Saturday, September 29, 2007

14th Week of SIP - Microbiology/Immunology

Hi all!

This is time for my sharing again. As I am currently attached to viral serology lab, I am learning different serological tests such as various enzyme immunoassays (EIA), hemeagglutination-inhibition tests (HI) and other specialized tests like immunoblots.

::Dengue In Focus

Since there were quit a number of dengue cases recently in Singapore, I think it would be useful to share how dengue is diagnosed.

Dengue virus can be isolated in the mosquito cell culture (C636 cell-lines) and specific dengue serotype can be identified using immunofluorescence method. Due to its specificity dengue isolation is the gold standard for dengue diagnosis. However viral isolation takes about 2 weeks.

Due to varies in its severity and undifferentiated symptoms, it is very important to diagnose dengue as early as possible and give appropriate treatments.

Therefore serodiagnosis of dengue which can detect dengue infection rapidly becomes the most common method. In this blog, I will be sharing about Dengue IgM Capture Enzyme-linked Immunosorbent Assay (ELISA).

::Dengue IgM ELISA


This particular dengue IgM kit uses Capture Enzyme-linked Immunosorbent method and specific dengue IgM is captured between anti-human IgM and commercial dengue antigen. The picture below clearly explains:

If O.D. reading is more than 1, it is considered as dengue IgM positive. Positive and negative control and calibrator added in each run for quality control. The assay is run 100% manually and takes about 4 hours.

Clinical Significance

Presence of dengue IgM shows that the patient is suffering from acute dengue infection as IgM are produced during the acute infection.

This test however cannot tell if the dengue infection is primary or secondary. Primary means this is the first time the patient is experiencing dengue infection. Secondary infection means patient had dengue before, but this time he/she is infected by dengue virus of different serotype from the first one.

Hemeagglutination-inhibition test is the only test which can determine the dengue infection is primary or secondary, acute, recent or recovering.

There are also commercially availiable rapid kits which uses various principles such as immunoblot, immunochromatographic, etc.

Well, I think this would be enough for this posting. Feel free to ask me if you have anything to clarify or wanna know more about dengue. All are welcome!

Thank you for reading. All the best for your SIP...6 weeks more yeah!


Ye Tun

Friday, September 21, 2007

13th week of SIP- Lab Techniques( Research)

Hi all, i think i miss out on my turn to blog one of those weeks.. so its ME again..
This time I m going to share with you a new assay I learnt: Co- Immunoprecipitation assay or in short, pull- down assay.

This assay works likes a protein- antibody reaction and with this assay, we can deduce whether are 2 proteins interacting with one another based on the Western Blot (WB) analysis which will be done after this.
For e.g: I wan to study if analog A binds to protein Z. normally, we will tag the analog A and protein Z with something so that we can detect them. Protein Z is tagged with the FLAG tag while HA tag for the TRX- analog and TRX proteins.
The analog A is attached to this protein scaffold (thioredoxine or TRX) to stabilize the structure.

I will have 3 reaction samples:
1. (-) Ctrl: Vector + Protein Z
2. (-) Ctrl: TRX + Protein Z
3. TRX- analog A + Protein Z
All this three DNA samples will be transfected into HeLa cells and then, we will lyse the Hela Cells to get the proteins translated.

After we lyse the cells and the cell Lysate are ready, we will save an aliquot mixed with protein dye without reducing agent for WB. The remaining will be topped up to 600- 1000ul with the lysis buffer.

Next, we will utilize beads. There are 2 types of beads: Protein G and A. however, I m using only the protein G. the protein G is attached to a solid matrix like agarose or sepharose and can bind to Ab. the beads will help to pulldown ab- protein complexes and when eluted, the complexes will detach from the beads and ready to load for WB analysis. (NB: Beads are hard to handle!!! So small and tiny and must be very careful not to suck them out in each step!)

Firstly, the beads will be pre-washed .
The lysate are then added to our pre-washed beads, and rotated at 4 °C for 1 hr. This step is called pre- clearing. We want to clear away those proteins in the lysate that will have an affinity to bind to the beads which can give non- specific bands under WB.
After that, the beads are spun down and the supernatant are transferred to another newly washed beads coupled with the Ab and rotated overnight at 4°C.
The next day, the beads are washed 5 times with wash buffer and eluted with 1 X TBS and protein dye. Proteins complexes that are bound together to the beads will therefore detach from the beads and can be loaded for WB analysis.

This assay often gives messy WB results. If we use a rabbit Ab for pulldown, we must use the mouse Ab for WB primary Ab and vice versa. The rabbit Ab will always give more background signal from WB than the mouse. Normally, I will also load the Lysate besides my pulldown in my WB. This allows me to compare and also troubleshoot whether my proteins are expressed in the Lysate or not. If my proteins expression is not detected in my lysate but I have a signal in my pulldown, it means that there might be something wrong in the experiment that can react to give a signal even my proteins are not around.
Any interaction can be seen based on the WB results and checked accordingly with the control to confirm that the experiment is working well and not due to any errors.
The success of pull down and protein expression will also depend upon the cell density for transfection and amount of DNA introduced into the cells.
So that’s all for the beads session. Feel free to ask me anything.

Hope you all are enjoying yourselves!!!

Ai Tee
TG 01

Sunday, September 16, 2007

12th Week of SIP- Microbiology

Hey all!

It’s my turn to blog again. This time, I will post on something I have learned in the Microbiology lab. It is the testing and detection of Neisseria Gonorrhoeae.

What is Neisseria Gonorrhoeae?

Neisseria gonorrhoeae
(also known as Gonococci) is a species of Gram-negative bacteria responsible for the sexually transmitted disease Gonorrhea.

How is it detected/tested?

  • To test for Neisseria gonorrhoeae, an endocervical swab is taken from the patient, then plated.
  • Neisseria species is isolated from the primary culture by plating on a TM plate (selective media).
  • Perform a gram-stain. Look for gram negative diplococcus (Neisseria gonorrhoeae) under the microscope.
  • To further test for the presence of Neisseria gonorrhoeae, Bacticard Neisseria commercial test kit is used.

Principle of Bacticard Neisseria:

  • It is used for the identification of pathogenic Neisseria species
  • Kit utilizes 4 chromogenic substrates impregnated on individual test circles to detect preformed enzymes associated with pathogenic Neisseria.
  • The enzymes b-galactosidase and butyrate esterase hydrolyze the substrates 5-bromo-4-chloro-3-indolyl-b-D-galactoside and 5-bromo-4-chloro-3-indolyl butyrate respectively to form a blue-green complex in the presence of oxygen.
  • Gamma-glutamyl aminopeptidase hydrolyzes gamma-glutanyl-b-naphthylamide and prolylaminopeptidase hydrolyzes L-proline-b-naphthylamide and reacts with the Bacticard Neisseria Colour Developer to form a red colour.

Procedure for using Bacticard Neisseria:

  • Rehydrate each test circle with 1 drop of Bacticard Neisseria Rehydrating Fluid.
  • Smear several colonies of the organism to be tested across each test circle.
  • Incubate at room temperature for 2 mins.
  • Observe IB test circle for a blue-green colour, indicating a positive test. If positive, proceed to identification. If negative, continue incubation for additional 13 mins.
  • After incubation, observe BGAL test circle for a blue-green colour indicating a positive test. If positive, proceed to identification. If negative, proceed by adding 1 drop of Colour Developer to the GLUT and PRO test circles.
  • Observe for the development of a pink to red colour at 30 secs. Proceed to identification.

Interpretation of results:

    • IB test
      • Positive - Colour change to blue-green after 2 mins.
      • Negative - No colour change.
    • BGAL test:
      • Positive - Colour change to blue-green within 15 mins.
      • Negative - No colour change.
    • GLUT and PRO test:
      • Positive - Colour change to pink or red within 30 secs after addition of Colour Developer.
      • Negative - No colour change.
    • Neisseria gonorrhoea should show negative for IB, BGAL and GLUT tests, but positive for PRO test.

That’s all folks! Continue to enjoy your SIP while it last.


Adrian Tan

Tuesday, September 11, 2007

11th Week of SIP- Clinical Chemistry

Hi people!

Due to some miscommunition and confusion between the ever-busy medical technologists, this posting has been delayed. Sorry for the inconvenience caused!

Do u guys still remember what test did Adrian share about in his previous post? Yes, thats right! G6PD screening! (see week's 6 posting for the test in detail.)

After reading his post, you will wonder what happens after the patient has been screened G6PD-deficient (no fluorescence) of intermediate (having weak fluorescence). Since his workplace does not perform the next confirmatory step, he has to send it over to the lab I work in for G6PD quantitation. G6PD quantitation is the measurement of the ACTUAL G6PD enzyme level of the patient, while the initial screening only indicates "deficient", "intermediate" or "normal".

In other words, the initial screen is only QUALITATIVE, while the confirmatory test is QUANTITATIVE.

Today, I shall put an ending to the G6PD testing story by explaining the QUANTITATIVE aspect of G6PD testing.

Glucose-6-Phosphate Dehydrogenase (G6PD) Quantitation, Blood

The screening test for G6PD is based on the following immunofluorescence principle:

Glucose-6-Phosphate + NADP+ ----( G6PD)----> Gluconate-6-phosphate + NADPH (Fluorescence) + H+

G6PD (Glucose-6-Phosphate Dehydrogenase) catalyzes the oxidation of G-6-P to Gluconate-6-phosphate and simultaneously reduces NADP to NADPH. The formation of NADPH is monitored under 340nm. The rate of NADPH formation is proportional to the G6PD activity.

Preparation of RBC haemolysate
1. Wash the EDTA blood 1 time with 0.9% saline.
2. Centrifuge to pack the cells at 3000rpm for 10 minutes.
3. Remove the saline and buffer coat completely.
4. Pipette 200 uL washed spun cells into 200 uL normal saline to obtain 1:1 RBC suspension. Mix well.
5. Pipette 50 uL of this 1:1 suspension into 250 uL of 1% saponin for lysis.
6. Mix well and stand for 2-80C for 10 minutes.
7. The haemolysate is ready for testing.

Aliquot the haemolysates onto autoanalyzer cups and load onto analyzer.

Clinical Significance
In addition to hemolytic anemia, G6PD deficient individuals can expect several other clinical manifestations of their condition. These include neonatal jaundice, abdominal and/or back pain, dizziness, headache, dyspnea (irregular breathing), and palpitations.

Treatments for neonatal jaundice and hemolytic anemia have existed for many years. These treatments insure that the body tissues will be provided with enough oxygen by the red blood cells. Infants with prolonged neonatal jaundice are placed under special lights, called bili-lights, which alleviate the jaundice. When an anemic episode occurs, individuals are treated with nasal oxygen and are placed on bed rest, which may afford symptomatic relief. Anemic individuals are sometimes treated with human haptoglobin products, and/or blood transfusions. In acute hemolytic anemia, patients are administered folic acid.

Last Words

Thats all folks! All the best for ur MP! Remember to get plenty of rest and water! :)

Kent Lieow

Sunday, September 9, 2007

10th Week of SIP- Lab Techniques (Research)

Hello all!
Hope you guys are enjoying work. Haha.

Anyway, today I'll be talking about the SOD Assay.

Superoxide Dismutase (SOD) Assay

What is SOD?
SOD is a metalloenzyme that catalyzes the dismutation of superoxide radicals (O2•-) into hydrogen peroxide (H2O2) and oxygen (O2).
Superoxide radicals are produced during metabolism in the mitochondria, muscle contraction, and as well as by NADPH oxidase during infections. Accumulation of these radicals would then lead to oxidative stress and eventually to diseases like Parkinson's disease, atherosclerosis etc.
Thus, with SOD present, it can defend against oxidative stress.

Other than SOD, Gluthathione peroxidase and Catalase also helps to protect against oxidative stress.

Liver supernatant will be used for this assay.

Principle of SOD Assay

The SOD Assay also uses tetrazolium salt to detect superoxide radicals. Xanthine oxidase present in the reaction mixture interacts with the radicals, which reduces the tetrazolium salt into formazan. The presence of SOD will reduce the amount of radicals, and in turn reduce the amount of formazan formed. Thus, increased levels of SOD, will lead to reduced levels of formazan.

After adding the respective reagents according to the protocol, abs is read using an ELISA plate reader at 450nm.

Fig. 1: Principle of SOD Assay

In other words: ↑SOD, ↓O2•-, ↓ formazan formed, ↓ Abs

That's it!
Take care guys~

Charmaine Tan