Science topic

Immunization - Science topic

Deliberate stimulation of the host's immune response. ACTIVE IMMUNIZATION involves administration of ANTIGENS or IMMUNOLOGIC ADJUVANTS. PASSIVE IMMUNIZATION involves administration of IMMUNE SERA or LYMPHOCYTES or their extracts (e.g., transfer factor, immune RNA) or transplantation of immunocompetent cell producing tissue (thymus or bone marrow).
Questions related to Immunization
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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?
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Thanks Gen! Is this 8 days optimum achieved after several immunization or only after 1 shot?
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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.
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Try  Beta propiolactone to 1/4000 . If you are working with small amounts , increase the inactivating dose. It is useful in such cases to dilute the GLP in sterile distilled water. Allow the mixture gently stirred for 2 hours at laboratory temperature and then overnight at 4 ° C . Intraperitoneally immunization of mice with recall 7 days after can give you satisfactory, I hope.
 Good continiation
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What is the correct way to evaluate T-cell responses after immunization?
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
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with anti CD3/25 you will test for the overall T cell potential to respond. This is not what you want. Therefore, re-stimulate with the immunogen and use elispot or IFN-g in the supernatant of 48h cultures as read-out.
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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.
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Hi, just a short thought: Helminths usually initiates a Th2 type immune response. The C57Bl/6 strain is not a very good Th2 responder strain in comparison to other inbred strains such as Balb/cJ or NIH/Ola, which I have also worked with in allergy studies. (Allergy is also Th2 response). For example - I used the Th2-promoting adjuvant aluminiumhydroxide, but the antibody response was much higher in the two latter strains compared to the C57Bl/6-strain. So as Frederic Beaudoin points to, it is very important to consider the type of immune response you want to study and from this select strain and adjuvant. Best regards, Jitka
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 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). 
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Hello Kevin,  
yes, both the choice of adjuvant and the immunogen can and will influence the cytokine profile of your immunization experiments. It is often valuable to initiallly investigate the effects of the antigen/immunizing agent alone to know its immune stimulating properties, and thereafter see how a added adjuvant can influence the response patterns. This basic knowledge is essential for understanding the impact of the different ingredients in your immunization procedure.
Also the localization where you give your immunogen and the dose you use of the immunizing agent will usually influence the cytokine pattern.
Best regards
Jorma H
How Rabies vaccine potency test is calculated?
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How Rabies vaccine potency test is calculated?
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Based on the NIH method, various dilutions of the rabies vaccine  immunized in mice(n=10) along with control mice, the immunization schedule 0, and 7 with appropirate adjuvant (alpo4) Human albumin, maltose stabilizers, after 14th day the immunized mice underwent Challenge using with the challenge virus strain(high viral titre) as well as the WHO standard /validated strain, baseded on the mortality the calculation was done by reed an Munch method, you may get this protocol in website
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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!
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Hi Lianh Liu,
Actually it does. 
we use OT-1 TCR Transgenic mice for the same purpose.
OT-2 TCR Tg mice are used for CD4+ T cell study and you do not need to immunize them as they are genetically engineered to constitutively express TCR that recognizes OVA MHCI or MHCII restricted antigens. 
The best thing is u can do it straightaway without any immunization i.e. no tedious and prolonged immunizations and one day experiment.
Best of Luck!!!
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I will appreciate very much your opinion
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Good  morning  María  Pilar do  you  have  more  information about the pigs  origin ?  ca yo please  send  or  post  this  information? (piggery, company animals genetically modified) is  mportant . thanks.
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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.
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polyacrylamide gel electrophoresis can be difficult to interpret when looking at isoenzyme specific peptide maps.
Glutaraldehyde should be removed .
Use rabbits rather than mice .
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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.
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Evaluation of Oral Immunization with Recombinant Avian Influenza Virus HA1 Displayed on the Lactococcus lactis Surface and Combined with the Mucosal Adjuvant Cholera Toxin Subunit B ▿
Han Lei,1 Zhina Sheng,2 Qian Ding,2 Jian Chen,2 Xiaohui Wei,2 Dominic Man-Kit Lam,3 and Yuhong Xu2,*
ABSTRACT
The development of safe and efficient avian influenza vaccines for human and animal uses is essential for preventing virulent outbreaks and pandemics worldwide. In this study, we constructed a recombinant (pgsA-HA1 gene fusion) Lactococcus lactis strain that expresses and displays the avian influenza virus HA1 antigens on its surface. The vectors were administered by oral delivery with or without the addition of cholera toxin subunit B (CTB). The resulting immune responses were analyzed, and the mice were eventually challenged with lethal doses of H5N1 viruses. Significant titers of hemagglutinin (HA)-specific serum IgG and fecal IgA were detected in the group that also received CTB. Cellular immunities were also shown in both cell proliferation and gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays. Most importantly, the mice that received the L. lactis pgsA-HA1 strain combined with CTB were completely protected from lethal challenge of the H5N1 virus. These findings support the further development of L. lactis-based avian influenza virus vaccines for human and animal uses.
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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...
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Thank you Mr Brijesh for your nice suggestions. I have already tried to grow clone in 2% FBS containing HT medium. Clones are growing very well but i could not find out the antibodies. So is it possible, increasing conc of FBS will stimulate the antibodies secretion.
Thank You 
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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.
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I think hybridoma technology is quite robust and has been useful to discover thousands of antibodies for different applications. For labs with expertise and good results using this method, it continues being the methodology od choice, particularly if the goal is to obtain antibodies for analytical purposes. Its main practical advantage is that, once the hybridoma clone is isolated, monoclonal antibody production in mouse ascites is simple, efficient and reproducible.
Phage display is much faster in the first steps- discovery of variable regions with the ability to bind the antigen, but the subsequent production of the soluble protein (not phage-displayed) as an antibody fragment in E.coli or as a fusion protein or whole antibody in mammalian cells can be challenging, depending on the particular variable region sequences.
Phage display is clearly advantageous when
1. You are attempting to simultaneously generate multiple binders against different antigens exploiting the high throughput potential of phage display.
2. You are interested in exploring human repertoires or repertoires from species that have not been widely used for hybridoma production .
3.You want to perform further genetic engineering of the binding sites, ie affinity maturation, specificity optimization, mutagenesis scanning.
I have good personal experience with both approaches, although my current field is phage display.   The choice of one or the other method depends not only on the final application, but mainly on the expertise of each lab. If you are a hybridoma or phage display expert, you will try to solve any problem with the tools you dominate. And both tools are quite useful and can solve many problems.
If you are not an expert and  just need to obtain a few antibodies that can be of mouse origin I would suggest to use hybridomas. On the other hand, if your project corresponds to one of the above mentioned applications for which phage display is advantageous, you should try to use phage display.
There are many recent publications about antibody phage display for sure, just have a look in pubmed.
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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?
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I am not sure which vaccines you tried, but these days most of the Rabies vaccines are recombinant or synthetic proteins. Therefore you should not expect any RNA or DNA in these vaccines. The old Sheep brain (attenuated) vaccine is no more in use. 
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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.
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I suspect there are three factors.
1) The immunity factor.  If women develop immunity  as children, they can protect the fetuses they carry just like rubella and many other diseases.
2) Viral drift to a strain that either transmits easier (as an STD) or has more pathology (microcephaly??)   See http://phys.org/news/2016-04-genetic-evolution-zika-virus.html for some of the genetic differences.
3) More effective human surveillance.  This is a disease with a majority of asymptomatic individuals.  Babies with microcephaly  months later.  Without advanced testing and surveillance, who would know?
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I would like to know a good adjuvant to combine with some peptides of 20aa long to immunize rabbits and after recovery of antibodies.
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Thank you Mario Cesar! It seems to be to induce, do you have any reference for this procedure?
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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.
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Also look at work by Julie Leask http://bmcpediatr.biomedcentral.com/articles/10.1186/1471-2431-12-154 she's done quite a bit of work in finding out why people don't vaccinate (often related to access to services) and how to improve or promote vaccination, she's active on twitter and other social networks @JulieLeask
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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?
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Serum does not contain free proteases but the only way to know the answer is to use your sera again. If  they  are not sterile, I think 10hrs at RT  (23°C probably) is not a long time for bacterial or yeast growth. Good luck
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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.
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My take on this question is that immunization assessment can be done via survey method which I think will be very difficult to conduct and is a costly and time consuming exercise. You should contact EDO office of District Swat and get data from DHIS. You can also get data from district coordinator EPI District Swat, he must be in the District health office.
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What peptide sequences for immunisation will suitable? How to find out? 
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Are you looking for antibodies that crossreact with the folded protein? In this case you look for antigen peptides that are accessible on the protein surface and somewhat conformationally flexible, that is, loop and turn regions on the protein. There are a number of sequence based methods that highlight potential epitopes, http://tools.immuneepitope.org/bcell/http://ailab.ist.psu.edu/bcpred/predict.htmlhttp://curie.utmb.edu/B-Cell.htmlhttp://www.cbs.dtu.dk/services/BepiPred/http://sysbio.unl.edu/SVMTriP/prediction.php
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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?
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Yes you can; however, there should be enough splenocytes in each spleen to assess the response to your stimulus whether this is an antigen or other activating agent. The power of having 5 replicates each stimulated under 3 different conditions (i.e. stimulated with medium alone as a negative control; a mitogen such as concanavalin A as a positive control and your test agent) will allow you to get a clear result with solid statistical analysis from this single experiment. I would, therefore, strongly advise analyzing each spleen separately.
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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
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Hi Marcos Vinícius Pereira Gondim , many thanks for the shared publication and for your time to answer and informative help.
Highly appreciated!
I will check this paper.
Best,
Ehab
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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?
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It certainly seems a very good idea to replace paper cards with electronic cards. In my country, we have established a national immunization registry allowing for the vaccination information on a specific child to be retrieved at each visit even when the paper cards are unenviable. you can find information on -
The Israel National Immunization Registry. May 2010. IMAJ 12(5):296-300. Project: Immunization Coverage
How to generate specific antibody for IHC purposes?
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Hi all, I am interested to generate specific antibody for IHC purposes. 1. What's the best way to immunize the animal? Denatured protein or peptide? 2. Is there any high throughput IHC screening strategy to screen for monoclonal clones? Thanks!
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https://www.novusbio.com/primary-antibody-selection-optimization
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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!
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for first part, the nanobodies are more flexible in thier reactivity, avoid specific hindrance, so less cross-reactive
for second part, to increase the antibodies affinity (avidity) and reduce the numaber f sourceo. However, for your knowlege you can make it from native source, but need a huge number of source
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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!
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Hello Anna.
I am working with tick-borne orbivirus. I´d like to increase its amount, because the titre is approx. 10 ^5. Could you share the protocol for PEG precipitation?
Thank you in advance.
Tomas.
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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 !
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Many lymphocyte clones with different antibodies or T cell receptors, could potentially recognize a given antigen, not all of them encounter the antigen at the same time. It depends on the location of the lymphocytes and the antigen in their traffic through the lymph nodes. Such ''random'' collisions between lymphocytes and antigen can occur in  different ways in different animals.
And as  the formation of antigen-specific receptors (by recombination) is in part also random, not all animals contain exactly the same antigen-specific receptors on their lymphocytes,    their repertoires are similar, but not identical. No immune system is totally the same as any other, so the responses can vary.
Its just a matter of understanding how the huge diversity of antigen-specific receptors is generated and the geography of immune responses. 
Im assuming that you don't have gross problems at your animal facilities like infections or so...that could of course disturb your results. Even in the best conditions, the individual responses are quite variable.
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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
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serum is more preferable because plasma contains fibrinogene which interfere the agglutination of Ab and Ag
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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)...
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The first group constituted up to 15 per cent of total blast cells. The staining was sometimes limited to small stippled dots over the nucleus or it was uniformly distributed
throughout the cytoplasm. Urso and Makinodan (1963) consider the stippled pattern to
correlate with the first appearance of antibody. This interpretation may well be correct
but we have certain reservations about the true intranuclear location of the antibody.
The cytoplasmic staining varied in intensity in different cells but it was particularly bright
in mitotic cells, which suggests that there was a progressive build-up of the antibody concentration in the cells as they progressed through the DNA synthetic and the G2 period
towards mitosis.
The blast cells then divide giving rise to cells of the intermediate type. These cells became labelled with [3H]T 4-6 hours after the injection of the isotope and their mean grain
count was about half the mean of the counts over the cells labelled at 1 hour which is the
order to be expected for progenitor and daughter cells. Occasionally these cells modified
their morphology very rapidly to assume the form of the mature plasma cell within 4-6
hours. In one experiment it was found that 70 per cent of the intermediate cells were
labelled 12 hours after a single dose of [3H]T, whilst only 22 per cent of the mature plasma
cells were labelled. It is possible that some of the intermediate cells did not differentiate
at this stage and entered a further division cycle. From the study of the post-divisional cells
formed during a 12 hour period it is apparent that the rate of change in morphology, i.e.
the change in nuclear cytoplasmic ratio and in the position of the nucleus in the cell is
variable. Cells with small eccentric nuclei were occasionally observed to be labelled within
this time which suggests that the morphology of the post-divisional cell is influenced by
factors other than its age. An unsolved problem is the nature of the pre-divisional cell
that gives rise to the small lymphocyte-like cell containing antibody. The majority of
antibody-containing mitotic cells were almost as large as blasts but occasionally much
smaller dividing cells have been observed and these might possibly have been the precursors of the small lymphocyte-like cells.
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which one is more appropriate for inducing AD (ICV injection of amyloid beta), immunization, Morris Water Maze test and hippocampus RNA extraction (RT-PCR)?
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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?
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Perhaps it would be better to leave the tag on your protein. During mAb screening, any anti-tag antibodies could be weeded out.
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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?
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Thanks dear Alim,
So aggregation is not suitable, what about the precipitation with methanol can we inject the mice with precipitated protein sample as an immunogene? 
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I want to carry out a cost effectiveness study on a participatory action research aimed at improve immunisation coverage.
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Greetings,
Much depends on the scope, size, and research design (and what is available from the granting agency).  In my previous health research, published in Environmental Justice 2013, (and in Local Environment in 2016), the costs were minimal (tiny incentives to complete f2f interviews, N=100 households, with a very high % response rate).  I added a small stipend for the community partner for all their time/labor, but they would have done it pro bono (because, perhaps, of my many years of pro bono leadership IN the community organization).  So the process was not totally measurable in cost/dollar terms.  And of course I shared findings with the community after the analysis was done.  Reciprocity makes the world go around. 
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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.
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In reality there are only articles that tend to show that seroconversion goes hand in hand with protection.
But the great majority of the works carried out all over the world lead to the opposite: an immune response giving no protection.
But these works are never published, without interest! What's the point!
For example: Influenza, HIV, Dengue, CMV, Leptospira, Pseudomonas, cancer cells ....
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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
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Hi Muhammed,
Maybe complement by saying what antibody class you are looking for?
What toxin is it? What route are you using to immunize? ip, im, iv, sc...
I've only actively sensitized with OVA with and without Alum, IP and SC. I don't measure titers, but I can see airway hyperreactivity when I do so and I've seen papers where they state the immunization route influences the proportion of IgG and IgE in serum.
I don't know about toxins, but you could check if there are commercial antibodies raised against this toxin and then just passively immunize so you have comparable levels in all mice.
Good luck!
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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
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yes for collecting antibody Scheherazade Sadegh-Nasseri 
!!
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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?
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The Centers for Disease Control and Prevention (CDC)- National Immunization Survey-Teen is a public domain survey conducted annually and may be helpful.  
I am a doctoral student proposing a qualitative study of adolescent parents knowledge, attitudes, and beliefs about adolescent immunizations specifically HPV.  I am seeking qualitative questions used in other studies however running into problems getting permission to use an existing survey.  
Any suggestions.
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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 
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Reddy, see attachement. The protocol described in the paper will give you the desired product though it is described for coupling to HRP. I used the protocol before and it was a success.
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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.
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we have tested several precipitation methods (methanol/chroroforme, ethanol, ...).
It seems, that ethanol precipitation works best for our "test protein". Simply add 9 volumes ice-cold ethanol (100%) to one volume of buffer containing the protein in 8M urea. Incubate at least 1h at -20°C and then spin down the precipitate. Wash the pellet with 90% ice-cold ethanol, remove the supernatant as good as possible and resuspend the pellet in a suitable buffer. We used PBS with 0,1% SDS - it worked perfect!
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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.
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Immunisation of high risk patients should be a matter of prioritisation if low ammount of vaccine is available.
Immunisation against seasonal influenza can be a very cost-efficient approach if a high percent of population is vaccinated. Herd-immunity can be achieved if we are targeting children [main reservoir of virus!], health-care workers, teachers and education-personell, public-relations personell, etc. If virus will not easily find a vulnerable target [that will act as a carrier for virus] we will protect also high-risk patients.
Effectiveness of influenza vaccine can be "damaged" by a miss-match between circulating virus and vaccinal strains. So I would be cautious to validate an anti-immunisation campaign solely on efficacy data!
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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.
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No upper limit has been established regarding the number of vaccines that can be administered in one visit. When giving several injections at a single visit, separate
2 intramuscular (IM) vaccines by at least 1 inch (2.5 cm) in the body
of the muscle to reduce the likelihood of local reactions overlapping.
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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?
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we have conducted a similar study. all variables listed in your question are right and studied in literature. how to measure, if you mean through conducting a study, it will be through a survey study for parents. this is usually what is done in such epidemiological studies. then you have to design your questionnaire. Have I got your question right?
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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?
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Darien,
several options: 1) paste the sequence of your protein into e.g. the SYFPEITHI algorithm and search for likely epitope candidates. Then have them synthesized and tested.
2) If the literature already proposes immundominant epitopes, have the synthesized and tested.
3) if you would like to be comprehensive, purchase a library of peptides, e.g. 15mers overlapping by 4 amino acids, that span the whole protein. Test this library by creating different peptide pools that allow you to dissect the immune response.
(The most comprehensive but also most expensive strategy.) For CD4 epitopes you may need to use longer peptides.
Read-out could be intracellular cytokine staining or ELISPOT .
Matthias
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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?
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Large, insoluble, or aggregated macromolecules generally are more immunogenic than small, soluble ones because the larger molecules are more readily phagocytosed and processed
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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.
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Thireshni, I have just realised I can add some papers to this repose so here goes.
If different amounts of oil adjuvant is added to an antigen will it affect the immune response?
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Oil adjuvant are known to act by depot action. I am wondering if the antigen is kept same with variable amount of adjuvant how will this affect the immune response?
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You will likely see lower efficacy if you do not use enough of your adjuvant to completely emulsify your antigen. It is likely that changes will affect your results, but exactly how they will be affected likely depends on your particular experiment. Here is a link to one paper that looks at this in more detail and they definitely see differences when varying SE: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485880/ In short, too much or too little is bad, try a few combos if possible. Good luck!
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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.
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The important number is the amount of the protein of interest in the conjugated product. In this way you can match the control group using similar amounts of un conjugated protein. And/or compare different doses.http://www.ncbi.nlm.nih.gov/pubmed/15024047
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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?
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I'm sorry but I do not have experience in your area.
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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?
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Not sure it is really different. Re immunization please find an interesting article below. Immunization rate was high, higher in children of pediatricians indeed.
Posfay-Barbe KM et al. How do physicians immunize their own children? Differences among pediatricians and nonpediatricians. Pediatrics. 2005 Nov;116(5):e623-33.
Results
Ninety-two percent of pediatricians followed the official immunization recommendations for their own children. In contrast, after controlling for gender, workplace, type of practice, and year of diploma, nonpediatricians were more likely not to have immunized their children against measles, mumps, hepatitis B, or Haemophilus influenzae type b. They more frequently postponed diphtheria-tetanus-pertussis (DTP) (OR: 4.5; 95% CI: 2.0-10.19) and measles-mumps-rubella (MMR) vaccination. Although projected immunization rates were higher than effective rates, 10% of nonpediatricians would still not follow the official immunization recommendations in 2004.
Conclusion
Ninety-three percent of the surveyed physicians agree with the current official vaccination recommendations and would apply them to their own children. However, the observation that 5% of nonpediatricians would not use Haemophilus influenzae type b vaccine if they had a child born in 2004 is unexpected and concerning. In contrast, both groups gave additional vaccines than those recommended to their own children. Among physicians in Switzerland interested in immunization, a significant proportion of nonpediatricians decline or delay the immunization of their own children with the recommended MMR- or DTP-based combination vaccines, which indicates that clarification of misconceptions such as fear of "immune overload" has not yet reached important targets among health care providers who thus are unlikely to answer parental concerns adequately
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Last week, I watched a talk show on TED Talk about Nanopatch for needle less vaccine delivery method. I am impressed.
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I saw this technology being presented by a famous company a few months ago and I was also impressed. However they are still dealing with the stability without cold chain. I think it can improve acceptance by society and later on improve compliance/vaccine coverage. One important thing to note though is that the price of the product may limit accessibility particularly in third world countries.
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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?
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Victor,
This will depend on the chemistry used to bind the peptides to KLH and the chemistry of the peptides. You need to determine the theoretical number of KLH surface binding sites. My guess if you are using Lysines as your coupling AA then there will be on the order of 10-20 tops for a molecule the size of KLH. Of course if you have denatured the KLH then more binding sites may be available. Then solubility of the final product may be an issue.
The peptide itself may also be able to bind in more than one configuration. If this is desirable then you don't worry about it. If it is not desirable then you will have to adjust your coupling chemistry so that only one configuration is achieved. This can frequently be done by adding an appropriate AA or other binding partner to one end of the peptide. In the end your will have to try a few empirical experiments to achieve the number of peptides/KLH that you desire. Generally more is better, but as you seem aware, not always. Also if you can make enough of your peptide-KLH material then you can just immunize with several doses and you don't have to be quite as optimal.
James
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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?
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Clara, thank you for the article.
It is very intresting and contains lots of useful references, but it doesn't completly fit the topic I am wondering about.
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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.
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Hi Avishekh
If you use killed bacilli it's better to freeze & thaw them (a concentrated pack of bacteria) then determine its protein concentration. Use 20 -30 ug protein with appropriate adjuvant. Subcutaneous immunization works well. Three times inoculation 2 weeks interval  will  usually results in high titer of Ab.
Good luck,
Soheila
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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.
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The health belief model as predictor of preventive health behaviors (Redman, 1980). It provides a complete theory regarding readiness to take health action. According to it, people are not likely to take health action unless they:
■ believe that they are susceptible to the disease in question;
■ believe that the disease would have serious effects on their lives if they should contract it;
■ are aware of certain actions that can be taken, and believe that these actions may reduce their likelihood of contracting the diseases or reduce the severity of it;
■ believe that the threat of taking the action is not as great as the threat of contacting the disease itself.
The model is based on the value expectancy approach to predicting behavior, which has a high cognitive value which motivates or acts a stimulus for change in behavior.
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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
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Hello Srinivas,
Though this is not my area of research, please check whether the attached article is of any help.
Best,
Debabani
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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?
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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. 
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Note that because of the high number of T cells there will be competition for antigen, which will limit their response compared to an adoptive transfer model. Responses wiill also be less robust/comparable between animals (http://www.ncbi.nlm.nih.gov/pubmed/11163194). I  recommend for your assay that you stick with the adoptive transfer model.
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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.
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It might be a ''before-after" study. Incidence rate of cholera using person-time determined before (risk in exposed) and after (risk in unexposed) vaccination campaign can be compared. The number of preventable cases is calculated as follows : number of person-time in exposed group * risk difference.  The etiologic fraction in exposed to assess the vaccine efficacy is : (RR-1)/RR = (number of preventable cases)/(number of cases in exposed group = (risk difference)/(risk in exposed).
NB : RR = risk ratio.
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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?
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vaccine vailure in the presence of "sufficient" antibody titres would probably indicate lack of neutralizing antibodies. This could be due to inefficient induction by the vaccine itself or by occurence of escape mutants / new strains. Thus, the first thing to check would be to sequence the viruses from the infected animals. The next task would be to validate the preence and titer of neutralizing antibodies. In more complicated situations vaccine failure may be due to coinfections which may be much more severe and where the vaccine doesnt provide enough protection.
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I want to know, in troubleshooting in the balb c immunization specially, what the difference between prebleeding and immunized mouse is?
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Take a look at the following manuscript
Konduri V1, Decker WK, Halpert MM, Gilbert B, Safdar A. Modeling dendritic cell vaccination for influenza prophylaxis: potential applications for niche populations.J Infect Dis. 2013 Jun 1;207(11):1764-72.
Abstract
BACKGROUND:
Cancer patients can exhibit negligible responses to prophylactic vaccinations, including influenza vaccination. To help address this issue, we developed in vitro and in vivo models of dendritic cell (DC) immunotherapy for the prevention of influenza virus infection.
METHODS:
Human cord blood (CB)-derived or mouse splenocyte-derived DCs were loaded with purified recombinant hemagglutinin (rHA). T-cell responses to HA-loaded CB-derived DCs were determined by ELISpot. Protective efficacy was determined by vaccination of BALB/c mice with a single injection of 10(6) autologous DCs. DC migration to peripheral lymphoid organs was verified by carboxyfluorescein succinimidyl ester staining, and HA-specific antibody titers were determined by enzyme-linked immunosorbent assay. Mice were then challenged intranasally with BALB/c-adapted A/New Caledonia influenza virus derived from four consecutive lung pool passages. Antigen-presenting cell (APC) dysfunction was modeled using the MAFIA transgenic system, in which the Csf1r promoter conditionally drives AP20178-inducible Fas.
RESULTS:
CB-derived human DCs were able to generate de novo T-cell responses against rHA, as determined by a system of rigorous controls. Mice vaccinated intraperitoneally developed HA titers detectable at serum dilutions of >1:1000. HA seroconverters survived virus challenge, whereas unvaccinated controls and vaccinated nonseroconverters lost weight and died. Furthermore, use of a model of APC-specific immunosuppression revealed that DC vaccination could generate HA-specific antibody titers under conditions in which protein vaccination could not.
CONCLUSIONS:
The model demonstrates that DC immunotherapy for the prevention of influenza is feasible, and studies are underway to determine whether populations of immunosuppressed individuals might ultimately benefit from the procedure.
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Due to higher salt concentraion the specific protein can aggregateed?
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If only they are smaller than your MW cut off dialysis tube they would dialyze out. Refer to th e link http://www.spectrumlabs.com/dialysis/FAQ.html for choosing the right pore size  
Is there anyone interested in conducting EPI coverage survey?
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I'm looking for individuals or organizations which are interested or have experience of conducting EPI coverage survey in different countries.
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Yes
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By family structure I mean nuclear and single parent family. Also by religiously I mean religious participation and the religious office. 
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truly i agree with the fact that type of family and religiosity effect the immunization pattern of children . there are several examples about the same. In many religions immunization is not permitted and type of family like joint family, one may skip the date and time of immunization, same could be with single parent family , due to excessive work the parent might skip the immunization schedule
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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
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Have you checked to ensure that the protein is routed to the cell surface using your selected vector? You should trial this is cell culture prior to immunization, if you have not done so already.
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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
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Please send me your research proposal at khaled.ali@med.au.edu.eg
Can OT-II cells become Foxp3+ T regulatory cells?
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I find the literature conflicting on this. Some people say that OT-II cells can express Foxp3. Others argue they don't. In my own hands, I could not induce Foxp3 in OT-II cells following OVA/CFA immunization using a Foxp3 GFP-OT-II mouse. Any ideas?
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Hi Mark, When you isolate CD4 T cells from the spleen or peripheral lymph nodes of OT II mice, there is a fraction of cells that already expresses Foxp3, as deteced by intracellular antibody staining for Foxp3. If you want to induce Foxp3 expression in Foxp3 negative OT II CD4 T cells I would try to stimulate them with TGFb under T cell receptor stimulatory conditions, maybe add some IL-2. I didn't try it with OT II cells but it worked in my hands with CD4 T cells on the B6 background.
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Why antibody-mediated immunity to Shigella dysentery requires several episodes of infection to get primed?
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I'm guessing a bit but as Shigella dysentry involves a  lot of toxin release rather than an inflammatory response say to Salmonella, there is insufficient invasion or persistence to lead to development of a sufficient adaptive response that leads to immunity
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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?
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ISSUE: Two samples comparison study
GROUP DEFINITION: Given that there are two sample groups. Group A drawn from the general population. These drawings are denotes as: Xa1, Xa2, …, Xan. Group B is selected from medical records file: Xb1, Xb2, …, Xbn. Now code A = 1 and B = 2 to avoid confusion. The following definitions apply:
(1)   SS1 = sum(Xi – X*1)2
This is the sum squared difference of group A, now called group 1.
(2)   SS2 = sum(Xi – X*2)2
This is the sum squared difference of group B, now called group 2.
The pooled variance for the two groups is given by:
(3)   S2 = (SS1 – SS2) / (n1 + n2 -2)
… where n1 = sample size of group 1 and n2 = sample size of group 2. The samples n1 and n2 do not have to be the same size.
   The following test statistic applies:
(4)   t = [(X*1 - X*2) – (mu1 – mu2)] / S(sqrt (n1 + n2 – 2))
   Here lies the answer to the question: which standard score should I use? The S in this equation is the square root of S2 defined above in (3). There is no separate S; there is only one S; this is called common standard deviation. In order to get the common standard, the standard deviation and square root of (n1 + n2 – 2) are used.
   Specify your confidence interval and compare the tobs to the standard. Say you use 95% confidence interval, then H0: tobs < t0.95 and HA: tobs > t0.95.
SAMPLE SIZE ADJUSTMENT: There is no need for sample size adjustment. The sample size does not have to be equal. The sample size for group A = n1 and the sample size for group B = n2. The total sample size: n = n1 + n2.
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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?
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Hi there are several protocols for immunization of mice, depends from the antigen you want to inoculate. Please give more details.
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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?
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Hi Fabiano,
Very interesting research.
I produced many monoclonals using short peptides (8-15AA). In my own expericne, it is difficult to indue immune response using short peptides. Therefore, they are nomrally conjugated to a protein crarrier such as Ovalbumin. Conjugated peptides are then made into water in oil immulsion using Complete Freund's adjuvant for one immunisation and incomplete FA in the next three immunisations. Final boost is used before spleen collection (attached table of immunisation protocol). On day 38, you run immune response test by ELISA using tail bleed. You test the immune response against the immunogen peptide by coating ELISA plates with the same peptide. However, the peptide used in ELISA is conjugated to another carriers to make sure you are not measuring the immmune respsonse against the carrier used in immunisation.
Just final note, make sure your run some bioinformatic on your peptides to make sure it has the necessary physico-chemical characterstics (e.g. antigenic,accessible and specific).
Good luck
Abdo