Comments on: Influenza hemagglutination inhibition assay

By: profvrr

profvrr — Fri, 03 Feb 2012 17:48:00 +0000

Log2.

By: Yashar

Yashar — Fri, 03 Feb 2012 09:09:00 +0000

How would you report HAI results? Is it log2 or log3?

By: Should we fear avian H5N1 influenza?

Should we fear avian H5N1 influenza? — Tue, 03 Jan 2012 15:16:50 +0000

[...] collected and analyzed for antibodies against several avian influenza viruses, including H5N1, by hemagglutination-inhibition and neutralization assays. The results indicate that 73 participants (9.1%) had antibody titers [...]

By: khairunnisa

khairunnisa — Fri, 04 Nov 2011 07:01:00 +0000

hello prof, I have some question regarding HI assay. Last week is my first time doing HI assay for my practical class. Could you mind to share some disadvantages of HI assay? I still wondering that for unknown sample, and the HI titre is high, how could we know that the high production of antibodies is due to vaccination or infection (secondary or past)?

By: How good is the influenza vaccine?

How good is the influenza vaccine? — Thu, 03 Nov 2011 10:50:08 +0000

[...] overestimation of the protective effect of vaccines. In many studies a four-fold increase in serum hemagglutinin antibodies were used to confirm infection. Immunization also increases these antibodies, making it difficult [...]

By: Pranav

Pranav — Mon, 29 Aug 2011 09:01:00 +0000

just finished doing HA test for TCF cultured influenza virus. Virus did not show haemagglutination although same sample cultured in egg is showing HA titre of 1:512. Any idea why this could have happened?

By: Haredy

Haredy — Fri, 26 Aug 2011 07:17:00 +0000

Dear Dr. Vincent
I have some problems doing the HA titer of influenza using the basic normal method, Although I am having a high plaque titer of influenza about 1×10^8 PFU/ml I can not get any activity for the HA assay. Is there a relationship between virus plaques and HA titer. I have also another questions, do you know why sometimes while doing the HA assay we get the blood spot at early dilution 1:2 and 1:4 and then it disappears on dilution
haredy

By: ANKYLOSING SPONDYLITIS RESEARCH | TNF Alert

ANKYLOSING SPONDYLITIS RESEARCH | TNF Alert — Fri, 05 Aug 2011 22:01:42 +0000

[...] score for RA patients, and Bath Ankylosing Spondylitis Disease Activity Index for AS patients. Hemagglutination inhibition (HI) antibodies were tested by a standard World Health Organization procedure. Response was defined as [...]

By: Rohannarayanm

Rohannarayanm — Fri, 22 Jul 2011 07:12:00 +0000

Hi..
Why is it that we use an acidic pH of 6.2 to 6.4 for arbo virus HA tests? I mean dats not wat happens in vivo right?

By: Matthias K

Matthias K — Wed, 15 Jun 2011 13:22:00 +0000

Thank you very much for the quick reply! I understand that logic – no reaction may be due to a drift (or even just found a new subtype…). But my question was rather for the opposite case: A drifted Influenzavirus can appear new to the human immunsytem when having no pre-existing antibodies (or antibodies against other Influenzaviruses don’t cross-react) – but how can it be, that it still can be subtyped e.g. as H1 (talking general, not for H1N1v). That is my main question, how can we already have test-sera against something “new”. Sure, logic thinking leads me to some assumptions, but it is hard to find out how this subtyping is done and I’d really would like to be sure, for personal interest. Thinking logically, I’d assume that the test sera must have a broader range of antibodies than those produced by humans. Is this just the point? That mixing together antibodies against hundreds of diefferent H1 just increases the possibility of a positive reaction? Thank you!!

By: profvrr

profvrr — Tue, 14 Jun 2011 21:09:00 +0000

The antibodies in test sera do not widely cross-react – you must use a
panel of sera, each against a specific viral subtype. If your viral
isolate does not react with any of the antisera, you may conclude that
it has drifted.

By: matthias k

matthias k — Tue, 14 Jun 2011 13:57:00 +0000

I mean, this is so contradictory to me – hope you can bring some light to this and thank you already in advance!

By: Matthias K

Matthias K — Tue, 14 Jun 2011 13:55:00 +0000

I have a question somehow related to this topic: As far as I know, Hemagglutinin was characterized and subtyped using double immunodiffusion. The antibodies used for that came from some selected stems. Antigenic drift cause minor chances in the hemagglutinin and therefore cause to “escape” the immunreaction (therefore also vaccines are updated from time to time). So if those antibodies don’t longer work due to altered binding sites (I have tried to read about the binding sites but it’s way to long to read through the papers dealing with it for me lacking in time and I had the impression I wouldn’t find my answer there anyway), how “minor” are the changes and how can it be on the other hand, that antibodies work quite fine to determine it as specific subtype. In other words: How can we have antibodies in test sera that widely cross-react on the one hand (which surely have been conservated from some animal somewhen) but on the other hand we see humans having no cross-immunity to different strains.

By: profvrr

profvrr — Tue, 04 Jan 2011 17:20:00 +0000

Sounds like you have pipetting problems. Make sure they are properly
calibrated – if you are using pipetmen.

By: Dreamerly

Dreamerly — Thu, 16 Dec 2010 08:30:00 +0000

Did you ever have problem about your HA titer(virus titer) in your HI assay???
I get the virus titer from HA 1:128 ,Iwant to dilute to 4HA in HI then i dilute virus in dilution 1:32.The virus back titration with HI assay don’t be 4HA….sometime less than but sometime more than….I don’t know how to solve this problem.

By: Milad_n_vet

Milad_n_vet — Wed, 03 Nov 2010 05:34:00 +0000

hi, i want an animation movie about HI test, would you plz help me to find it out.

By: DML

DML — Thu, 23 Sep 2010 19:12:48 +0000

Hello!!!
WOW this blog is incredibly helpful!!
I'm performing this assay and you mentioned that a fixed amount of HA (virus/vaccine) is used. The protocol I'm using does 4HAU and pretty much every paper I run into does the same. That fixed standard amount is because 40 HI titer is associated with 50% reduction of “Flu” risk??

Thanks.

By: Pandemika

Pandemika — Wed, 01 Sep 2010 20:13:15 +0000

Hi profvrr,

1st congratulation for your blog… very interesting!

2nd I have a question regarding the HI assay.

I'm currently analysing serum with the HI and MN (microneutralisation) assay… I was wondering what kind of AB I would detect with the 2 tests. Both tests detects AB raised against the receptor binding site of the HA but I thought that the MN assay would cover a broader range of AB (against other flu AG such as the NA, NP etc) and other parts of the HA (e.g. AB against the HA cleavage site…). An expert told me the opposite so that I'm a bit lost… What's your opinion about that …

Thanks for you reply

By: profvrr

profvrr — Tue, 20 Apr 2010 03:16:12 +0000

The responses to natural infection and immunization with inactivated
vaccine are quite different. The latter has little viral RNA and
internal proteins such as NP and therefore doesn't induce good
inflammation. The response is likely less cross-reactive as a
consequence. Flumist should be better but hasn't been used long enough
to know.

By: mggibson

mggibson — Tue, 20 Apr 2010 02:39:38 +0000

The following lab can do influenza HA titers as well as the hemagglutination inhibition test and other flu testing services for non-clinical purposes. www.virapur.com

By: profvrr

profvrr — Mon, 19 Apr 2010 20:16:12 +0000

By: mggibson

mggibson — Mon, 19 Apr 2010 19:39:38 +0000

The following lab can do influenza HA titers as well as the hemagglutination inhibition test and other flu testing services for non-clinical purposes. www.virapur.com

By: fbedoya

fbedoya — Sun, 18 Apr 2010 20:45:49 +0000

Thank you for the great information posted! I can't imagine how much time this demands from you…
I have always wondered if infection and vaccination produce the same kind of antibody immunity (in terms of amounts, isotypes, receptor-binding capacity, etc). Also, if they are different, can this be an issue targeted to achieve better protection and/or increase cross-reactivity of Abs produced?
Many thanks

By: William Lees

William Lees — Fri, 09 Apr 2010 14:23:25 +0000

Many thanks for this clear explanation of the HI assay.

I wonder if you could explain why in some cases the HI assay results show a higher heterologous titer than homologous, or refer me to any recent papers on the subject?

By: nath

nath — Sun, 04 Apr 2010 04:07:40 +0000

Great information.

I was wondering what the difference is between seroprotection (so in this case a HA inhibition of at least 1:40) and seroconversion? Can seroconversion values be used the measure the efficacy of influenza vaccination?

Also, I went through your posts on adaptive immunity. Is it possible to measure the effectiveness of the vaccine not just using humoral response but also T-cell activation? If so, how can this be done.

I also have one last question. Do you think the influenza vaccine (which is currently annually recommended in COPD patients) would be less effective in COPD patients because they may have a weakened immune system?

Thank you very much

By: profvrr

profvrr — Thu, 01 Apr 2010 16:27:18 +0000

HI assay is done to determine the level of specific viral antibodies
in serum. In humans, this information is useful for many reasons,
including determining the extent of infection of a population with a
particular virus strain, or to test vaccines. In animal models
measurement of viral antibodies provides information on experimental
virus infection or vaccine efficacy.

By: arslantariq1990

arslantariq1990 — Thu, 01 Apr 2010 15:30:41 +0000

why we perform HI test…..commercial benefits , diagnostic benefits, etc

By: Mold Removal

Mold Removal — Thu, 01 Apr 2010 09:09:18 +0000

For determination of viral titers, serial dilutions of influenza virus were ….. Comparison.

By: profvrr

profvrr — Tue, 23 Mar 2010 14:34:05 +0000

That is correct, the HI assay will not provide information on the
presence of antibodies against the viral neuraminidase (NA) protein.
Those antibodies can be identified by using a neuramindase-inhibition
(NI) assay. The NA assay measures the ability of the enzyme to release
sialic acids from a substrate; and the NI assay measures inhibition of
the NA by antibodies in serum.

By: huang

huang — Tue, 23 Mar 2010 09:03:54 +0000

Thank you for this useful information!
As you showed that HA assay is a good way to get the approximate titer of hemagglutinin domain-contained virus such as Influenza. However, the serotype of anti-flu antibody should fully recognized H and N; even the sera can inhibit the HA in assay, it still can't give the information regarding the specificity to N. So, HI assay can preliminarily screen the H-positive serum but not N-serotype. Is it right?

By: kannant

kannant — Thu, 18 Feb 2010 16:46:23 +0000

I read your post with immense interest.

Since the CDC (or FDA) uses HI antibody titers as a measure for evaluating a vaccine for approval, your post implies that only vaccines that induce antibodies targeting the receptor binding region can be approved by CDC/FDA. Is this true? Recently, several broadly neutralizing antibodies were discovered (F10, CR6261). However, these antibodies target the membrane proximal region of HA. So does this mean that they can only be used as therapeutic antibodies?

Can you also post the guidelines that the CDC/FDA use in order to approve a vaccine? Also, could you provide the relevant information in the case of a drug approval?

By: Chandranand

Chandranand — Fri, 12 Feb 2010 01:25:02 +0000

Thnx 4 d valuable info

By: Protection against 2009 influenza H1N1 by immunization with 1918-like and classical swine viruses

Thu, 11 Feb 2010 20:21:57 +0000

[...] cross-react with the 2009 H1N1 strain. This possibility was confirmed by examining mouse sera by hemagglutinin inhibition assay. Sera from animals that were immunized with 1918 virus-like particles, SW/30 or NJ/76 had [...]

By: Lylalala

Lylalala — Wed, 13 Jan 2010 13:24:54 +0000

Amazing information, very useful for the understanding of this concept, thank you!

By: Jeanie

Jeanie — Wed, 16 Dec 2009 16:40:52 +0000

I talked to my local health dept- they are to work on it but there doesn't seem to be one yet as of a couple weeks ago. Focus has been all about getting the vaccine out and delivered. All the best.

By: marioszarvou

marioszarvou — Wed, 16 Dec 2009 04:47:12 +0000

Do you happen to know of any FDA approved commercially available kit for the estimation of
IgG antibodies in human serum

By: marioszarvou

marioszarvou — Wed, 16 Dec 2009 04:44:33 +0000

Do you happen to know of any FDA approved commercially available kit for the estimation of H1N1 IgG antibodies in human serum?

By: Kos D

Kos D — Sun, 06 Dec 2009 21:39:22 +0000

Fantastic!!! I have a job innterview tomorrow and this has been SO helpful!!!

By: Jeanie

Jeanie — Wed, 18 Nov 2009 18:07:14 +0000

Thanks much! Can you call me 408.353.2014-when you can.

By: peterb

peterb — Tue, 17 Nov 2009 19:51:29 +0000

I believe there are many in the community who have contracted the novel H1N1 swine flu infection but not had confirmatory testing at the time of acute infection. They may, subsequent to recovery, be faced with the recommendation (particularly if in a high-risk demographic) to uptake the vaccine to protect self (if they actually had some other non-novel H1N1 illness) or general community or specific groups with which they have contact (at work, etc).
To take the vaccine if one has already acquired natural immunity offers no benefit that I can perceive to anyone (other perhaps than allaying anxiety of health/other administrators – but even this is spurious relief, and increasing use of a commercialized health care product). Not having the vaccine if truly not needed would limit wasteful use of a (still) scarce and important resource, direct such use to the more appropriately targeted, enhance to the public the credibility of health care authorities and fulfil more satisfyingly principles of parsimonious distribution of public and individual health care in a time of epidemic.
Finally although the likelihood and severity of adverse reactions to the novel H1N1 vaccine (in the non-allergic) is reported as low, the avoidance of such is surely a desirable goal in those who do not need the vaccine.
The above argues for the ready availability of post hoc serological testing but my question is whether a (presumably single) “convalescent” titre would provide adequate sensitivity and specificity for the diagnosis of novel H1N1 swine flu after the fact ?

By: u2star

u2star — Fri, 13 Nov 2009 20:40:18 +0000

Sounds like the biggest threat is getting the H1N1 virus in the younger population (cdc recommends 2 vaccinations for children under 9 and antibody response takes time remember!) I would vaccinate if it were my child as soon as the vaccine is made available. Where do you live? I listened to a great radio program on NPR the other day with a panel of doctors discussing the virus/vaccine. I'll post a link when I find it.

Best Wishes
Diane

My 2 year old niece received her 1st H1N1 vaccine yesterday. She slept quite a bit more than usual the first 24 hours but other than that she is doing well. Long term effects unknown.

By: u2star

u2star — Fri, 13 Nov 2009 20:34:08 +0000

Jeanie-

I can't seem to find a lab that will do the "titer" just the PCR for the H1N1 influenza virus (and not the antibodies).
I am still looking and will keep you posted if I find anything. I am checking with my allergist on Monday to see what he says. My internal medicine doctor is of no help in finding a lab to do this.

Sincerely and stay well
Diane

By: Jeanie

Jeanie — Fri, 13 Nov 2009 20:21:31 +0000

I am attempting the same thing you are- did you get it done and how?

By: ale

ale — Tue, 10 Nov 2009 13:38:42 +0000

could you please suggest also an outsourcer for flu challenge studies in mice?

By: profvrr

profvrr — Mon, 09 Nov 2009 14:35:09 +0000

An allergist is a good option. Check
http://www.acaai.org/LocateAllergist/ to find one.

By: u2star

u2star — Sat, 07 Nov 2009 14:01:54 +0000

Thanks for the reply. My physician said they will test me but I have to find a lab. Any suggestions?

By: profvrr

profvrr — Thu, 05 Nov 2009 00:25:02 +0000

You should have your son tested for egg allergies before receiving the
vaccine. In those without allergies the side effects include injection
site soreness, fever, aches, chills. The vaccine should not be given
to those with egg allergies.

By: Kim

Kim — Wed, 04 Nov 2009 05:05:16 +0000

Do you think that if we use different kind of blood like turkey, horse, human blood
will it give a better results?

By: Patricia Barral

Patricia Barral — Mon, 02 Nov 2009 23:57:43 +0000

I have a 9 year old son who's about to get the vaccine at his school. There hasn't been many cases of H1N1 virus in the area we live. My question is:

Should we wait until there's more of a threat to vaccinate our son?
He has allergies ( grass, trees, mold, dust mites, cats,etc…..).
I have to make my decision before 11/10/09. What are the side effects?

By: Being older is a good defense against 2009 H1N1 influenza virus

Being older is a good defense against 2009 H1N1 influenza virus — Mon, 02 Nov 2009 22:31:16 +0000

[...] the guinea pigs inoculated with the seasonal H1N1 or H3N2 strains did not contain antibodies with hemagglutination-inhibition (HI) activity against 2009 H1N1 virus. One explanation for the protective response is that the initial [...]