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SLE346 Pernicious Anaemia Practical Report

Practical Report for pernicious anaemia practical Practical Report for...
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Molecular Basis of Disease (SLE346)

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SLE346 Molecular Basis of Disease

Diagnosis of pernicious anaemia in mice sera samples through the presence

of gastric H+/K+-ATPase autoantibodies

Georgia Armstrong

219198683

Wednesday 1pm

Word count: 2500

INTRODUCTION:

Vitamin B 12 deficiency can be caused by malabsorption of the vitamin, dietary insufficiency, atrophic gastritis, underproduction of intrinsic factor, or problems in uptake in the ileum related to disease, resection, or bacterial overgrowth, drug-nutrient interactions, and genetic effects (O'Leary & Samman 2010). One of the most common causes is pernicious anaemia (PA), which appears in chronic atrophic gastritis, and is usually only detected in the later stages of the disease. Chronic atrophic gastritis is characterised by loss of gastric mucosal folds and gastric mucosa thinning, with Type A, or the autoimmune variant, involving the fundus and body of the stomach. Type A is associated with PA, as autoantibodies are produced against intrinsic factor and the proton pump of parietal cells found in the gastric glands. Intrinsic factor, a 60 kDa glycoprotein produced by gastric parietal cells, is responsible for the binding of vitamin B 12 in the diet. The destruction of parietal cells by the body causes a decrease in intrinsic factor production, and autoantibodies against intrinsic factor prevent the formation of a complex between vitamin B 12 and intrinsic factor that is crucial for absorption, and thus a deficiency of the vitamin. The lack of B 12 impairs erythropoiesis, thus leading to anaemia. Furthermore, the antigen that the parietal cell autoantibodies recognise is the gastric H+/K+-ATPase, which is a proton pump that possesses catalytic α and glycoprotein β subunits with sizes of 95 kDa and 60-90 kDa, respectively. The pump is responsible for secreting hydrogen ions in the exchange for potassium ions (Toh et al. 1997).

Diagnosis of PA using the presence of intrinsic factor autoantibodies has only been successful in 40- 60% of PA patients due to its insensitivity. However, the use of the mechanism behind the gastric H+/K+-ATPase, in that either or both of the subunits will bind to the autoantibodies produced, is a much more effective diagnosis tool, and in early stages of the disease, can detect PA in 80-90% of patients (Ammouri et al. 2020).

The aim of this report was to determine which patients suffered from PA based on the presence of autoantibodies against the gastric H+/K+-ATPase in mice serum samples. In order to achieve this, two methods were utilised. The first method involved the use of an SDS-PAGE to separate proteins and Western blot to produce bands that can be visualised and analysed, and the second method involved immunohistochemical staining to detect the presence of the parietal cell antigens. It was hypothesised that the patients with PA would possess a band at 95 kDa, representing the α subunit, and brown staining of DAB substrate in parietal cells.

METHOD:

SDS-PAGE and Western blotting

Electrophoresis

An electrophoresis tank was prepared, with 12% polyacrylamide gel placed into it and its reservoirs filled with Tris-glycine running buffer (25 mM Tris, pH 8, 0 M Glycine, 0% SDS). The stomach protein sample to be analysed was prepared by adding 6μL of 5x SDS-sample buffer (0 M Tris, pH 6, 25% Glycerol, 10% SDS, 5% 2-beta mercaptoethanol, 0% Bromophenol blue) to 24μL of pre-diluted 2μg/μL mouse stomach protein. This was then mixed well by pipetting up and down to ensure homogeneity of the solution. 5μL of the BIO-RAD Precision Plus Protein™ WesternC™ Blotting Molecular Weight Marker #161- 0318 was dispensed into the first well, and 20 μL of the mouse protein sample was pipetted into each of the next six wells of the electrophoresis tank. The tank was then connected to a power supply, set to 200V, and run for 40 minutes to an hour. The gel was assessed every 10 minutes to ensure the buffer level in the inner chamber remained higher than the short gel plate.

Transfer

Upon completion of the electrophoresis, the power supply was switched off and the gel was removed, with careful consideration of the position of the protein marker. The gel cassette was then run under water to cool it. The cassette was pried open using the cassette-opening tool, and the plastic gel wedge was used to cut the wells off the edge of the gel. The orientation of the gel and position of the protein marker was noted by cutting off the top corner of the gel where the marker was located. The gel was then removed from the plate by submerging the plate into a tray of transfer buffer, and gently detaching the edges of the gel with the plastic gel wedge and rocking the container back and forth to loosen the gel from the plate. The detached gel was removed and set aside.

An Invitrogen iBlot was then set up for the transfer of the proteins to a nitrocellulose membrane. The lid of the iBlot was opened and the Anode (bottom) stack was unwrapped and placed onto the blotting surface, ensuring alignment to the gel barriers. The previously-run gel was then placed onto the anode stack. A piece of iBlot filter paper was soaked in deionised water and placed on top of the gel in the iBlot, and a blotting roller was used to remove any air bubbles between the layers. The Cathode (top) stack was unwrapped and had its tray removed before being placed and aligned on top of the filter paper, with the metallic side facing upwards. The blotting roller was again used to remove any air bubbles. The disposable sponge was then placed into the inside of the lid, with the metal contact located in the upper right corner, before the lid was closed, and the latch was secured. “Program 3” was selected, the check time was set to 7 minutes, and the transfer began. Once the transfer had finished, the power supply was turned off and the transfer stack was disassembled. The nitrocellulose membrane was placed into a container filled with 50mL of 0% Ponceau S stain in 1% acetic acid, which was then placed onto a rocker for 1 minute. The stain was then poured off and the membrane was rinsed with deionised water to aid visualisation of protein bands and confirm the transfer of proteins. The water was poured off and the membrane was labelled with a ball-point pen, with the first lane labelled “M” for the marker, the next two lanes labelled with a “+” and “-“, respectively, and the next four wells labelled “P1”, “P2”, “P1”, and “P2”, respectively. The lanes were then cut into individual strips with scissors, resulting in a total of 6 strips, and were rinsed briefly in 50mL of 0 NaOH on the rocker to remove the stain. The NaOH was discarded, and the

45 minutes. The slides were then washed three times for 2 minutes each in PBS and rinsed in water. 100μL of DAB substrate was added to each section and incubated for 5 to 10 minutes. They were rinsed in PBS three times for 1 minute each, and dipped in haematoxylin for 3 seconds, before being washed off in water for 30 seconds. They were immersed in ethanol for 2 minutes and airdried, before being covered with DPX mounting medium and a cover slip and viewed under the microscope.

RESULTS:

Figure 2. Histological section of mouse stomach tissue Image captured at x40 magnification of mouse stomach tissue section treated with haematoxylin and eosin. Labels indicate the features of the stomach.

Figure 1. Diagram of histological section of mouse stomach tissue Sketch of an image captured at x40 magnification of mouse stomach tissue section treated with haematoxylin and eosin. Labels indicate the features of the stomach.

Figure 4. Histological section of mouse stomach tissue Image captured at x 100 magnification of mouse stomach tissue section treated with haematoxylin and eosin. Labels indicate the features of the stomach.

Figure 3. Drawing of a section of mouse stomach tissue Image captured at x 100 magnification of mouse stomach tissue section treated with haematoxylin and eosin. Labels indicate the features of the stomach.

Gastric pit Gastric mucosa

Muscularis mucosal layer

Submucosa

Mucous secreting cells

Gastric pit

Parietal cell

Chief cell

The initial staining of the mouse stomach section to determine morphology using haematoxylin and eosin (Figures 1-4) produced purple staining of the gastric mucosa and gastric pits, pink staining of the muscularis mucosal layer, and pink/red staining of the submucosa. The mucous secreting cells appeared as unstained cells grouped together, whilst chief cells appeared dark purple and parietal cells had their nucleus stained purple and their cytoplasm stained pink.

Figure 5. Positive control for PA immuno-peroxidase staining of mouse stomach Image captured at x100 magnification of a mouse stomach tissue section as a positive control for pernicious anaemia. The parietal cells have been stained brown.

Figure 6. Demonstration positive control for PA immuno- peroxidase staining of mouse stomach Image captured at x100 magnification of the demonstration positive control for pernicious anaemia in mouse stomach tissue. The parietal cells have been stained brown.

Figure 7. Western blot of patient samples Image of western blot results captured using a Chemidoc. The first strip holds a BIO-RAD Precision Plus Protein™ WesternC™ Blotting Molecular Weight Marker #161-0318, the second and third lanes contain positive and negative controls, and lanes 4 and 6 possess patient 1 sera, whilst lanes 5 and 7 possess patient 2 sera. The molecular marker has been labelled. (Source: School of Life and Environmental Sciences (2021)).

  1. kDa

  2. kDa

  3. kDa

  4. kDa

  5. kDa

  6. kDa

  7. kDa

  8. kDa

REFERENCES:

Alhajj M & Farhana A (2021) ‘Enzyme Linked Immunosorbent Assay’, StatPearls [Internet].

Ammouri W, Harmouche H, Khibri H, Benkirane S, Azlarab M, Tazi ZM, Maamar M & Adnaoui M (2020) ‘Pernicious Anaemia: Mechanisms, Diagnosis, and Management’, EMJ Hematol US , 1(1):71- 80.

Hanukoglu I (1990) ‘Elimination of non-specific binding in western blots from non-reducing gels’, Journal of biochemical and biophysical methods , 21(1):65–68, doi/10.1016/0165- 022x(90)90046-f

Karlsson FA, Burman P, Lööf L, Olsson M, Scheynius A & Mårdh S (1987) ‘Enzyme-linked immunosorbent assay of H+,K+-ATPase, the parietal cell antigen’, Clinical and experimental immunology , 70(3):604–610.

Kumar R, Xiavour S, Latha S, Kumar V & Sukumaran (2014) ‘Anti-Human IgG-Horseradish Peroxidase Conjugate Preparation and its Use in ELISA and Western Blotting Experiments’, Journal of Chromatography & Separation Techniques , 5:1-20.

O'Leary F & Samman S (2010) ‘Vitamin B12 in health and disease’, Nutrients , 2(3):299–316. doi/10.3390/nu

School of Life and Environmental Sciences (LES) (2021) SLE346 Molecular Basis of Disease ‐ Practical Manual 20 21 , Waurn Ponds Ed., Deakin University Press.

Toh, B-H, van Driel IR, & Gleeson PA (1997) ‘Pernicious Anemia’, The New England Journal of Medicine , 337:1441 – 1448, DOI: 10/NEJM

Ward JM & Rehg JE (2014) ‘Rodent Immunohistochemistry: Pitfalls and Troubleshooting’, Veterinary Pathology , 51(1): 88 – 101 , doi/10.1177/

QUESTIONS:

a) What is the expected size of the protein being detected by the sera from pernicious anaemia patients?

The expected size is 95 kDa (Toh et al. 1997).

b) Which subunit of the proton pump does this correspond to?

This corresponds to the α subunit of the H+/K+-ATPase proton pump (Toh et al. 1997).

c) Describe another method that could be used to detect and quantitate the levels of anti-proton pump antibodies in patient’s sera?

An Enzyme-linked Immunosorbent Assay (ELISA) can be carried out to determine levels of autoantibodies against the H+/K+-ATPase proton pump (Karlsson et al. 1987). An ELISA is a plate- based technique and involves the detection of the autoantibodies in the patient sample by their binding to H+/K+-ATPase antigens. A measurement is obtained through detection of a signal produced by direct or secondary tags attached to the antibody (Alhajj M & Farhana A 2021).

d) The secondary antibody that was used in the Western and immuno-histochemistry was a sheep anti-human Ig-HRP conjugate. What is the antigen that this antibody is specific for? What animal was this antibody raised in? What is the enzyme that is conjugated to the antibody?

The antigen that anti-human Ig-HRP conjugate is specific for is the human immunoglobulin G. This antibody was raised in a sheep, and the enzyme that is conjugated to it is horseradish peroxidase (Kumar et al. 2014).

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SLE346 Pernicious Anaemia Practical Report

Course: Molecular Basis of Disease (SLE346)

13 Documents
Students shared 13 documents in this course

University: Deakin University

Was this document helpful?
SLE346 Molecular Basis of Disease
Diagnosis of pernicious anaemia in mice sera samples through the presence
of gastric H+/K+-ATPase autoantibodies
Georgia Armstrong
219198683
Wednesday 1pm
Word count: 2500
INTRODUCTION:
Vitamin B12 deficiency can be caused by malabsorption of the vitamin, dietary insufficiency, atrophic
gastritis, underproduction of intrinsic factor, or problems in uptake in the ileum related to disease,
resection, or bacterial overgrowth, drug-nutrient interactions, and genetic effects (O'Leary &
Samman 2010). One of the most common causes is pernicious anaemia (PA), which appears in
chronic atrophic gastritis, and is usually only detected in the later stages of the disease. Chronic
atrophic gastritis is characterised by loss of gastric mucosal folds and gastric mucosa thinning, with
Type A, or the autoimmune variant, involving the fundus and body of the stomach. Type A is
associated with PA, as autoantibodies are produced against intrinsic factor and the proton pump of
parietal cells found in the gastric glands. Intrinsic factor, a 60 kDa glycoprotein produced by gastric
parietal cells, is responsible for the binding of vitamin B12 in the diet. The destruction of parietal cells
by the body causes a decrease in intrinsic factor production, and autoantibodies against intrinsic
factor prevent the formation of a complex between vitamin B12 and intrinsic factor that is crucial for
absorption, and thus a deficiency of the vitamin. The lack of B12 impairs erythropoiesis, thus leading
to anaemia. Furthermore, the antigen that the parietal cell autoantibodies recognise is the gastric
H+/K+-ATPase, which is a proton pump that possesses catalytic α and glycoprotein β subunits with
sizes of 95 kDa and 60-90 kDa, respectively. The pump is responsible for secreting hydrogen ions in
the exchange for potassium ions (Toh et al. 1997).
Diagnosis of PA using the presence of intrinsic factor autoantibodies has only been successful in 40-
60% of PA patients due to its insensitivity. However, the use of the mechanism behind the gastric
H+/K+-ATPase, in that either or both of the subunits will bind to the autoantibodies produced, is a
much more effective diagnosis tool, and in early stages of the disease, can detect PA in 80-90% of
patients (Ammouri et al. 2020).
The aim of this report was to determine which patients suffered from PA based on the presence of
autoantibodies against the gastric H+/K+-ATPase in mice serum samples. In order to achieve this, two
methods were utilised. The first method involved the use of an SDS-PAGE to separate proteins and
Western blot to produce bands that can be visualised and analysed, and the second method
involved immunohistochemical staining to detect the presence of the parietal cell antigens. It was
hypothesised that the patients with PA would possess a band at 95 kDa, representing the α subunit,
and brown staining of DAB substrate in parietal cells.

Students also viewed

Related documents