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Postby Buzznerd123 on February 11th, 2015, 12:19 pm

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Last edited by Buzznerd123 on May 1st, 2016, 11:44 am, edited 1 time in total.
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Buzznerd123
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Re: Autoantibody discovery

Postby Little Lost on February 11th, 2015, 9:08 pm

The road is lonnnnnggggggggg........

Will give you an example of discovery through protein microarray, followed by a working example of the technical approaches being currently used to identify auto antibodies on MS.


First there has to be some evidence of an abnormal humoral immune response (excessive antibody production), in the pathogenesis of the disease being studied. Then they set about identify immunogenic targets recognized by the humoral immune response.

Traditional autoantibody detection technologies involved time consuming approaches such as Western blot and ELISA of antibody repertoire in the target tissue of the disease being investigated . More recent methods such as comprehensive phage display, fluid-phase immunoassays, and antigen microarrays are being utilised.

Take one example i.e.. the use of protein microarrays. These arrays are simply protein microchips which contain functionally active proteins, in all their modified states, immobilised on a surface. The chips contain literally thousands of different human proteins all labelled (ProtoArrays) . You can get arrays of target tissues if you are looking at a particular disease, such as human liver, or more general arrays.

Identification of autoantibody ( in serum and plasma or CSF etc being tested) occurs when it binds and interacts with its antigen i.e. a human self protein on the microchip. Then they start investigating if it is statistically significant between patients ( i.e. what percentage of sufferers have this autoantibody, what percentage of healthy controls carry it, what stage of disease does it appear etc etc.

Of course antigen / antibody interactions are specific to pH , protein configuration ( dimerisation etc), cross reactions so microarrays are useful but other methods are used also.

Here is a typical life science advertisment for antibody specific profiling ( though this is a generic advert for specifcity not autoantibody, it does gives you the general idea).

Quote :
ProtoArray® Human Protein Microarrays are an excellent method to use for testing antibody specificity. Using this method you can profile your antibody against 9,000 functional proteins and identify possible cross reactivity. Since the identity of every protein on the array is known, you can immediately identify any proteins bound by your antibody.

http://www.lifetechnologies.com/uk/en/h ... iling.html

I thought I would also give you a working example. It is a paper exampling many of the methods used to try to find possible autoantibodies in MS. ( including the protein antigen microarrays we just discussed),.

For MS disease serum, CSF, neuronal, axonal and glial compounds have all been analyzed as autoantibody targets in MS. The controls are healthy individuals.

The methods are quite complex but if you interested you can look the technology up. The article covers methods of autoantibody profiling including antigen arrays, immunoblotting, various phage expression library screening systems etc etc.


http://www.sciencedirect.com/science/ar ... 7209000330


Lastly just wanted to add. The novel methods being developed to detect new autoantibodies will hopefully aid the diagnosis of certain conditions through the generation of reliable disease markers. However whether most autoantibodies are pathogenic and able to promote disease is still debatable. Many auto antibodies seem to be against neoantigens ( I.e. self antigens that are only exposed because of the disease process itself, so immune system undergoing central tolerance during embryogenesis would not have been educated that this antigen was a self protein). For example bone erosion due to chronic late damage to joints in RA. Damage exposes various auto antigens, antigens the immune system was never meant to see, so resulting immune reaction and generation of auto antibodies to such antigens clearly comes after pathology, and hence these auto antibodies are not the root cause of RA.

Hope this helps answer something
Hx
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Re: Autoantibody discovery

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