Abstract
Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor-acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.
| Original language | English |
|---|---|
| Pages (from-to) | 13-17 |
| Number of pages | 5 |
| Journal | Analytical Biochemistry |
| Volume | 425 |
| Issue number | 1 |
| DOIs | |
| State | Published - 1 Jun 2012 |
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Keywords
- Fluorescence
- Proteins
- Resonance energy transfer
Cite this
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Engineering resonance energy transfer for advanced immunoassays : The case of celiac disease. / D'Auria, Sabato; Apicella, Elisa; Staiano, Maria; Di Giovanni, Stefano; Ruggiero, Giuseppe; Rossi, Mauro; Sarkar, Pabak; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt.
In: Analytical Biochemistry, Vol. 425, No. 1, 01.06.2012, p. 13-17.Research output: Contribution to journal › Article
TY - JOUR
T1 - Engineering resonance energy transfer for advanced immunoassays
T2 - The case of celiac disease
AU - D'Auria, Sabato
AU - Apicella, Elisa
AU - Staiano, Maria
AU - Di Giovanni, Stefano
AU - Ruggiero, Giuseppe
AU - Rossi, Mauro
AU - Sarkar, Pabak
AU - Luchowski, Rafal
AU - Gryczynski, Ignacy
AU - Gryczynski, Zygmunt
PY - 2012/6/1
Y1 - 2012/6/1
N2 - Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor-acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.
AB - Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor-acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.
KW - Fluorescence
KW - Proteins
KW - Resonance energy transfer
UR - http://www.scopus.com/inward/record.url?scp=84859367665&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2012.02.035
DO - 10.1016/j.ab.2012.02.035
M3 - Article
C2 - 22394617
AN - SCOPUS:84859367665
VL - 425
SP - 13
EP - 17
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -