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
Principle
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!
Cheers
Ye Tun
~TG01
20 comments:
Aloha Yetun! Interesting post yeah
Clear explanation indeed, but i would like to ask why hemeagglutination-inhibition test is not performed initially since it can also diagnose positive dengue patients and at the same time differentiate between primary and secondary infections?
And yes 6 more weeks to the ending point! =D
Chaur Lee
Hi Chaur Lee
Yup...hemeagglutination-inhibition test is not performed initially, because it needs paried serum (acute and convalescent)to be able to diagnosed dengue and the interval between acute and convalescent serum is at least 7 days.
HI test actually measures the total dengue antibody in the serum without differentiating between IgM & IgG. In order to diagnose acute dengue infection by HI, the paired serum must show at least 4-fold rise in antibody titre.
According to WHO guideline for HI, if the patient has acute primary dengue infection, the antibody titre will rise 4-fold but the titre would be less than 2560. If the patient has acute secondary dengue infection, 4-fold rise in antibody titre will be seen and the titre would be more than 2560.
So HI test has limitations that one serum alone cannot determine the patient condition, paired serum collected at least 7 days interval is needed, HI test procedure is also quite complicated and takes 2 days to perform. Cross-reactivity among dengue strains and other falviviruses are also common.
So HI test is not very suitable for rapid diagnosis of dengue when the patient is first presented with symptoms. It is though very useful to monitor the patient condition.
So what's more important is to know the patient has acute dengue infection as early as possible, then the doctor can closely monitor the patient condition and give appropriate treatment.
Cheers
Ye Tun
Hi ye tun,
i am slow in understanding the differences between the HI and the dengue IgM Capture ELISA, are they the same concept or 2 different matters?
Ching Wei
hi casper cheng hong here,
can u explain alittle on the principle of the new rapid detection kit for dengue. thanks see ya soon!
Chenghong
Hey Yetun,
Hey interesting post!
Regarding the primary and secondary infection, from what we learnt in AIMM before, if the person is infected the 2nd time, there will be more IgG present rite, then why can't the ELISA kit also incorporate 'IgG capture ELISA' too?
Meaning if the person has a lot of IgG, its secondary lor, :))
Tanx!
Nisha
TG02
0503254E
Hi Ching Wei
Haha. Principle of HI and dengue IgM ELISA are different. ELISA, as we know, uses the antibodies and enzymes to detect targeted antigen or antibody.
Hemeagglutination-inhibition test uses the principle that dengue viruses are able to agglutinate the RBCs. If the antibodies against the dengue virus antigens are present in the serum, they will bind to antigens and prevent the antigens from agglutinating the RBCs.
So, if dengue antibodies are present in the serum, hemeaggluination is inhibited. If there is little or no dengue antibodies, hemeagglutination will be observed.
I think this will clarify principles of HI and ELISA.
Cheers
~Ye Tun
Hi Cheng Hong
The new dengue rapid kit, I am evaluating as part of my MP, uses the principle of immunochromatography and membrane diffusion by capillary movement. It's about the same as the dengue rapid test Doreen mentioned in her blog (BMT Journal)posted on 5 Aug.
Um...how good is the new test kit is still under evaluation, I will tell you when I finished...haha.
Enjoy yeah!
~Ye Tun
Hi Nisha
Um..Ok.
To answer your question, I have to briefly explain about the immune response to dengue infection.
When the person gets dengue infection for the first time (primary infection) by a particular strain of dengue virus, IgM will be produced in a large amount (which can be detected by dengue IgM immunoassays).
A few days later (about 1 week), IgG will be produced but not in very high level (but still able to be detected by immunoassays) and IgG will remain for life.
In secondary dengue infection, IgG level will shoot-up to very high level. IgM usually will appear in low amount.
One important thing is that all four serotypes of dengue virus have significant cross-reactivity among them as well as with other flaviviruses.
Ok..now..what is the significant of dengue IgG immunoassay?
1)Immunoassay will detect IgG as long as IgG is present in the serum. So whether it is primary or seconday or past infection, IgG will be detected.
2)One may argue that high IgG level reading can be considered as secondary infection. But the issue is how high means 'high' and how low is 'low' since there is no established standard. IgG detected may not even be of dengue infection due to its cross-reactivity with other flaviviruses.
3)Therefore detection of dengue IgG does not play a significant role in the diagnosis of dengue infection.
4)In primary dengue infection, IgG assay may be useful to detect seroconversion though.
Therefore IgG assays are not performed for diagnostic purposes, they are used to detect previous exposure to dengue or in seroprevalence studies.
I think this would be enough to the question....haha :)
Cheers
~Ye Tun
Hi Ye Tun,
If the patient only have IgG, it will be diagnosed as dengue negative?
Pei Shan
Hi Pei Shan
If the patient is suffering from acute dengue infection, there should be dengue IgM produced and it will be detected by ELISA.
If the patient have only IgG, it can be due to past dengue infection or dengue IgM might have disappeared due to late collection time (usually dengue IgM level drops 2 weeks after the infection) or in a few cases of secondary infection, IgM may not appear.
But If we tested negative for dengue IgM, we would report as "dengue IgM negative".
Cheers
~Ye Tun
Hi Ye Tun
So does it mean that both ELISA and HI are done for one patient sample?
Ying Ying
Hi Ye tun =)
What is the physical appearence of the hemeagglutination-inhibition test results for patients with primary or secondary, acute, recent infection or recovering from dengue?
Eugene Wong
TG02
Hi Ying Ying
Um..actually it depends on the request of the doctor or the patient...e.g. specimen from patient A is received and IgM is requested, we will only do IgM, if HI is requested, we will do HI, if both are requested, then we will do both tests on the specimen.
But for HI test, we will wait untill the paired (both acute and convalescent)serum has been received and run HI in pair.
Cheers
Ye Tun
Hi Eugene
It's quite difficult to discribe the physical appearance of hemeagglutination-inhibition test result in words...
When there is hemagglutination, RBCs clump together and looks like red-coloured precipitates at the base of the microwell.
When hemagglutination is inhibited, the RBCs will just settle down at the base of the well and big red dot is seen.
Check out the link below to see the picture:
http://www.gekkeikan.co.jp/RD/research/img/new001/fig05.jpg
hemagglutination looks like the positive control well in the picture and hemagglutination-inhibition looks like the negative control well.
The HI titre is the dilution when hemeagglutination-inhibition is started to seen.
Sorry...I don't have picture/photo of the real dengue HI test...
Hope this will help :)
Cheers
~Ye Tun
hey.. juz wondering.. since its a immmunoassay ritez.. are there any interference?
Jia Hao
Hi Jia Hao
Um..based on what I learnt...
Hyperlipemic, heat-inactivated, hemolysed, or contaminated sera may cause errors in result.
Since the test is done manually, there may be human errors. But in diagnostic lab, all the test procedures are standardized, validated and documented and, if the lab personal performs the test according to the procedure, there shouldn't be any problem.
One thing to take note in performing all immunoassays detecting IgM is, IgG and rheumatoid factors in the serum must be preabsorbed with appropriate reagent (e.g. anti-IgG) so that there will be no interference by those non-specific immunoglobulins in detecting specific IgM.
Cheers
~Ye Tun
hey hi
jus asking out of curiosity.. how does immunochromatographic work in the diagnosis of dengue??
Vinodhini
TGO2
Hi Vino
In general, immunochromatography is the development of colour through reactions of antibodies and antigens. To give example for the use of immunochromatography in rapid diagnosis of dengue, I have to refer to the posting of Doreen in her blog (BMTjouranl) on 5 Aug.
Below is the quote taken from Doreen's blog:
"Serum antibodies of the IgM/IgG class, when present, bind to the anti-human IgM/IgG immobilized in 2 lines across the test strip. The colloidal gold-labeled antiflavivirus monoclonal forms complexes with the dengue antigen that is captured by dengue specific IgM/IgG in the patient’s serum. These complexes are visualized as pink/purple line(s)".
Cheers
Ye Tun
hi;)
what does O.D stand for? optical density? so if the O.D is less than 1, doesnt that mean that the antibody is still present and that the person has been infected? why is it only after it hits 1 then its considered positive?
cass
TG02
Hi Cass!
Sorry for late reply...
Actually O.D. reading given by the spectrophotometer is not the direct O.D. reading of dengue IgM in the specimen.
This particular test kit from a manufacturer, includes the cut-off calibrator to determine the cut-off point to interpret as positive or negative. The manufacturer said if the ratio of the specimen O.D to the cut-off calibrator O.D is equal to or greater than 1, the specimen should be interpreted as dengue IgM positive.
But the spectophotometer in the lab was programmed such a way that it can automatically calulate the ratio. Therefore when the ELISA strip are read by spectophotometer, it will give the ratio automatically, instead of the actual O.D reading of the specimen.
Method of interpretation of the result may vary for different test kits by other manufacturers.
Cheers :)
Ye Tun
~TG01
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