Building an Enzyme-Linked Immunosorbent Assay for Measuring Mouse IgE Reactive to Peanut Proteins

Tasneem Ali Author

04/03/2021 Added

34 Plays

Description

An Enzyme-Linked Immunosorbennt Assay (ELISA) is an assay used to detect antibodies present in the blood. In a peanut-specific ELISA, we want to measure IgE antibodies that are specific to peanut.

Searchable Transcript

Search:

[00:00:02.220]Hello. My name is Tasneem Ali and I am a senior biological science major.
[00:00:07.230]I conducted my research under Dr.
[00:00:09.210]Amanda Raymer T of the department of food sciences. Today,
[00:00:12.720]I will be presenting to you about the research that I worked on,
[00:00:15.660]which is building an enzyme linked immuno absorbent assay for measuring mouse,
[00:00:19.620]IgE, reactive.
[00:00:20.640]To proteins. Food.
[00:00:22.920]Allergies are on the rise around the world.
[00:00:24.960]Many investigators have shown a link between the microbiome and the development
[00:00:28.620]of food allergies. However,
[00:00:30.450]additional studies are needed to determine if there is a causal relationship.
[00:00:34.830]Our lab is investigating whether microbiome interventions early in life can
[00:00:39.210]impact production of peanut protein, reactive IgE antibodies. However,
[00:00:43.500]current Eliza kits are limited in their specificity to the wider food allergens.
[00:00:49.740]Our.
[00:00:49.980]Bodies contain specialized white blood cells called B lymphocytes or B cells
[00:00:54.360]that produce antibodies antibodies work by recognizing specific proteins.
[00:00:58.740]For example,
[00:00:59.400]proteins that can be found on the surfaces of viruses and bacteria and Eliza in
[00:01:03.780]is, is an assay test in order to detect antibodies present in the blood and it
[00:01:08.730]peanut specific Eliza.
[00:01:09.990]We want to measure IgE antibodies that are specific to peanut rather than using
[00:01:14.010]a general Eliza that detects all IgE antibodies regardless of their specificity,
[00:01:19.440]in order to measure how well the model recapitulates and allergic phenotype and
[00:01:23.820]Eliza measuring peanut specific IgE is needed.
[00:01:27.150]We designed the assay from scratch by looking in the literature at other similar
[00:01:30.600]design peanut specific Eliza's.
[00:01:33.330]The goal was to develop a peanut specific IgE Eliza that could be used to test
[00:01:37.890]serum samples from two mouse studies to measure the levels of peanut specific
[00:01:41.280]IgE present. However,
[00:01:43.470]the overall hypothesis for this project is that a probiotic,
[00:01:46.530]bifidobacteria longum, subspecies and fantasies can continue wait.
[00:01:50.460]The severity of peanut allergy in germ-free mice, given an infant.
[00:01:53.630]Microbiome.
[00:01:56.670]First step in building an Eliza is to make a plan and find the appropriate
[00:02:00.080]reagents sandwich Eliza's was chosen based off of a general IgE kit.
[00:02:04.760]I sandwich Eliza is one of the most common types of Eliza's used by researchers.
[00:02:08.930]Sandwich Eliza's are highly specific and are normally two to five times more
[00:02:13.040]sensitive than a director indirect Eliza. For example,
[00:02:17.360]when we look at this graph,
[00:02:18.800]figure one a shows us the steps of a sandwich. Eliza,
[00:02:23.240]the antibodies are layered.
[00:02:24.470]As you can see around the specific target molecule and a sandwich Eliza,
[00:02:28.400]this Eliza measures antigens between layers of capture antibodies and detection
[00:02:32.720]antibodies. Figure three B shows the light reaction
[00:02:38.690]observed after adding TMB substrate.
[00:02:41.000]To a solution.
[00:02:43.340]Second, the struct Avidan HRP and IgE concentration or detection,
[00:02:47.840]body and coding antigen concentration.
[00:02:50.330]So our reagents that were used for this assay need to be optimized.
[00:02:54.770]So in these figures, there are the plate maps for region optimization.
[00:02:58.310]We tested assay and titration experiments of serum samples with high IgE
[00:03:02.290]determined from a total IgE Eliza kit from a previous experiment.
[00:03:06.160]In order to know the concentrations of reagents to use,
[00:03:08.950]we had to optimize the strength of stroke evidence, the coding antigen,
[00:03:12.670]and the detection antibody here in figure two a it shows us the plating scheme
[00:03:17.140]for testing the best combination of strep Abbot in HRP and IgG detection,
[00:03:21.940]antibody strep Avadyne was tested at a range of four micrograms
[00:03:26.890]per milliliter to 0.2 micrograms per milliliter.
[00:03:31.000]I showed him figure two,
[00:03:32.050]a above the optimal kombucha concentration was 0.2 micrograms per
[00:03:36.670]milliliter using this optimal concentration of stripped Avenue and IgG D
[00:03:41.290]previously determined.
[00:03:42.400]We next saw to optimize coding antigen concentration figure two B shows us
[00:03:47.380]the plating scheme for determining the best concentration of peanut extract.
[00:03:51.370]We tested out a range of one to 10 micrograms per milliliter of peanut extra as
[00:03:56.170]shown in the figure to be below.
[00:03:59.440]It was determined that five micrograms per milliliter was the best combination
[00:04:03.040]for this reagent.
[00:04:07.020]However.
[00:04:08.490]Based off of a general IgE Eliza kit,
[00:04:11.370]we were able to infer while pilot studies would result in the highest peanut
[00:04:15.300]reactive IgE.
[00:04:16.740]The two studies were peanuts specific IgE pilot one and pilot two
[00:04:21.690]would share one common treatment.
[00:04:23.610]Both studies included a group of mice that received five milligrams at peanut
[00:04:27.810]flour with 10 milligrams of corn toxin after a dose of sodium bicarbonate each
[00:04:32.490]week for five weeks. However, we didn't know which dilution factor.
[00:04:35.880]Doctors to use for this.
[00:04:40.320]Titrating.
[00:04:40.890]The samples allows us to clearly see how the response changes according to how
[00:04:44.640]we dilute the sample.
[00:04:46.020]The dilutions use for each sample are shown on the X axis of these graphs.
[00:04:50.610]This was important because of larger dilution factors.
[00:04:53.010]Wouldn't show the magnitude of difference between the treatments as well as the
[00:04:55.950]lower dilution factors are peanut specific IgE titration curves show that we can
[00:05:00.870]dilute out the response,
[00:05:02.340]which validates that sensitive citation strategy does induce allergic response
[00:05:06.720]to peanut. As you can see in figure three, figure three,
[00:05:10.110]a and C were made by applying a nonlinear regression line of best fit to the
[00:05:14.880]average values in each treatment.
[00:05:16.620]In order to show how variable the responses were.
[00:05:18.840]We also graph the curves for each individual mouse as shown in figure three B
[00:05:23.250]and figure 3d.
[00:05:24.840]This graph shows which strategy is best for the highest peanut IgE figure three,
[00:05:29.670]a specifically as the first pilot study,
[00:05:32.280]we tested two different doses of peanut flour,
[00:05:34.860]one milligram and five milligrams in combination with 10 micrograms of Koran
[00:05:39.090]talks.
[00:05:39.390]And given 30 minutes after a dose of sodium bicarbonate average values per
[00:05:43.470]dilution are shown for each treatment.
[00:05:45.540]Figure three B shows the individual mouse curves from the pilot.
[00:05:49.140]One study figure three C is the second pilot study.
[00:05:53.220]We tested treatments that varied in route and substance frequency of
[00:05:56.310]sensitization and challenge.
[00:05:58.160]The average values per dilution are shown for each treatment figure.
[00:06:01.970]3d shows the individual mouse curves from the second study.
[00:06:05.390]There are approximately seven,
[00:06:06.770]six to 10 mice per treatment when looking at these graphs for both pilot
[00:06:11.390]studies and when comparing the two doses of one milligram and five milligrams,
[00:06:14.960]the five milligram was the best meaning using a higher dose of peanut
[00:06:19.790]is better for inducing peanut specific IgE
[00:06:25.220]in conclusion,
[00:06:26.570]food allergies are Abrams immune responses in our bodies that occur after eating
[00:06:30.470]certain foods,
[00:06:31.340]the process of building and choosing an Eliza depends on what you are trying to
[00:06:34.850]measure in terms of our project.
[00:06:36.710]It was determined that we wanted to measure our response to a specific allergen.
[00:06:40.520]So that is why we use a peanut specific Eliza rather than using a general as
[00:06:44.850]Eliza kit.
[00:06:45.800]We were able to compare two pilot studies that showed high total IgE to
[00:06:49.400]determine the best strategy for sensitizing.
[00:06:51.830]My subpoena Eliza is can also be used to detect target molecules in other
[00:06:56.510]bodily fluids like urine saliva salmon, and sell super namings.
[00:07:01.550]There are broader applications for Eliza that include clinical diagnosis,
[00:07:04.820]for example, HIV,
[00:07:06.350]which uses an Eliza test in order to measure anti HIV molecules from blood
[00:07:10.280]samples.
[00:07:13.730]This work was made possible by Dr.
[00:07:15.590]Amanda rammer T who gave me the opportunity to be in this lab, Morgan, Kate,
[00:07:19.830]the PhD student,
[00:07:20.690]who helped me through every step of the way my entire lab and my entire lab team
[00:07:24.620]for always being willing to help, whether it being inside or outside of the lab.
[00:07:28.580]And you care for funding this project,
[00:07:30.680]thank you to each and every one of you for making this project possible.
[00:07:35.380]Yeah.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

Building an Enzyme-Linked Immunosorbent Assay for Measuring Mouse IgE Reactive to Peanut Proteins

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments