1 swine with 3 booster

Suggestion please

I would like to immunize different groups of OT-1 mice with SIINFEKL and some variants we have made to determine if there is a difference in T-cell response. The OT-1 model is attractive because all the CD8 T-cells are specific. They are great in vitro tools or for adoptive transfer, but can you directly immunize an OT-1 mouse? The alternative is to use B6 mice, but we have a lot of OT-1 on hand and I'd get a lot more cells from them. 

I stained mouse lymph nodes with pd-1,cxcr5,cd4 and cd44 for t follicular helper cells and cd19,cd38,cd73 and af488 conjugated to an antigen that I originally immunized the mice with. I collected the samples at day0, day5 and day42. My mice were 6 weeks old at the time of immunization, is it typical to not see much staining for pd-1? And I also did not see many cells staining for cd73 at day 42. How long does it typically take for memory b cells to develop?

I would like to know what is the best approach between prevented number of cases for every 1000 vaccinated individuals and the decreasing lethality of cholera in the vaccinated region when it comes to assess the Oral Cholera Vaccine Efficacy during a mass vaccination campaign in Sub Saharan Africa.

We are seeing plenty of cases of vaccine failure as outbreaks of velogenic Newcastle disease and Infectious Bronchitis which are reported in in vaccinated flocks. How do you think should one investigate failures? If outbreaks occur in presence of sufficient antibody titres. Will virus isolation and sero-neutralization in ovo be enough. Or should one do challenge tests in vaccinated birds? 

Any suggestions?

I want to know, in troubleshooting in the balb c immunization specially, what the difference between prebleeding and immunized mouse is?

Due to higher salt concentraion the specific protein can aggregateed?

Dear Researchers,

I'm looking for literature on how to analyse IgG1, IgG2, IgG1/IgG2 ratio and IgM response after vaccination in Cattle. Appreciate if u can direct me to relevant literature. You can also share your knowledge on this. 

Thanks

I'm working on polyclonal antibody production through genetic immunization in rabbits and after 5 month of immunization I got nothing! I need to know is there a problem with Tolerance induction following genetic immunization? Is there anyone who has faced similar problems?

Thanks

I want to make some emperical studies on child and issues such as child mortality, immunization etc using demographic and health survey data. I can analyses dat a as I'm student of statistics. I would like if some one with knowledge of public health collaborate with me as co-oauthour in these studies.

Regards

Atta from Pakistan

Why antibody-mediated immunity to Shigella dysentery requires several episodes of infection to get primed?

I am using direct standardization to compare immunization rates of two groups.

I will age adjust the groups (13-18 years old). Group A immunization rates were determined using random telephone dialing survey methods of 13-18 years old (State Level). Group B immunization rates will be determined using actual records of Group B immunization history records for 13-18 years old (County level).

What denominator should I use to standardize? What standard population should I use?  Should I adjust Group B sample size to the sample size used for Group A?

We are working on a xenogeneous anticancer vaccine (a protein extract of avian-origin cells). Once we performed an experiment in prophylactic settings: there were three vaccinations (one per week). Then 30 days after the LAST vaccination the mice were challenged with Lewis Lung Carcinoma Cells. In the vaccinated group we observed the statistically significant inhibition of tumour growth and a decrease in metastasis formation compared to the unvaccinated group and, to a lesser extent, to mice immunized with antigens of Lewis Lung Carcinoma cells. I started to write an article, and stated that the studied vaccine elicited 1) a specific antitumour response and 2) an immune memory. My tutor objected these, saying that it is a false conclusion. So, my questions are: 1) if he is right, what else besides immune memory may provide the observed antitumour effect? 2) Is there a good reference to read about studies into prophylactic settings of anticancer vaccines? Which effects of vaccine application do they reveal?

By family structure I mean nuclear and single parent family. Also by religiously I mean religious participation and the religious office. 

Presenty, people link hesitance to child immunization with the distance of the health center, availability of vaccines, the child sickness, busy mothers, health workers lack of interpersonal communication with parents. How can we measure the confidence in child immunization?

I am doing a research study on the impact of parental attitudinal barriers on Infleunza immunization in the pediactric population. Does anyone have or know of a tool I can convert into survey monkey on facebook with consented permission?

Can somebody help me, if I want to inject an antigen into a mouse for immunization to produce the monoclonal antibody, how much of the antigen do I have to inject?

I inoculated 2 x 10^6 dose of P.aeruginosa through intraperitoneal route. I also did further repeated immunisation on the same mice after a week. However, I could not find significant differences in IgG in serum as well as peritoneal fluid of control as well as single immunised and repeatedly immunised mice. I wonder what the dose should be? I would be grateful for your suggestions.

Occasionally I get some non-specific reaction in ELISA. I belive that this colud be due to there may some antibodies for the carrier protein. To avoid the interferences in ELISA it would be better to use coating antigen should be different one than used for immunization. I have antibodies Aflatoxin B1-BSA conjugate and prefer to use Aflatoxin B1-KLH as coating conjugate in the indirect competitive ELISA. Looking forward for any comment or suggestions 

I have three synthetic peptides. Their size are between 15 and 20 aa. I would like to couple them with KLH to rise the immune response in my immunization on mouses and rats. I was looking for the correct ratio and the aproximated amounts of synthetic peptide and KLH that I have to use but I've not found clear information about how can I estimate it. Does anybody know how can I do?

Last week, I watched a talk show on TED Talk about Nanopatch for needle less vaccine delivery method. I am impressed.

I conjugated my 25 KD protein to Anti DEC-205 mAb by polysaccharide groups in Ab structure. I didn't have SMCC or Sulfo-SMCC then I used this protocol for conjugation. Now I have smears with high molecular weight ( > 200 KD). I must immunize my mice by this conjugated product. How much of this product is suitable for immunization? Which of them is important? The amount of protein in conjugated product or amount of whole conjugated product? In other words is it necessary to calculate the amount of protein in conjugated product? If i want to immunize my mice with 5 micrograms, this 5 micrograms is the net protein in conjugated product or is the amount of Ab-protein product? I sent the western-blot picture of my conjugated product.

An interesting situation one commonly encounters. While conversing with many physicians, it inadvertently slips from their ends that they often fall behind on immunizing their children, and even the mode of treatment of illnesses are quite different from what is being practiced on patients e.g drug regimens, investigations etc. What has been your experience?. Doctors advise patients effectively- but do they fall short when it comes to their own kin?

What's the immunogenic characteristics of an insoluble protein, such as a membrane protein? Can we use the precipitated insoluble protein for the immunization of a rabbit, what's the result if someone did it this way?

I immunized mice with a recombinant protein and I would like to characterize them against which T cell epitopes the immune response is directed. Which techniques could serve me with this purpose?

Too many vaccines added to the routine vaccination schidule which might require giving two months old child up to 4 injections even with the combined vaccine injection.

Recent CDC report estimates the effectiviness of influenza vaccine by 62%. And we are douting this percent in our area, as most of our patients who got the vaccine reported to have several influenza episodes after vaccination. I think this vaccine should be given to certain group of high risk patients only.

the protein sample will be used for immunization of mice or rabbits, therefore no urea or other chaotrophic chemicals should be present. Precipitation is a benefit and used for concentration of diluted samples, but precipitate should be easy to homogenize in buffer like PBS.

Our immunization schedule is: 3 s.c. shots of antigen on Alum (d0, d14, d28)

I would like to know if anybody has tested the re-appearance of memory T cells in blood in a time course experiment.

Is there an optimal time point after last immunization for testing T cell reactivity with e.g. an ELISpot assay?

Are the kinetics the same for mice and humans?