Pernicious anaemia is a form of anaemia that occurs when the body is unable to absorb vitamin B-12 from the small intestines. Since vitamin B-12 is an essential vitamin in the body, humans obtain it from a diet that is rich in vitamin B-12. The digestion mechanism releases vitamin B-12 from the proteins, which consequently binds to intrinsic factor, a protein that parietal cells of the stomach secrete, to form a complex that the small intestines readily absorb (Lahner & Annibale 2009).
The absence of the intrinsic factor owing to genetic or autoimmune disorder causes deficiency of vitamin B-12 in the body and consequently causes the emergence of pernicious anaemia (Irvine 1965). According to Toh, Van Driel, and Gleeson (1997), an autoimmune disorder is responsible for the occurrence of pernicious anaemia because autoantibodies attack parietal cells and affect the secretion of intrinsic factors and the absorption of vitamin B-12. Hence, pathogenesis and pathophysiology of pernicious anaemia emanate from the autoimmune disorder, which affects the integrity of the parietal cells, the secretion of intrinsic factors, and the absorption of vitamin B-12.
Pathogenesis and pathophysiology of pernicious anaemia indicate that the absence of intrinsic factors and malabsorption of vitamin B-12 owing to the defects in parietal cells cause pernicious anaemia. Toh et al. (1997) state that autoantibodies bind to gastric H+/K+–ATPase, which is a pump made of a 100KD catalytic α-subunit and glycoprotein β-subunit (60-90KD), and hamper the functioning of the parietal cells in the stomach. To enhance the understanding of pathogenesis and pathophysiology of pernicious anaemia, this report discusses the morphology of the stomach about the occurrence of pernicious anaemia, examines diagnosis of stomach samples using the western blot technique, and detection of antibodies in a serum sample using staining technique of immunohistochemistry.
The report aims to find out if patient serum samples have antibodies to the gastric proton pump using western blot and immunohistochemistry techniques. To undertake an accurate diagnosis of pernicious anaemia among patients, the report first aims to examine the morphology of a healthy stomach.
The procedure of Examining Morphology of the Stomach
The stomach samples were prepared in slides for examination and placed in the safety cabinet. The students incubated the slides in xylene for two minutes and further incubated them in ethanol for another two minutes. Water was then used to rinse the slides for 30 seconds before and after incubating them in haematoxylin. After staining and washing the slides, they were temporarily put in 1% acid alcohol and rinsed well with tap water to remove the alcohol.
The stained slides were placed for 30 seconds in Scott’s tap to incubate, and thereafter, rinsed before staining them with eosin for 4 minutes. The slides were subsequently placed for 30 seconds in 90% and 100% ethanol to fix them. Following the fixation, the slides were put on ethanol for 2 minutes and air-dried immediately. To prepare the slide for mounting, a drop of DPX medium was smeared on the fixed slide, enclosed with a coverslip, and viewed under the microscope using a magnification of ×400.
Two diagrams were drawn focusing on the stomach wall and the gastric gland using A4 papers and a pencil.
The procedure of Preparing SDS-PAGE and Western Blot
In the preparation of the stomach specimen, 48µL of protein sample was added into a microfuge containing 12µL of SDS-sample. Molecular weight markers for the assessment of the molecular weights were prepared in advance. To sediment the molecular markers and protein sample, the solutions were spun in a centrifuge for a few minutes. Next, lane 1 of the prepared SDS-PAGE in electrophoresis apparatus was loaded with 10μL of the molecular weight markers. Just like molecular weight markers, subsequent wells of the SDS-PAGE were loaded with 25μL of the protein sample. The electrophoresis apparatus was then switched on and left to run for 45 minutes at the power of 200 volts.
After undergoing the electrophoresis process, the apparatus was switched off and dismantled, and then, the gel was put on a clean plate with the assistance of the demonstrator. To assess the separation of proteins according to their weights, the gel was systematically set and placed in the iBlot device. 10uL of distilled water was used in the placement of one gel in the iBlot device, which processed it for 7 minutes, and another gel was put in nitrocellulose film. For the detection of the protein to occur, a 50mL of 0.1% Ponceau solution was used to stain the nitrocellulose membrane by allowing it to stay for 1 minute, a period that was sufficient for protein transformation to occur.
The labelling of the nitrocellulose membrane was done to make sure that the strips have proteins that detect sera of patients. A pair of scissors was used to cut lanes, which were consequently rinsed using NaOH to remove all stains. 50mL of TBS was used to wash the membrane before storing it for the subsequent practical in 10mL of Tris-buffer at 40C.
Membranes were removed from storage and rinsed with deionised water. Three main steps were performed after the wash. First, the blocking step was performed using 5mL of 5% skimmed milk. Then, 2.5mL of serum samples or the positive and the negative controls were used in incubation as a primary antibody. Finally, the secondary antibody incubation using 5mL of labelled antibody was done. The same device used for the protein transfer (iBlot) was used again to perform the previous three steps. Consequently, membrane strips were removed from the device and washed with TBS 3 times for five minutes. The demonstrator took the strips to the Chemidoc for imaging and uploaded the results on CloudDeakin.
The procedure of Preparing Immuno-Peroxidase of Mouse Stomach
Following the preceding steps, 100μL of serum was incubated for 20 minutes. PBS was used in washing the slides and 50μL of antibodies conjugated with horseradish peroxidase (anti-human Ig HRPO) was put and left to incubate for three-quarters of an hour. Consequently, the slides were washed with PBS and rinsed with water before adding 100µL of DAB and incubating them for 10 minutes. Before soaking of slides in haematoxylin for 3 seconds, PBS was used to wash them. Next, the water was used in rinsing the slides before incubating them in ethanol for 2 minutes. Ultimately, DPX was smeared on the slides and viewed under the microscope at the magnification of ×400.
Results: Morphology of the Stomach
Low Power Diagram of Stomach Wall
High Power Diagram of Gastric Gland
Results: Western Blotting
Western Blot (Unsuccessful Results, group 5)
Western Blot (technical staff results)
Results: Immuno-Peroxidase Staining of Mouse Stomach Section
High Power Diagram of Gastric Gland
Table 1. Immunochemistry Results
|Sample||Positive control||Negative control||Patient 1||Patient 2|
Table 1: Shows the results of different serum samples after applying Immuno-peroxidase staining.
+ To indicate a patient positive for pernicious anaemia
– To indicate a patient positive for pernicious anaemia
Discussion of Stomach’s Morphology
Figure 1depicts sub-mucosal and mucosal layers of the mouse stomach, which have been stained with eosin and haematoxylin, while figure 2 depicts the gastric gland. These parts determine the major morphology of the mouse stomach. Irvine (1965) notes that atrophy of the mucosal cells, such as parietal cells, is the major characteristic of pernicious anaemia that is evident among patients. Parietal cells, which secrete intrinsic factors and gastric acid, facilitate the absorption of vitamin B-12 in the ileum. Essentially, parietal cells have the H+/K+-ATPase pump, which consists of beta and alpha subunits (Rhoades & Bell 2008).
Figure 2 clearly shows that parietal cells have pink stains, while chief cells have dark-pink stains. Chief cells mainly secrete pepsinogen, an enzyme that is integral in the catabolism of proteins (Toh et al. 1997). Hence, the morphology of the stomach determines the physiology of parietal cells and the catabolism of proteins.
Discussion of Western Blot
To establish the existence of pernicious among patients, a western blot technique proved effective, and the findings were presented on CloudDeakin. However, the western blot was not successful because it yielded spurious results. While some membranes have no bands, others have uneven or patchy spots as figure 3 shows. Any defects in antibodies, antigens, or buffers interfere with the occurrence of definite bands. The use of inappropriate primary and/or secondary antibodies makes bands not appear or become vague. As the concentration of antibodies influences the appearance of bands, the use of very low concentration makes the signal to be invisible.
Likewise, very low concentration or lack of the antigen causes the probing to be invisible. In this view, there is a need to use a different antigen to detect whether the invisibility of bands is due to antigens in the sample or primary and/or secondary antibodies used.
Moreover, procedures such as the washing of membranes affect the visibility of bands because prolonged washing diminishes the appearance of signals. The purity of buffers also determines the appearance of bands for contamination contributes to the invisibility of bands (Lahner & Annibale 2009). Thus, when undertaking the procedure of western blot, one should ensure that buffers like PBS, TBST, and ECL are free from any contaminants.
The procedure involved in the transfer of proteins is very delicate because it is prone to partial transfer and interference by air bubbles (Andres & Serraj 2012). Patchy or uneven spots on the blot usually occur due to the improper transfer of proteins. When gel and membranes trap air bubbles, the film appears dark. Hence, to prevent the formation of bubbles, it is recommendable that one should use a shaker during incubation to provide even distribution of particles.
The technical staff prepared the results, which are fully demonstrated in figure 4. According to Lahner and Annibale (2009), H+/K+-ATPase is the main autoantigen that triggers autoimmune disorder, which leads to the destruction of parietal cells and annihilation of the intrinsic factor. The autoantibodies to the parietal cells attack H+/K+-ATPase and affect the functioning of the parietal cells, thus, leading to the occurrence of pernicious anaemia (Andres & Serraj 2012).
Structurally, the expected size of the autoantigen (gastric H+/K+–ATPase) is approximately 160 to 190KD, depending on the size of protein subunits that it contains. Toh et al. (1997) state that autoantigen comprises of 100KD catalytic α-subunit and glycoprotein β-subunit (60-90KD). These subunits determine the nature of an autoimmune disorder that affects parietal cells and intrinsic factors.
Analysis of the western blot results shows that the fragment detected is approximately 70KD. Since the fragment detected corresponds to the molecular marker of 70KD, it implies that the fragment is a glycosylated β- subunit of the H+/K+-ATPase pump. Toh et al. (1997) assert that H+/K+-ATPase is the only autoantigen that autoantibodies to parietal cells recognize in the event of pernicious anaemia, and therefore, it is important in the diagnosis of pernicious anaemia using western blot.
The stained strip of patient 1, as indicated in figure 4, shows that it is positive and the approximate size of the subunit bound by the autoantibody is 70KD, which is the glycosylated β-subunit of the H+/K+-ATPase pump. Hence, the western blot findings do not only confirm that the patient is positive for pernicious anaemia, but also they show that the autoantigen subunit is glycosylated β- subunit of the H+/K+-ATPase pump.
Enzyme-linked immunosorbent assay (ELISA) is another method that can be used to detect anti-proton pump antibodies, which are in sera of patients with pernicious anaemia. Lahner and Annibale (2009) recognize H+/K+-ATPase as the main autoantigen that is present in the sera of patients with pernicious anaemia because it triggers autoimmune disorder, which destroys parietal cells and prevents secretion of intrinsic factors.
By using the ELISA method, the autoantigen is immobilised on the microtitre plate and the sample of serum containing anti-proton pump antibodies is added and incubated with the immobilised antigen. A sample of serum that has antibodies specific to the antigens of the proton pump, will either bind to β or α subunits of the proton pump. Excess antibodies and other proteins in the serum are then washed and secondary antibody, which is linked to an enzyme and is specific to the primary antibody, is added to the washed microtitre plate (Sugiu et al. 2006). The addition of the chromogenic substrate produces a change in colouration, which indicates the presence of proton pump antibodies.
To quantify the amount of proton pump antibodies in sera using the ELISA method, the assessment of the intensity of colour, electrical signal, or fluorescence is necessary. Sugiu et al. (2006) indicate that a spectrophotometer is applicable in measuring the intensity of the colour produced in terms of absorbance or illuminance because it accurately quantifies the amount of proton pump antibodies. Hence, the intensity of colour or fluorescence is very important in the quantification of proton pump antibodies.
The sheep anti-human Ig-HRP conjugate is a secondary antibody that indirectly links to the H+/K+ ATPase antigen via the primary antibody. Chevrier, Chateauneuf, Guerin, and Lemieux (2004) note that anti-human Ig-HRP is an antibody that is specific to human Ig. Production of the antibody occurs when a sheep is immunised with human Ig and an immunological response occurs. Since anti-human Ig is a secondary antibody in western blot and immunohistochemistry, it is conjugated to horseradish peroxidase, which is an enzyme that catalyses chromogenic substrates.
Discussion: Immuno-peroxidase staining of mouse stomach section
This part of the report focuses on the immuno-histology of the mouse stomach section with a view of establishing whether a patient is positive or negative for pernicious anaemia. Antibodies can be spotted in the majority of patients with pernicious anaemia as they attach to proton pumps in parietal cells (Irvine 1965). In this case, the experiment applied secondary anti-human Ig antibodies to human Ig of the unknown patients, labelled P1 and P2. Based on the experiment, the results show no brown stain, and thus, indicate that antibody-antigen attachment on proton pumps (parietal cells) for patient 2 did not occur.
Therefore, patient 2 is negative for pernicious anaemia. In contrast, the existence of brown stain indicates the existence of antibody-antigen attachment on proton pumps (parietal cells) for patient 1. This means that patient 1 is positive for pernicious anaemia.
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