The Immune System Response To Allergy: 1212577

Introduction

The human immune system has been referred to the well-organized system in which the body takes a series of actions to provide resistance to any types of external toxins or on an initiating infection. The main function of the human immune system is to protect the human body against the infectious agents like bacteria, viruses and various other microorganisms which have bee reported to be pathogenic in nature. The main system which contains the lymphoid tissues and channels, the tissues have been found to be free from the unwanted extracellular liquid from the periphery through the thoracic duct (Body Defenses). Both innate and adaptive immune system has been found to play an important role in allergic reactions which is initiated in response to the allergens. However, it has been found that both the immune systems respond in a different manner in allergic reactions. Human adaptive immune system has been found to fight against bacterial antigens and toxins which produces a secondary immune response. The innate immune system has been found to act as the first line of defence against the pathogens and helps in sensitizing and catalyzing procedure for allergic reactions associated with a tree nut allergy (Charles, Janeway, Travers, Walport, & Shlomchik, 2001). TN (Tree Nut) allergy has been found to be one of the most dangerous and common allergies which can be very serious if not treated at an early stage. The main symptoms of nut allergy include nausea, anaphylaxis, difficulty in swallowing, shortening of breath, itching eyes, mouth, skin and redness. Tree nuts which are included in this category are pecan, walnut, hazel, walnuts, pine, cashew, macadamia nuts, coconut and almonds (Clark, 2007). However, there is no evidence for allergy associated with shea nuts, coconut oil and butter since they do not cause any type of hypersensitivity as their precursor nuts do. Almond and other tree nuts have been found to be present among the most commonly consumed food across various countries and have been found to result in cases of anaphylaxis in many children. Anaphylaxis has been observed as the life threatening and acute allergic reaction which involves the coordination of more than one organ in the body. As soon as a three nut protein is recognised as antigen, anaphylaxis occurs and mediators such as basophils and mast cells are released just after the ingestion of tree nuts. The response of human immune system is clinically manifested by leukotrienes, histamines and prostaglandins which results in the occurrence of pathophysiologic effects including increased vascular permeability, smooth muscle contraction, nervous system stimulation and vasodilation. The prevalence of tree nut allergy has been found to vary by geographical zone, age and the prevalence has been found to increase in the children (Zhu et al., 2016). More specifically, it can be stated that Cor a 9, which is an 11S globulin component of hazelnut is responsible for sIgE induced hazelnut allergy in human beings which consume the same. The cashew allergen responsible for causing allergy in human beings is Ana o 2 which is a legumin like 11S globulin and Ana o 3, which is a 2S albumin. Jug r 1 component of walnut allergen has been found to exceptionally cross react with Car I 1 2S albumin and Car i 4 11S legumin in order to generate a double blind anaphylaxis reaction which is very much deadly in case of people who are sensitive to the allergen. Components of almond, such as Pru du 6 (11S globulin), Pru du 3 (LTP), and Pru d 4 (profilin), have been recognized, however very little clinical information is available about them. Tree nuts have been reported to induce PFS (pollen food syndrome) which causes an allergy mediated by IgE which is caused by the cross-reactivity of pollens and homologous food allergens. Skin prick test, oral food challenges (OFCs), atopy patch tests (APTs) and allergen-specific serum test have been found to be the most used diagnostic tests for the identification of TN induced allergy in an individual (Sciani & Pimenta, 2017). According to various observations, it can be stated that hypersensitivity to almonds has been found to increase the chances for the occurrence of tree nut allergies among the human population (Thompson, 2015). Two classes of allergic reactions have been identified. One of the classes is mediated by IgE (immunoglobulin E) and the other reactions are mediated by the non-IgE that has a delayed response period which is compared to the IgE group (Suresh & Mosser, 2013). TN allergy has been stated to be an example of a food allergy which is orchestrated by IgE proteins. After the interaction of IgE with cell surface receptors, the interaction occurs followed by an inflammation that triggers the allergen release, After the interaction of IgE allergen, the process of inflammation occurs which triggers the release of WBC (white blood cells) in order to affect the site associated with prolonged symptoms (Geiselhart, Hoffmann-Sommergruber & Bublin, 2018). Late risk factors have been found to consist of delayed onset of symptoms including beta blockade and inadequate dose of epinephrine administration, which may fail to act against the opposing allergen. According to many research studies, it has been found that IgE to Ara h 2 has been shown to be associated with almond allergies as confirmed by OFC. However, cross-sensitisation between nuts is very much lower, as stated by many research studies. The nut allergy tests confirmed that IgE mediated tree nut allergy had a prevalence rate of 2% and sometimes the prevalence percentage fell down to 0.05 to 4.9%. According to the reports of 2011, the prevalence percentage of nut allergy was 1.76% in the UK (Sympayrac 2011). However, it has been observed that the prevalence rate of nut allergy has increased by two times since the past 10 years. This paper will analyse the response of the immune system to tree nut allergy by following the procedure stated in the following sections.  

Aim and Objectives

The aim of this experiment is to analyse the immune response to tree nut allergy.

The objectives of this experiment are:

1. To separate the proteins present in Almond nuts by SDS-Page gel.

2. To stain the gel and identify the protein bands of interest.

3. Running the samples on mass spectrophotometry- Protein mass spectrophotometry.

4. To analyse the proteins from the observation of mass spectrophotometry.

Methodology

Materials required

The materials required for this experiment is given in the following table:

Chemicals:

10% trichloroacetic acid in acetone containing 0.07% 2-mercaptoethanol, Acetone containing 0.07% 2-mercaptoethanol, 7M Urea, 2M thiourea, 4% (w/v) CHAPS, 2% (w/v) DTT, Bovine Serum Albumin (BSA), Coomassie Brilliant Blue, Ethanol, Phosphoric acid, H2O, Bradford reagent (see recipe in method), BSA, Acrylamide 30%, Concentrated hydrochloric acid (HCL), 1,5 M Tris (pH 8.8), 1M Tris (pH 6.8), 20% Sodium dodecyl sulfate (SDS), Tetramethyethylenediamine (TEMED), 10% Ammonium persulphate solution (APS), Mass Spectrometry: trypsin solution, Acetonitrile, Matrix solution

Physical equipments:

Pipettes, Eppendorf, Fume cupboard, Vortex, Centrifuge, 96-well microplate reader set at 595nm absorbance, Vacuum concentrator, MALDI plate

Sample: Almond

The laboratory method has been divided into several steps, the first being “Protein extraction”. There are three main stages of this procedure.

 Protein extraction

The almonds were roasted at 100 degrees for five-time intervals. The five-time intervals are 0, 15, 30, 45 and 60 minutes respectively. The almonds were crushed and different nuts were separately blended and then transferred into the mortar. The total amount were beaten with a pestle in order to form a finer substance which will make the process of protein extraction easier. Some of the almonds from each of the sample were extracted and weighted to 100 mg. This amount was then transferred to an Eppendorf (1.5 mL) and 1mL of extraction solution was added to it. The extraction solution was prepared with 10% TCA (Tri-Chloro-Acetic acid) inside cold acetone which contains 0.07% (v/v) beta-mercaptoethanol. Equation for the recipe:  = 5ml

 =  = 0.035

The extraction solution was freshly prepared and stored in the freezer at -20 degrees. Trichloroacetic acid (TCA) in acetone containing 2-mercaptoethanol is used to precipitate proteins during the sample preparation and using TCA OR acetone alone. The samples were stored overnight at 4-degree centigrade after the addition of TCA. The time was allowed for protein separation in order to change the protein solubility technique. After the process of overnight precipitation of protein, sample centrifugation at 15,000 xg for 4-degree centigrade was done for 15 minutes. The washing solution is made up of acetone containing 0.07% (v/v) 2-mercaptoethanol stored at -20oC. To work-out the recipe for 50ml from 100ml of the washing solution needed for the experiment.

Equation: = 0.035

The purpose of adding the washing solution is to remove the TCA extensively with acetone or ethanol. Prolonged exposure to this low pH solution may lead to some protein degradation or modification. After washing the samples, the liquid was discarded which was lying above the sample residue after the precipitation and then placed the samples into a fume cupboard and the pellets were allowed to air dry in order remove the residual acetone.

  • The samples were stored at -80 degree centigrade freezer unless it was ready to be used for the next step.

Protein solubilisation

The next step of this experiment was to solubilise in 500 l solutions and stored at -2 degree centigrade. The solution has been found to increase the solubility of a poorly water-soluble substance associated with a surface agent. The solubilisation calculations have been shown below for the respective reagents and chemicals-

(i) Urea = 42

(ii) Thiourea = 15.224

(iii) CHAPS = 245.952

(iv) DTT = 30.8506 ÷2=15.4253  in 50ml

Urea and thiourea can improve solubilisation of the hydrophobic proteins. Samples that have both urea and thiourea can be used in SDS PAGE when diluted with SDS PAGE sample buffer. In this case, the protein solutions may not be above 37oC. DTT is used as a reducing agent to reduce disulphide bonds of proteins and prevent intramolecular and intermolecular disulphide bonds forming between cysteine residues of proteins.

 Bradford assay

Bradford protein assay is widely used to work-out the concentration of the total protein found in each of the samples. It is commonly used because of its spontaneous and suitable protocol as well as its comparative sensitivity for protein quantitation. The use of this assay is fairly accurate for most proteins but with the exception of smaller primary polypeptides such as the enzyme ribonuclease.

Only 10 micro-litres of each sample was used as the Bradford reagent’s volume was reduced from its normal value of 1mL. Bradford reagent was prepared by using sodium phosphoric acid, ethanol, Coomassie blue and H2O and 50 mL of methanol. These chemicals were poured into 100 mL of 85% H3PO4 and the solution was mixed gently giving rise to a final solution of 500 mL with H20. After the dye was dissolved, the precipitates were removed with the help of a filter paper in order to get rid of the precipitates before the same solution was used again. The solution was kept in a dark bottle at 4-degree centigrade.

Standard assay process

The plates were read at a wavelength of 595 nm. The format of the used plates were 96 wells. The protocol followed for this experiment is known as Ed’s protocol. An aliquot of protein standard at 1 mg/Ml was used to make six different protein standard dilutions by using BSA (bovine serum albumin) and water. The diluted and unspecified protein samples were found to obtain 100 .

Proteins Standards
 Protein standard dilutions
SampleBovine Serum Albumin (BSA) mg/mlDistilled H2O ml
10100
22080
34060
46040
58020
61000

The plates were read at a wavelength of 595 nm. The format of the used plates were 96 wells. The protocol followed for this experiment is known as Ed’s protocol. An aliquot of protein standard at 1 mg/Ml was used to make six different protein standard dilutions by using BSA (bovine serum albumin) and water. The diluted and unspecified protein samples were found to obtain 100 .

Table 1: Standard dilution

Source: Created by an author (Ed’s protocol)

A mixture of 20  of the BSA and H2O dilution was pipetted into the well plate and 100 microliter Bradford reagent was added to it. The absorbance was read at 595 nm. Each of the sample protein standards was then duplicated. Two blanks were set in A1 and B1 on the 96 well plates. The Blanks were made up of 10 water and 200  of the Bradford reagent. The next reading started from C1 for 0 minutes reading, D1 again for 0 minutes and C2 and D2 wells for 60 minutes. The duplicates were then assayed for each of the samples and the same volume was added to each of the unknown protein samples to the necessary wells. The same wells contained the same Bradford reagent volume. 1.0 of each sample and 200  of Bradford to each well. The sample and reagent were mixed by using a Pasteur pipette in up and down motion. A fresh pipette tip was used for each sample to avoid contamination by the Bradford reagent in each well. The plate was then incubated at five minutes at room temperature and then the plates were read in a plate reader set to 595 nm. The mean absorbance reading was noted from the protein standards and used to work out the standard curve. This curve was found to turn along with the mean value observed from the Bradford reagent and the sample mixture absorbance. This process helped in deciding the amount of protein to be used in the following step of this research.

Gel electrophoresis

Chemicals and reagents

10 ml of 10% separating gel- 4.1ml H2O, 3.3 ml acrylamide (30%), 2.5 ml 1.5 M tris (pH 8.8), 50 microliter SDS (20%), 100 microliter Ammonium Persulfate, APS (10%), 10 microliter TEMED 2.7 ml H2O

4 ml of 6% stacking gel- 0.8 ml acrylamide (30%), 0.5 ml 1 M tris (pH 6.8), 20 microliter SDS (20%), 40 microliter Ammonium Persulfate, APS (10%), 4 microliter TEMED, Protein samples, Isopropanol, Distilled water, Running buffer, Coomassie staining solution, Destain solution, SDS-PAGE ladder

Physical equipments: Mini glass beakers, Pasteur pipettes, Gel cassette, Casting Strands, Plastic combs, Clamping frame and electrode assembly, Glass plates, Disposable weighing boats, Block heater. All the materials were collected according to Morrison Lab’s protocol SDS-PAGE recipes.

Five individual glass plates sandwich were prepared and the small plates and large plates were placed together with the smaller plate facing outwards and the larger plate backwards. Thus, the plates were placed in a pattern such that the two plates were aligned into the casting frame and then onto the casting stand. Agar solution was added to the bottom of the gel and the plates were sealed which prevented the leaking of gels during the process of pouring. 1.5 % agar solution was stored in an Erlenmeyer flask and the top was sealed with aluminium foil at room temperature. After adding the agar solution to the bottom, it was left to dry. There are different recipes for making the separating gel and I used 10% separating gel recipe. Five different gels were used for each of the nut samples and each gel was added up to 10 mL. The materials were added to Pyrex glass beaker step by step which was listed as given in the protocol with water which went first. Next, acrylamide, SDS, APS, Tris and TEMED were added. TEMED was added at last in order to prevent a pre-drying of the solution. After the agarose gel dried, APS and TEMED were poured into the beaker and were mixed for three to five seconds and they were poured into the separating gel mixture. After the gel solidified, isopropanol was poured over the separating gel and washed with isopropanol before five seconds with distilled water. Paper towel was used to dry out the remaining liquid at the top of the gel. In the next step, stacking gel was made. The same procedure was followed for the preparation of a stacking gel solution. The stacking gel solution was then mixed and Pasteur pipette was used to pour stacking gel on the separating gel and then a comp was put into it. Polymerisation usually takes about half an hour and the gel was left in the refrigerator overnight to dry. After the polymerisation process was over, the gels were taken out of the fridge and the samples were taken out of the freezer which leaves it to turn into a molten state by using lukewarm water. 10 microliter sample buffer was pipetted into different volumes of nut samples inside different tubes. The first was to pipette 10 ul of the sample buffer and then 10 ul of the 0-minute nut samples into an Eppendorf tube, then 10 ul of the sample buffer and 7.5 ul of the 0-minute nut sample into another tube, 10 ul of the buffer sample and 5 ul of the 0-minutes nut sample, 10 ul of the sample buffer and 2.5 ul of the 0-minutes nut sample into tube. The same process was repeated for the same volume of sample buffer and the same volume of nut sample. Each tube was labelled as 0 minutes at 10 microliters and 0 minutes at 2.5 microliters. These tubes were boiled for five minutes at 1000 degree centigrade in a block heater- QBDI grant. The SDS ladder was not placed to boil in the blocker heater. Instead, I went on to set up the running equipment for the SDS –PAGE gels. In order to start assembling of gels inside a Bio-Rad MINI-PROTEAN, the PROTEAN was prepared and assembled according to the running module by opening up the win clamps on either of the sides. The gels were loaded onto the running module with the shorter plates facing inside into the electrode assembly module. The plastic combs were then removed from the gel and the running buffer was added into the inner chambers. Then the samples were loaded into the gel according to the sequence given after this line. The first nut sample (0 minutes) containing 10 microliter nut sample was pipetted into the first lane. Then 20 microliter was pipetted into the third lane from the left followed by 17.5 microliter sample mixture of 0 minutes into the third lane, 15 microliter of sample mixture to the fourth lane and 2.5 microliter sample mixture also to the fourth lane from left. The same addition process was followed for all the time intervals (15, 30, 45 and 60 minutes) nut samples. After different volumes of nut samples were put into the gel, the gel box was covered with a lid which makes sure that the coloured electrodes on the lid match the electrode posts on the core and the gels were left to run. The power supply was set to 100 volts and it was increased to 200 volts when the samples were running halfway to the end through the gel. After the run was completed, the power supply was turned off and the lids were removed. After the completion of the run, the wing clamps were opened from either side of the gel and spatula was used to open the sandwich carefully in order to break the plate. Coomasie blue stain was put into the several trays and then they were placed onto the gel and each tray of staining. The next step was known as de-staying step and the de-stain was poured into different trays and it was kept overnight unless the background was clear. The bands were found to appear in the tray and the gels were kept in a detainer for two hours unless the background was clear.

Preparation of proteins for matrix-assisted laser desorption (MALDI) Mass Spectrometry data analysis

Zero to fifteen minutes of samples were used for this step. The last column gel bands were cut from all the samples and put in separate tubes after adding them to 50 microliter acetonitrile. These were then dried in a vacuum concentrator before adding 50 µl of trypsin solution (1mg/ml trypsin in 50mM ammonium bicarbonate) and left at room temperature for approximately 16 hours. The supernatant was then removed into a new tube and sonicated for 15 minutes. The supernatant was then removed and added with the previous supernatant and this step was further repeated for two more times in order to give a final volume of 200 microliters for each of the sample. The supernatant was removed and added to the previous supernatant and this step was repeated 2 more times giving a final volume of 200 µl for each sample. This was then dried in a vacuum concentrator and re-solubilised in 10 µl of matrix solution (10mg/ml hydroxycinnamic acid in 50/50 acetonitrile /0.1% trifluoroacetic acid in water) and 1 µl of this was added to a MALDI plate. Each sample on the MALDI plate had a calibrant mixture on the adjacent spot and the MALDI analysis utilised a Voyager DE-STR mass spectrometer in positive reflectron mode with a voltage of 20kV and the laser energy was manually adjusted as required for each sample and calibrant. The resulting mass spectra were externally calibrated using the calibration mixture to ensure mass accuracy and the peptide mass fingerprint was submitted to MS-Fit for protein identification with a mass tolerance of 10ppm and a minimum of 1 peptide identified.

Results

Standard deviation calculation

Protein StandardsTriplicate readings of absorbance(595nm)MeanStandard Deviation
Bovine Serum albumin (BSA) 1ug/ulH20 ulReading 1Reading 2Reading 3
0 ug1000.2150.1850.2220.20730.01965536
20800.4540.4530.4790.4620.01473092
40600.5210.4740.3660.45360.07947536
60400.5310.4590.5580.5160.05117617
80200.3860.6020.5460.51130.1120952
10000.5560.5340.6300.5730.05029248

Table 6: Result table 1 (standard curve of protein)

Source: Created by the author

Graph of the standard curve

Fig A: Standard curve of BSA plotted from the first table (X axis shows the concentration of BSA) and Y axis shows the Absorbance values. A best fit line has been drawn for the obtained standard curve to linearize the results). This curve was prepared by plotting the concentrations of protein in the X axis and the mean absorbance values on the Y axis from table 6.

Source: Microsoft Excel

The R2 value calculated with Microsoft excel data analysis is found to be 0.72. The results have been shown below-

SUMMARY OUTPUT        
         
Regression Statistics       
Multiple R0.849874386       
R Square0.722286472       
Adjusted R Square0.65285809       
Standard Error0.075550855       
Observations6       
         
ANOVA        
 dfSSMSFSignificance F   
Regression10.0593820.05938210.403330.032115   
Residual40.0228320.005708     
Total50.082213      
         
 CoefficientsStandard Errort StatP-valueLower 95%Upper 95%Lower 95.0%Upper 95.0%
Intercept0.3082380950.054685.6371580.0048740.1564230.4600530.1564230.460053
X Variable 10.0029125710.0009033.225420.0321150.0004050.005420.0004050.00542

The above stated R2 value has been found to be 0.7 that is well above 0.5. This means that the points in the graph are very close to the best fit line which is also evident from the figure. Thus, it can be stated that, in trial 1, there was a constant increase in absorbance for the increase in protein concentration while performing the experiment for standard curve.  

Samples (temperatures) oCTriplicate readings of absorbance (595nm)MeanProtein concentrationProtein concentration required for SDS page gel   100 ugThe final concentration of Protein added to the gels. 100ugConcentrations added100ug
Reading 1Reading 2Reading 3    
00.9720.9971.0200.9963231.697674411.584883728.6319389
151.3471.1341.1021.1943277.74418613.88720937.2008717
300.5800.6860.5670.611142.09302337.10465116314.0752914
450.6770.4530.4010.5130118.67441865.9337209316.8528317
600.4660.4120.2120.36384.418604654.22093023323.6914624

 

Table 7: Result table 2 (unknown protein concentration determination)

Source: Created by the author

Time of roasting vs protein concentration curve trial 1:

Fig B: Time vs protein concentration curve (before adding to gel)- This curve is has a proper increasing nature which proves that as the time of roasting increases, the concentration of protein also increass. This increase has been observed due to the increase in time of roasting the protein samples (nuts) before loading them onto the gel. As time went on increasing, almond proteins broke up opening their aromatic side chains which showed a high protein concentration also and thus resulted in an increase in linear increase of the curve and overall optical density of the protein solution.  

Source: Microsoft Excel

The R2 value of the curve has been found to be 0.88. The results of the calculation has been shown below:

SUMMARY OUTPUT        
         
Regression Statistics       
Multiple R0.935560539       
R Square0.875273523       
Adjusted R Square0.833698031       
Standard Error9.671859065       
Observations5       
         
ANOVA        
 dfSSMSFSignificance F   
Regression11969.3651969.36542721.052630.019445483   
Residual3280.634693.54485777     
Total42250      
         
 CoefficientsStandard Errort StatP-valueLower 95%Upper 95%Lower 95.0%Upper 95.0%
Intercept-16.608315111.0406-1.5042943820.229556-51.7444371718.52780697-51.7444371718.52780697
X Variable 13.2822757110.7153554.5883146770.0194451.0056955415.5588558821.0056955415.558855882

The R2 value has been found to be well above 0.5 which shows that the points are very close to the regression best fit line. Thus, it can be stated that as the temperature increases, there is a constant increase in the protein concentration or the concentration of roasted almonds added to the gel.

Determination of unknown protein concentration in unknown samples

Samples (minutes roasted) oCDuplicate readings of absorbance (595nm)MeanProtein concentrationProtein concentration required for SDS page gel
00.2170.2070.21249.302325582.4651
150.8280.7660.797185.34883729.2674
301.2501.0991.1745273.139534913.657
451.1420.8120.977227.029302311.36
600.7251.2200.9725226.162790711.308

 

Time of roasting vs protein concentration curve trial 2:

Fig C: Time vs concentration curve (duplicate trial)- This curve showed that as the Time value of protein solution went on increasing, so did the concentration of protein in the gel. The best fit line has been shown with black dotted line. This graph was plotted by using the Roasting time on the Y axis and protein concentration values on X axis from the above table

Source: Microsoft Excel

The R2 value has been found to be 0.99 which is well above 0.5. This shows that the points are very close to the best fit line and protein concentration increases linearly with the time of roasting. The results of R value calculation has been shown below-

SUMMARY OUTPUT       
         
Regression Statistics       
Multiple R0.999999999       
R Square0.999999998       
Adjusted R Square0.999999997       
Standard Error1.9056E-05       
Observations5       
         
ANOVA        
 dfSSMSFSignificance F   
Regression10.5435506990.54355069914968386433.8081E-14   
Residual31.0894E-093.63132E-10     
Total40.5435507      
         
 CoefficientsStandard Errort StatP-valueLower 95%Upper 95%Lower 95.0%Upper 95.0%
Intercept1.71907E-062.30021E-050.0747352730.945129788-7.1484E-057.49221E-05-7.1484E-057.49221E-05
X Variable 10.0860009662.22288E-0638688.9993.8081E-140.0859938910.086008040.0859938910.08600804
         
         
        

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