SLRC-MOLBIO-2022-07
Moderator: Fatima Diane Villaseran
Secretary: Lelin Amorin
Members:
Michael Arman Metran
Marey Gold Labajo
Reyva Jane Brigildo
Principles of Dot Blot Assay
Case Vignette
An 18-year-old female patient applied to a university hospital with complaints of fever and
fatigue beginning 15 days ago accompanied by headache, weakness, palpitation, abdominal pain,
and diarrhea a week later. The patient was then transferred to the Infectious Diseases Department.
o
On her physical examination, temperature was 39 C, pulse rate: 92/min, rhythmic, breath rate:
24/min, blood pressure: 100/60 mmHg. She had apathic confusion, her sclerae were subicteric, her
skin and conjunctivae pale, her lips and tongue dry and tongue rusty. She had a painful
hepatomegaly which exceeds the costal ridge 1 cm and she had no lymphadenopathy.
3
In laboratory examinations: leukocytes 2.500/mm , (with differential: 47% lymphocytes,
3
43% polymorphonuclear leukocytes, and 10% monocytes), erythrocytes: 3.360.000/mm , Hb: 9.9
3
gr/dl, Htc: 27.8%, platelets: 31.000/mm , ESR: 30 mm/h, PT: 16.3 sc (INR: 1.36), PTT: 32 sc,
CRP: 75 mg/dl. In blood biochemistry: AST: 161 U/ L, ALT: 67 U/L, total bilirubin: 2.05 mg/dl,
direct bilirubin: 1.65 mg/dl, LDH: 1250 U/L, CK: 898 U/L, BUN: 19 mg/dl, creatinine: 0.8 mg/dl,
Na: 131 mEq/l, K: 2.52 mEq/l, Ca: 6.1 mg/dl, Mg: 1.51 mg/dl. G6PD deficiency was not
determined. Occult blood in stool was ++ positive, there were sparse leukocytes and erythrocytes
in stool examination. Thyroid hormones were in normal range. In abdominal ultrasonography, only
pathological finding was hepatomegaly (liver size: 160 mm).
The patient was hospitalized with a preliminary diagnosis of typhoid fever with
complications such as hypocalcemia, hypopotassemia, pancytopenia, intestinal hemorrhage, and
hepatitis. After samples were taken for microbiological analyses, oral treatment of antibiotics was
initiated by means of nasogastric tube and electrolyte replacement was maintained. After 8 hours
of her admission, discordance between pulse and fever disappeared and pulse rate increased to
o
116/min while fever was 38 C. Tachycardia was attributed to hypopotassemia.
In Gruber- Widal test, TO antibody was 1:200 and TH antibody 1:100. After 2 days of
admission, S. typhi was grown in blood culture. Salmonella did not grow on urine and stool
cultures. In antibiotic susceptibility testing of the isolated strain, there was only moderate
resistance to ceftriaxone, but sensitive to ciprofloxacin, chloramphenicol and co-trimoxazole.
Fever disappeared at the third day of her admission and electrolytes came to normal at the fifth
day. Repeated Gruber-Widal test revealed that TO antibodies increased to 1:800 and TH antibodies
to 1:200 one week later. Her platelets came to 199.000 and also her leukopenia improved. Hb level
gradually reduced to 7.6 gr/dl after admission and remained at that level. She was given 2 units of
whole blood, occult blood positivity in stool disappeared after 10 days of admission. After 2 weeks
of antibiotic treatment, the patient was discharged with full recovery.
Learning Outcomes
The students must be able to:
1. Determine the causative agent for typhoid fever and its pathogenesis.
● Typhoid fever, also known as enteric fever, is a communicable disease, found more likely
in man and occurs due to systemic infection mainly by Salmonella typhi organisms, a gramnegative bacterium.
● Environmental exposure to Salmonella typhi may be more frequent in males, presumably
due to sex-linked differences in hygiene practices and dining-out behavior. But, it
predominantly affects the children of school-age or younger.
● These diseases are spread through sewage contamination of food or water and through
person-to-person contact. People who are currently ill and people who have recovered but
are still passing the bacteria in their feces can spread Salmonella Typhi. Thus, it is through
the fecal–oral route.
● It is an acute generalized infection of the reticuloendothelial system, intestinal lymphoid
tissue, and the gallbladder.
● It is a potentially fatal multisystemic illness that causes nearly 220,000 deaths annually and
22 million illnesses per year.
● Isolation of Salmonella from blood, urine or stool is the most reliable means of confirming
an infection. Blood culture is regarded as the gold standard for diagnosis and carry 70-75%
diagnostic yield in the first week of illness
2. Discuss on the principles and analysis of the Dot Blot technique/assay.
● DNA and RNA can be more quickly analyzed using dot blots or slot blots. These
procedures are applied to:
- Expression
- Mutation
- Amplification/deletion analyses
Blots
Techniques in which target or probe sequences are immobilized on a solid matrix.
Dot Blot Technique
● It follows a similar principle to Western or Southern blotting, except that the sample is not
blotted from a gel. Instead, samples are dotted directly onto a membrane before being
probed for detection.
Principle: multiple samples are immobilized in a geometric array on a nitrocellulose or nylon
membrane
● Dot blot is a method for detecting biological samples like proteins or nucleic acids.
● Samples are dotted directly on to a membrane before being probed for detection.
● The target DNA or RNA is deposited directly on the membrane by means of various
devices, some with vacuum systems.
● A pipet can be used for procedures testing only a few samples.
● For dot blots, the target is deposited in a circle or dot.
● Dot blots are useful for multiple qualitative analyses where many targets are being
compared, such as in mutational screening.
● Performed most efficiently in less complex samples, such as cloned plasmids, PCR
products, or selected mRNA preparations.
● It is important that the probe hybridization conditions be optimized because crosshybridizations cannot be definitively distinguished from true target identification.
● A negative control serves as the baseline for interpretation of this assay.
Applications of Dot Blot
- Monitoring labeling efficiency
- Estimating protein concentration
- Comparing antibody performance
Dot Blot Analysis
Using:
-
ImageJ
UN-SCAN-IT gel Analysis Software
ImageJ
● ImageJ plugin used to measure microarray image stacks. Use the control panel that appears
to define the ROI (circle or rectangle) grid.
● There are two built-in methods for analyzing a dot blot in ImageJ. ImageJ is an image
analysis program extensively used in the biological sciences and beyond.
1. To treat each row as a horizontal "lane" and use ImageJ's gel analysis function.
2. To subtract the background and measure the integrated density of each dot.
UN-SCAN-IT gel Analysis Software
●
The UN-SCAN-IT gel Analysis Software turns your scanner into a gel densitometer and
allows you to automatically analyze gel electrophoresis images.
YouTube: Gel Analysis – Dot Blot Analysis Tutorial
3. Point out the problems encountered using this method.
● High background on the blot
-
often caused by too high concentration of the antibody, which can bind to PVDF
membranes
- the buffers used may be too old
- film overexposed or became wet during exposure
- short blocking time or washing intensity
● Weak or no signal from the blot
- can be caused by low concentration of antibody or antigen
- lack of protein and this can come from poor sample preparation
● Non-specific binding
- Short blocking time can lead to non-specific binding by the primary and secondary
antibodies
- Too much time in the blocking buffer can lead to poor binding
4. Explore other immunoassays in determining the cause of this clinical condition.
●
Immunoassay
o is a test that relies on biochemistry to measure the presence and/or concentration
of an analyte. The analyte can be large proteins, antibodies that a person has
produced as a result of an infection or small molecules.
● First immunoassay formats
o based on the agglutination reaction
o characterized either by gel formation in a liquid phase or as an opaque band in an
agar plate assay.
● Agglutination
o only occurs when the right amounts of antibody and antigen are present.
- Positive agglutination test - appears cloudy
- Negative result
⮚ is characterized by a ‘button’ at the bottom of the reaction vessel that is
formed by non-reacted particles.
⮚ may indicate the mismatch of antibody to antigen or it may be obtained
from either excess antigen or excess antibody.
● Agglutination immunoassays
o are commonly used for the detection of microbial pathogen antigens in blood
serum as they provide rapid results with a minimum of equipment.
● Enzyme linked immunosorbent assays (ELISAs)
o These are routinely used as diagnostic methods that detect antigens of infectious
agents or antibodies against them in bodily fluid.
●
Immobilization
o is achieved either directly, or indirectly by the use of a coating antibody (also
called capture antibody), which actively traps antigen in the solid phase.
●
Enzyme immunosorbent assays can be classified into:
o Direct (e.g. double antibody sandwich)
o Indirect (e.g. triple antibody sandwich) ELISAs
●
Double antibody sandwich ( DAS) ELISA
o (also called direct ELISA) is probably the most widely used immunochemical
technique in diagnostics.
o used extensively in horticulture and agriculture to ensure that plant material is free
of virus.
o Principle: is that the antigen is immobilized on a solid phase by a primary coating
antibody and detected with a second antibody that has been labeled with a marker
enzyme
o Marker enzyme used is usually either:
▪ horseradish peroxidase
▪ alkaline phosphatase
● In some systems, the enzyme is replaced with a radioactive label and this format is
known as the immunoradiometric assay ( IRMA)
●
Triple antibody sandwich ( TAS) ELISA,
o also known as indirect ELISA,
o is a method often used to identify antibodies against pathogens in patient blood to
diagnose infection.
o The test works well for the diagnosis of HBV infection and is also used to ensure
that blood donations given for transfusion are free from this virus.
●
Competitive ELISA
o is used when the antigen is small and has only one epitope.
o is routinely used for testing blood samples for thyroxine, a hormone that is
responsible for regulating metabolic rate; a deficiency (hypothyroidism) or excess
(hyperthyroidism) results in retarded or accelerated metabolism, respectively.
o
provides an accurate measure of the circulating level of the hormone compared to
a standard curve of known dilutions.
o involves enzyme-labelled probes, but radioactive labelling is done as well, and
this form of competitive ELISA is known as radioimmunoassay ( RIA)
o Principle: is based on the competition between the natural unlabelled antigen to
be tested for a labeled (e.g. enzyme-conjugated) form of the antigen which is the
detection reagent.
o
Competitive ELISA using conjugated antibody - can also be used to quantify
levels of circulating antibody in test serum
5. Know how the test method is being done in the clinical laboratory.
Dot blot is a quick and easy method for detecting biological samples like proteins or nucleic acids.
It follows a similar principle to western blotting and southern blotting except the sample is not
blotted from a gel, instead samples are dotted directly onto a membrane before being probed for
detection. Because dot blot is so simple it's used to support many different applications, these
include monitoring labeling efficiency with various probes, estimating the concentration of a
specific protein in a sample, and comparing the performance of different antibodies.
As an example, let's consider how a dot blot would be used to determine the labeling efficiency
where a sample has been labeled with biotin.
1.
This involves comparing the labeled sample to a known biotinylated reference
standard
2.
First, make serial dilutions of the test sample and the reference standard ensuring
that both are diluted in exactly the same way.
3.
A 10-fold serial dilution is a good place to start as it gives a wide range of sample
concentrations.
a.
To make a 10-fold serial dilution, aliquot 90 microliters of buffer into each
tube.
b.
Then take 10 microliters of the test sample and add it to the first tube.
c.
Mix thoroughly before transferring 10 microliters to the next tube.
d. Repeating this for all the tubes in the series performs the same process for the
biotinylated reference standard.
4.
Next, prepare the membrane according to the manufacturer's specifications then take
small aliquots of your serial diluted test sample and dot them on the membrane surface.
5.
The dots should be uniform in volume and size, usually one microliter or even 0.5
microliters is sufficient and should be spaced carefully to avoid overlap dot.
6.
The serial dilutions of the reference standard onto the membrane in the same way
placing the dots adjacent to the test sample dots.
7.
Then allow the membrane to air dry for a few minutes.
8.
The membrane is now ready for detection.
9.
In this example the biotin labeled sample can be detected with any streptavidin or
avidin labeled conjugate such as streptavidin alkaline phosphatase or avidin horseradish
peroxidase.
10. A typical detection workflow begins with 30 minutes incubation in a blocking solution
to minimize any background signal followed by two 15-minute washes in TBST (TrisBuffered Saline with Tween 20).
11. Next, the membrane is incubated for 30 minutes with a suitable conjugate diluted in
the blocking solution before two further 15-minute washes in TBST (Tris-Buffered Saline
with Tween 20).
12. Finally, the membrane is transferred to an appropriate enzyme substrate for color
development. This might be BCIP/NBT (5-Bromo-4-Chloro-3-Indolyl-Phosphate/Nitro
Blue Tetrazolium) for alkaline phosphatase detection or DAB (3,3′-Diaminobenzidine) to
detect peroxidase.
13. Once the signal is developed, compare the intensity of the test sample dots to those
of the reference standard.
14.
This provides a semi-quantitative assessment of the labeling efficiency.
YouTube: Dot Blot Tutorial
6. Compare and contrast Widal Test and the Dot Blot assay in the detection of the disease’s
certain causative agent.
Similarities of Widal Test and Dot Blot Assay:
● Both is used as early detection modalities
● Relies on the same principle that many immunological techniques rely on: the recognition
and binding of an antigen by an antibody, like IgG & IgM.
● Commercially and Readily available
● Affordable tests
● Simple to Administer
Difference of Widal Test and Dot Blot Assay:
Widal Test
● Sensitivity and Specificity is Lower vs. Dot Blot Assay
● Detection Rate is Lower in case presenting before 7 days and after 7 days of symptoms.
Dot Blot Assay
●
●
Greater Sensitivity and Specificity
Detection Rate is Higher in case presenting before 7 days and after 7 days of symptoms.
In conclusion, the efficacy of the rapid diagnostic test (Dot Elisa) over the Widal test for both early
and late diagnosis of typhoid fever, which has a high sensitivity as well as specificity for
identification of the causative agents.
7. Provide instructional and informative materials, such as videos and illustrations, regarding
the said method in the flow of the discussion.
YouTube:
Dot Blot Tutorial
Gel Analysis – Dot Blot Analysis Tutorial
References:
Hassan, et al.: Comparative Study between WIDAL and DOT ELISA in the Diagnosis of Typhoid
Fever
Hofmann, A. & Clokie, S. (2018). Wilson and Walker's: Principle and Techniques of
Biochemistry and Molecular Biology. Cambridge University Press. 8th Ed
Dot Blot Tutorial. Vector Laboratories Inc. December 20, 2022. YouTube
https://www.youtube.com/watch?v=wO6dWx0bczQ
Gel Analysis – Dot Blot Analysis Tutorial Silk Scientific. March 31, 2019. YouTube.
https://youtu.be/GNGBCuNDkUo
Shen, C. (2019). Characterization of Nucleic Acids and Proteins. In Diagnostic Molecular Biology
(pp. 265-267).
R&D Editors (2006) UN-SCAN-IT gel 6.1 https://www.rdworldonline.com/un-scan-it-gel-6-1/
Griffin, D.M. (2021). Bench Tips: Dot Blots. BenchSci. Retrieved May 1, 2022, from
https://blog.benchsci.com/dot-blot-bench-tips