Hi Alix,

It seems not feasible to use mAb to detect viroids (constitute with ssRNA). See a description from this book:

Microbial Plant Pathogens-Detection and Disease Diagnosis:

Viral and Viroid Pathogens, Vol.3

Viruses, with the primitive structural features are capable of causing diseases in all life forms on the earth. With limited morphological characteristics of the virus particles and lack of physiological functions, the need for the development of techniques based on the characteristics of the viral proteins and genomic nucleic acids was realized in order to detect, identify, differentiate and quantify viruses in the infected plants/planting materials with or without symptoms of infection. Immunoassays based on the antigenic properties of viral capsid proteins have been successfully applied for the detection of viruses in the symptomatic and asymptomatic crop and weed host plant species as well as in the vectors that are involved in the transmission of the viruses infecting crop plants. Development of monoclonal antibody technology has enhanced the sensitivity and specificity of immunoassays especially applied for the differentiation of strains of viruses with different virulence levels. Nucleic acid-based techniques have been demonstrated to have higher levels of sensitivity, specificity, reliability, reproducibility and rapidity. The viroids with only an RNA molecule have been detected by techniques based on the biological characteristics in certain cases. Application of immunoassays for viroid detection is not feasible because of the absence of a protein component that is present in the viruses. Nucleic acid-based techniques are the most reliable and sensitive tests for detection, identification and differentiation of viroids present in plants and planting materials. With the aim of providing a comprehensive knowledge of detection of viruses and viroids and diagnosis of the diseases caused by them, information distilled from extensive literature search is presented to fulfil the requirements of the researchers, teachers and graduate students of various disciplines of biological sciences. Inclusion of numerous protocols in appropriate chapters as appendix is a unique feature of this volume to enable the scientists to select the suitable methods for the various investigations.

Hi Alex, well I am not an expert on viroids, but while it is true that there are antibodies against DNA, I do not know how specific they are, but there are other molecules that can bind to DNA, RNA or proteins. Aptamers. Aptamers are sequences of nucleic acids that form secondary structures and can bind with great specificity to their ligands. There are several articles published in Nature. There are libraries of aptamers from where you could identify the one that has greater specificity to your viroid. Greetings from Mexico City

Thank you Jesus, 

I will look at aptamer library. My question was caused by one proposal about viroid-specific antibodies generation. I am rather pessimistic about success of such work and believe that the problem can be solved by other means - just like you said...

Best wishes Alex

Dear Alex Ignatov

The answer you add it in the project

Pls. find the attached files

http://onlinelibrary.wiley.com/wol1/doi/10.1111/ppa.12624/full

http://www.sciencedirect.com/science/article/pii/S0168945214001083

https://www.ncbi.nlm.nih.gov/pubmed/22678585

http://www.annualreviews.org/doi/abs/10.1146/annurev-phyto-080614-120030?journalCode=phyto

https://www.ncbi.nlm.nih.gov/pubmed/7695839

Pearson, M. N., Clover, G. R. G., Guy, P. L., Fletcher J. D. and Beever, R. E. 2006. A review of the plant virus, viroid and mollicute records for New Zealand. Australas. Plant Pathol. 35: 217-252.

http://www.apsnet.org/publications/apsnetfeatures/Pages/Viroids.aspx

http://onlinelibrary.wiley.com/doi/10.1111/ppa.12624/abstract

Hoping this will be helpful

Regards

Prof. Houda Kawas

also

http://nar.oxfordjournals.org/content/22/15/2951.full.pdf

Thank you Prof. Kawas!

You most welcome

Houda

Hi Alix,

It seems not feasible to use mAb to detect viroids (constitute with ssRNA). See a description from this book:

Microbial Plant Pathogens-Detection and Disease Diagnosis:

Viral and Viroid Pathogens, Vol.3

Viruses, with the primitive structural features are capable of causing diseases in all life forms on the earth. With limited morphological characteristics of the virus particles and lack of physiological functions, the need for the development of techniques based on the characteristics of the viral proteins and genomic nucleic acids was realized in order to detect, identify, differentiate and quantify viruses in the infected plants/planting materials with or without symptoms of infection. Immunoassays based on the antigenic properties of viral capsid proteins have been successfully applied for the detection of viruses in the symptomatic and asymptomatic crop and weed host plant species as well as in the vectors that are involved in the transmission of the viruses infecting crop plants. Development of monoclonal antibody technology has enhanced the sensitivity and specificity of immunoassays especially applied for the differentiation of strains of viruses with different virulence levels. Nucleic acid-based techniques have been demonstrated to have higher levels of sensitivity, specificity, reliability, reproducibility and rapidity. The viroids with only an RNA molecule have been detected by techniques based on the biological characteristics in certain cases. Application of immunoassays for viroid detection is not feasible because of the absence of a protein component that is present in the viruses. Nucleic acid-based techniques are the most reliable and sensitive tests for detection, identification and differentiation of viroids present in plants and planting materials. With the aim of providing a comprehensive knowledge of detection of viruses and viroids and diagnosis of the diseases caused by them, information distilled from extensive literature search is presented to fulfil the requirements of the researchers, teachers and graduate students of various disciplines of biological sciences. Inclusion of numerous protocols in appropriate chapters as appendix is a unique feature of this volume to enable the scientists to select the suitable methods for the various investigations.

@Yuan-Yeu, Thank you a lot! Thanks to everybody who replied to my question. I am completely on the side of pessimistic view on serology application for viroid detection, and my main task - to justify such position, serving as reviewer of proposal devoted to antibodies development for PSTVd. I was afraid that this proposal might be based on some unknown to me information, but, I see that this problem cannot be solved so far.

Alex

@ Alex,

I am curious: if nucleic acid-based techniques are the most reliable and sensitive tests for detection (as mentioned above), why has such a protocol trying to develop immunoassay way for detection? Is any motivation behind it? Is because immunoassay kit easier to carry to fields for detection work?

The group of researchers has developed multiplex strip antibody-based assay for several potato diseases (viruses and bacteria). they want to add viroid as well. The assay is not sensitive enough for seed lot certification or pathogen detection in minitubers, but could be used for confirmation of pathogen present in symptomatic plants.  I guess they targeted field diagnostic by unskilled staff. 

@ Alex,

Now I understand.

The antibodies do be produced and commercialized from single-stranded RNA virus (ex. +ssRNA). See this website: [ http://www.sinobiological.com/+ssRNA%20Virus_antibody_rssrna-virus.html ]. I am wondering whether ssRNA from viroids are different from those from +ssRNA viruses?

Hi Alex,

Not sure whether one can develop antibodies against viroids or any ss-nucleotide molecule. The nucleotides are not very immunogenic. But there is one antibody (mab J2. Link is pasted below for the reference. ) for dsRNA. 

But one can try phage display method for screening binders. They are pretty useful to find binders against molecules which are not even immunogenic. 

Here is the link for Mab-J2

https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-7-264#Sec10

I agree with Lav, you can use phage display, If you carefully select antibody use antibody phage library you may end up getting a specific antibody against viroid. Good part is that you will have a clone which is easy to process and get bulk amount of antibody also if you use human phage library your antibody can be directly used for human use too.

@All, I highly appreciate your help! 

Alex

History of antibodies against nucleic acids has a long tail, too long even to cite publications on the topic here. 

First successes with nucleotides and their derivates changed quickly for fails due to the finding of low immunogenicity of the high molecular weight nucleic acids. The last have properties rather of haptens.

The project meets two challenges:  i) proteins composed of 20 different amino acids, but nucleic acids in general content four different nucleotides only. Thus, specificity of antibodies against nucleic acids looks degenerative in comparison with the specificity of Abs to proteins; ii) weak immune response to nucleic acids in animals because of too much risk of autoimmune diseases.

Despite of these limitations, we are working and hope to solve the task.

@ all

Pls. have a look at

https://academic.oup.com/nar/article/38/7/2378/3100622

https://academic.oup.com/nar/article/30/15/3349/1077134

Best regards

Houda

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861895/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541879/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634539/

https://academic.oup.com/nar/article/30/15/3349/1077134

https://academic.oup.com/nar/article/38/7/2378/3100622

I appreciate much your recommendations. I know a little about aptamers and their use, two neighbors in door deal with aptamers in our Institute. Detection of viroids is easy with DNA probes. But we need antibodies!

Hi Yervand,

Prof. Lukács, who was working in the lab of Detlev Riesner at the time (late 1980's early 1990s), tried very hard, using all the adjuvant tricks and mouse strain differences she could find but without success.

The anti-dsRNA antibodies that scicons now sells (J2 etc) were the result. She did produce some antibodies that bound to viroids but the cross-reaction with other forms of RNA was always so great that they were not useful for discriminatory purposes.

Hi Peter,

Thank you for this interesting details of the work behind J2.

I would also imagine it difficult to get a specific antibody against viroid RNA, which is a circular covalently closed molecule with dsRNA regions along with hairpin loops and hammerheads.

On the other side, viroids during their replication, produce dsRNA which can be detected with J2 and I am not aware of any publication yet, which has tested viroid infected plants for the presence of dsRNA with J2 (eg. size of the bands)

Thanks all!

I appreciate very much all references you sent.

We raised antiserum to the PSTVd transcript. It works well for the specific transcript detection in the range 1-10 ng and detects both: transcript added to the total prep of RNA isolated from the uninfected potato, and PSTVd in the infected potato with a low sensitivity yet.The most complicated is blocking stage of the diagnostic protocol. Isolation of the PSTVd-specific polyclonal antibodies is ahead.