A Practical Approach 2
A Practical Approach 1
A Practical Approach to Diagnose Pernicious Anemia
Student’s Name
Student’s Practical Partner Name
OBJECTIVE OF THE PRACTICAL
The disarray of Pernicious anemia is one of the usual types of cobalamin dearth which are auto-immu and comes with numerous immunological as well as environmental etiologies causing the disease. Several advances have been taken place to understand the molecular biology as well as pathogenesis of the disorder and hence, diagnosis of this disease has remained a challenging issue for the clinicians as it addresses complex and diverse presentations. The proposed scientific, practical record aims to determine the presence of antibodies against gastric proton pumps in the blood serum samples of the patients by means of molecular biology techniques like Western blotting and IHC (immuno-histochemistry) and diagnose whether the patients exhibit the disease of pernicious anemia. To execute western blotting procedure, the primary objective of the study would be to carry out SDS-PAGE of the stomach proteins of a mouse for analyzing numerous protein products and their respective sizes on a polyacryl-amyl gel. Further, Western blotting would be conducted for validating the results.
Introduction
Pernicious anemia (PA) is a chronic inflammatory auto-immune disorder which is characterized by chronic infiltration in the regions of a gastric mucosal layer and subsequently there is a reduced production of zymogenic cells as well as parietal cells. There are various immunological etiologies responsible for triggering immune responses in the patients suffering from PA, for example, IF (intrinsic factors) and the subunits (α & β) of H+ and Na+/K+ Pumps that behave as the antigens for the antibodies against parietal cells (Toh et al., 2010). The requirement of IF in our body is for the absorption of a dietary vitamin cobalamin, i.e. vitamin B12. Therefore, this type of anemia is known as pernicious anemia as it results in the reduced production of IF as parietal cells are being lost in the body. Other physiological pathogenesis associated with PA are macrocytosis, hyperhomocysteinemia, and damage of the nervous system (Lahner, 2009). Thus, there is a requirement for an early diagnosis of the patients who might be carrying the symptoms of premalignant lesions, hence, require effective monitoring. The autoimmune disease of gastritis associated with PA potentially creates a problem in the main region of the digestive system and such patients are observed with hyperplasia of the cells that produce gastrin. This causes an abnormally high concentration of gastrin enzyme and consequently low concentration of HCl and pepsinogen in the blood serum of the patients (Andres and Serraj, 2012).
The diagnosis of PA in the following study is on two practical analytical approaches. The first approach would be to isolate gastric proton pump antibodies (proteins) from the stomach of the mouse with the help of SDS-PAGE. This technique makes use of the solubilizing nature of SDS which is an anionic detergent that effectively binds to the hydrophobic areas of the protein mixtures, providing an overall negative charge to the entire sample; and 2-β mercapto-ethanol which is a reducing agent that disrupts all the sulphide bonds present in the sample. Finally, in the presence of electric field, the proteins start migrating towards the positive electrode (anode) and get separated on the basis of their molecular weight. The expected size of the protein detected by immunoblotting is 65-70 kD by the serum samples obtained from pernicious anemia patients (Tun AM, 1985).
Further, the separated proteins are transferred to nitrocellulose membrane electrophoretically which is further probed with the antibodies patient serum samples comprising of antibodies and a secondary antibody conjugated with the enzymatic probe is used to detect the presence or absence of color. This technique is termed as Western Blotting. The results can be further visualized under Chemi-doc system (Toh et al.,1997).
The second approach is to make use of the technique known as IHC (Immuno-histochemistry) for detecting whether a specific antigen is present in the tissue sample or not or for detecting the presence of specific antibodies from the sample of the patients. For the current study, this technique helps in analyzing the position of anti- virus injection against pump in proton in the samples of the serum of the people.
The approach which is used to quantitate and finds the protocol of antibodies is automated chemiluminescent immunoassay which makes use of the automated antigen or antibody along with a molecule that is capable of emitting light at the time of the chemical reaction. The amount of light emitted is directly proportional to the quantity of antigen-antibody complex formation (Datta and Dasgupta, 2003).
METHODOLOGY SECTION
SDS PAGE mouse stomach proteins:
Initially, the pre-cast polyacrylamide gels were kept in the electrophoretic tank, and the tank was filled with Tris-glycine buffer (tank buffer) to run the gel. 100 µL of mouse stomach protein sample was added to 25 µL of 5X SDS gel loading sample buffer. The sample was mixed appropriately by pipetting until it appeared homogeneous in texture. The tube was then sealed and incubated at room temperature until use. The first well of the gel was filled with the protein marker dye of set range of molecular weight ranging from 2 kb to 200 kb weight. Further, 20 µL of the prepared sample was aliquoted into the rest of the wells of the gel. The equipment was linked to the power pack and the tube was running at 200 V for 60 mins and observed until the yellow tab which reached the end of the gel.
Transfer to Nitrocellulose Membrane:
Once, the gel is reached through the yellow gel, this turned off the power and the gel was taken from the electrophoretic gel plate carefully observing and keeping the creativity of the protein marker noted on the gel. The gel tube was sealed off by prying the gel plates in a part and with the help of the opening tool. The top stacking gel portion of the gel was removed with the use of the wedge and the upper left corner was given a slight cut in order to remember the position of the marker. Now, in order to remove the gel from the gel plate, the plate was kept immersed in a tray containing tank buffer and gently the gel was detached from the plate with the use of wedge. Further, for tranferring the proteins from the gel to nitro-cellulose membrane, a Turbo-blot transfer cassette was set up. For this, first of all, all the required materials such as pieces of extra thick paper and nitrocellulose membrane were sinked in the tank buffer for certain time period. To the transfer cassette, first a blotting paper was kept, bottom ion reservoir stack, then the nitrocellulose membrane, then gel, then the top filter slack and after that the cassette was closed. The gel was aligned in the correct orientation.
Figure1: Illustration of transfer slack arrangement
The lid was placed on the cassette and it was locked and it was inserted into TurboBlot transfer apparatus and was run at 50V for 10 minutes. This turned off the power supply. Further, the nitrocellulose membrane with the transferred proteins was stained with 40 mL 0.1% Ponceau stain and allowed it to the rocker for 1 min. The membrane was then peeled off for 3 successive washing steps with distilled water until protein bands were seen. The bands appeared straight with no smear formation. Further, the lanes on the membrane were marked out with the help of the pencil in order to cut the strips for western blotting.
Figure 2: Illustration of marking of lanes on nitrocellulose membrane
After this, the layer was further peel off briefly with 70 mL 0.1 M NaOH and kept on the plate and observed till the color goes. The layer was further peel off in the presence of 70 mL TBS. Further, the blocking solution (5% skim milk powder dissolved in TBS) was added to the layer to block all the non-specific proteins.
Western Blotting:
The layer was then cut off in 6 layer as there were 8 patient serum samples were available and 2 controls (positive control and negative control) were kept. The patient’s samples were named as P1, P2, P3, P4, P5 and P6. The gel samples were previously sufficiently diluted with distilled water (1:100). The strips were carefully kept on a parafilm piece and 150 µL of serum sample was loaded onto the strips. Another cut of parafilm was kept over the membrane in order to prevent it from drying out. The entire strip was now covered with patient’s serum sample containing the gastin antibodies ( antibodies against the subunits of proton pumps behave as the antigen for secondary antibody). This apparatus was incubated for 1 hour on the rocker at RT. Now, after the incubation, the strips were subjected to 3 consecutive washing steps for 10 minutes each in TBST solution with subsequent rocking. After this step, the strip was placed over a fresh piece of parafilm and 150 µL of sufficiently diluted (1:2000), HRPO enzyme conjugated, , sheep anti-human secondary antibody (which was raised in sheep using human IgM) was added evenly onto it with the help of a pipettor. This experiment was performed under low light conditions and the strip was incubated for 1 hour on rocker. This was further followed by 3 consecutive washing steps of 10 minutes each in TBS on a shaker. The strips were finally taken to the chemi-doc dark room where they were incubated first in the freshly made LumiLight substrate for about 5 minutes. After that, the strips as well as the marker was aligned on parafilm piece. The parafilm was kept under the chemi-doc instrument and light emitting from the substrate was detected and recorded. The image obtained from the instrument was then posted on CloudDeakin.
Immuno-histochemistry:
The slides of mouse stomach sections were first incubated in xylene for 5 min. Further, the slide was kept in ethanol for 5 minutes. It was then rinsed for 1 minute under the tap water. The slide was then transferred to a Sequenza Coverplate™ clip and was inserted into the rack. 100 µL of 0.5% H2O2 prepared in PBS was added to the slide for removing the endogenous peroxidase activity and the slide was incubated for 30 mins at RT on a shaker. The slide was then washed in PBS for 2 mins. Further, in order to remove non-specific binding of the proteins, the slide was further incubated with 150µL of 1% BSA (prepared in PBS) for 30 mins. Then 250 µL of patient’s diluted serum sample (1:100) was then added to the slide section by the use of the pipettor and inclubated for overnight at 40C.
Figure 3: Illustration of Thermo Shandon Coverplate™ System
The next day, slides were subjected to consequent 3 washing steps of 5 minutes each in 500 µL of PBS. After that, HRPO conjugate sheep anti-human secondary antibody (1:100) was added to the section of the slide and incubated for 1 hour on a shaker at RT. This was followed by 3 successive washing steps of 5 minutes each. Further, 300 µL of DAB solution was added to the sections and incubated for 10 minutes at RT. The layers were then peeled 3 times for 2 minutes each with 200 µL of PBST. The bad slides were peeled for 30 sec in water and excessive stain was removed by agitation. Further, the slides were incubated in ethanol for 1 minute and mounted with a small drop of Limolene and examined under the microscope.
References
Toh, B., Chan, J., Kyaw, T. and Alderuccio, F. (2010). Cutting Edge Issues in Autoimmune Gastritis. Clinical Reviews in Allergy & Immunology, 42(3), pp.269-278.
Lahner, E. (2009). Pernicious anemia: New insights from a gastroenterological point of view. World Journal of Gastroenterology, 15(41), p.5121.
Andres, E. and Serraj (2012). Optimal management of pernicious anemia. Journal of Blood Medicine, p.97.
Toh, B.H., van Driel, I.R. and Gleeson, P.A. (1997). Pernicious anaemia. New England Journal of Medicine, 337, p1441-1448
Tun AM, e. (1985). Pernicious anemia: Fundamental and practical aspects in diagnosis. – PubMed – NCBI. [online] Ncbi.nlm.nih.gov. Available at: https://www.ncbi.nlm.nih.gov/pubmed/28164751 [Accessed 18 Aug. 2017].
Datta, P. and Dasgupta, A. (2003). Evaluation of an automated chemiluminescent immunoassay for complexed PSA on the Bayer ACS:180? system. Journal of Clinical Laboratory Analysis, 17(5), pp.174-178.