Difference between revisions of "Team:Freiburg/Results/Diagnostics"

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We obtained the sequence for an immunogenic <a href="http://parts.igem.org/Part:BBa_K1621006"target="_blank"><i>Salmonella</i> Typhimurium antigen</a> and a corresponding <a href="http://parts.igem.org/Part:BBa_K1621006"target="_blank">anti-<i>S.</i> Typhimurium antibody</a> from Prof. Dr. Hust's laboratory. Both His-tagged proteins were successfully expressed in <em>E. coli</em> and spotted on a PDITC surface. In an iRIf measurement we analyzed the binding between <i>S.</i> Typhimurium antigen and antibody (figure 1). The measurement was a great success. When the <i>S.</i> Typhimurium antibody was flushed over the chip a distinct shift in the binding curve for the <i>S.</i> Typhimurium antigen was detectable, whereas the negative control showed no binding event (figure 2). Moreover the obtained iRIF result was validated with a standardized method. The purified antigen was analyzed by a 12.5% SDS-PAGE. Afterwards a <a href="https://2015.igem.org/Team:Freiburg/Protocols/Western_Blot">Western Blot</a> with the self-purified antibody was performed. This antibody contains a c-Myc tag that is not present in the antigen. Therefore we used as secondary antibody an anti-c-Myc antibody derieved from goat. For chemilumineszenz detection an anti-goat HRP was used. Also in the conventional method the binding of the anti-<i>S.</i> Typhimurium antibody to the corresponding antigen is detectable (figure 3 (B)). The presence of both proteins was also validated by an Western Blot with anti-his conjugated antibody (figure 3 (A)).
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We obtained the sequence for an immunogenic <a href="http://parts.igem.org/Part:BBa_K1621006"target="_blank"><i>Salmonella</i> Typhimurium antigen</a> and a corresponding <a href="http://parts.igem.org/Part:BBa_K1621006"target="_blank">anti-<i>S.</i> Typhimurium antibody</a> from Prof. Dr. Hust's laboratory. Both His-tagged proteins were successfully expressed in <i>E. coli</i> and spotted on a PDITC surface. In an iRIf measurement we analyzed the binding between <i>S.</i> Typhimurium antigen and antibody (figure 1). The measurement was a great success. When the <i>S.</i> Typhimurium antibody was flushed over the chip a distinct shift in the binding curve for the <i>S.</i> Typhimurium antigen spot was detectable, whereas the negative control showed no binding event (figure 2). Moreover, the obtained iRIF result was validated with a standardized method. The purified antigen was analyzed by a 12.5% SDS-PAGE. Afterwards a <a href="https://2015.igem.org/Team:Freiburg/Protocols/Western_Blot">Western Blot</a> was performed using the self-purified antibody. This antibody is genetically fused to a c-Myc tag that is not present in the antigen. Therefore, we used an anti-c-Myc antibody derived from goat as secondary antibody. For detection via chemiluminescence an anti-goat HRP was used. The conventional method confirmed the binding of the anti-<i>S.</i> Typhimurium antibody to the corresponding antigen (figure 3 (B)). The presence of both proteins was additionally validated by Western Blot with an anti-His conjugated antibody (figure 3 (A)).
 
     With this measurement we demonstrated that our system is able to detect a specific antigen-antibody binding.  
 
     With this measurement we demonstrated that our system is able to detect a specific antigen-antibody binding.  
 
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             <strong>Figure 1: Quotient picture indicating changes of layer thickness caused by antibody binding. Positive control: GFP-His; negative control: biotinylated BSA.</strong>  
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             <strong>Figure 1: Quotient picture indicating changes of layer thickness caused by antibody binding at the <i>Salmonella</i> Typhimurium antigen spot. Positive control: GFP-His; negative control: biotinylated BSA.</strong>  
 
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             <strong>Figure 2: Relative light intensity at a spot related to the background in the anti-<i>S.</i> Typhimurium iRIf measurement</strong>  
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             <strong>Figure 2: Change of relative light intensity at a spot related to the background while flushing the slide with anti-<i>S.</i> Typhimurium </strong>  
 
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             <strong>Figure 3: Western Blot of <i>S.</i> Typhimurium antigen (DHAD) and anti-<i>S.</i> Typhimurium antibody (anti-DHAD, scFv).</strong>  (A) Western Blot of His-tagged <i>S.</i> Typhimurium antigen as well as the corresponding scFv with anti-His HRP conjugate. The expected molecular weight of DHAD is 63 kDa and 30 kDa for the scFv, respectively. (B) Western Blot of <i>S.</i> Typhimurium antigen with the purified <i>S.</i> Typhimurium scFv; the purified antibody was used in a 1:100 dilution. The scFv is c-Myc tagged. Anti-c-Myc antibody (1:1000; rabbit) for the detection of c-Myc tagged scFv was used in a second step. For chemilumineszenz detection anti-rabbit HRP antibody (1:5000) was used.
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             <strong>Figure 3: Western Blot of <i>S.</i> Typhimurium antigen (DHAD) and anti-<i>S.</i> Typhimurium antibody (anti-DHAD, scFv).</strong>  (A) Western Blot of His-tagged <i>S.</i> Typhimurium antigen as well as the corresponding scFv using anti-His HRP conjugate. The expected molecular weights of the antigen and the scFv are 63 kDa and 30 kDa, respectively. (B) Western Blot of <i>S.</i> Typhimurium antigen using the purified <i>S.</i> Typhimurium scFv; the purified antibody was used in a 1:100 dilution. The scFv is c-Myc tagged. Anti-c-Myc antibody (1:1000; rabbit) for the detection of c-Myc tagged scFv was used in a second step. For detection via chemiluminecence anti-rabbit HRP antibody (1:5000) was used.
 
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     Another important result during the establishment of our diagnostic device was the immobilization of three proteins on one single slide (GFP, a rabbit- and a mouse-derived antibody). In this experiment the slide was flushed with three different antibodies, one after the other, each compatible to one of the immobilized proeins. In three different outputs, dependent on the antibody, we received a highly specific binding at each spot. The corresponding binding curve shows the relative light intensity. Each binding event is highly specifc (figure 4). Additionally, the quotient pictures show a distinct binding of the antibody to the related protein (figure 5). This confirmed specific binding events of antibodies to proteins in our setup. With this promising result we were one step further along our diagnostic application.  
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     Another important experiment on our way to the establishment of the DiaCHIP was the immobilization of three proteins on one single slide (GFP, a rabbit- and a mouse-derived antibody). In this experiment the slide was sequentially flushed with three different antibodies, each specifically binding to one of the immobilized proteins. In three different outputs, dependent on the antibody, we received a highly specific binding at each spot. The corresponding binding curve shows the changes in relative light intensity at each spot. The occuring binding events are specific, except a slight cross-reactivity of the anti-mouse antibody (figure 4). Quotient pictures additionally visualize distinct binding of the respective antibodies to the corresponding protein (figure 5). This confirmed specific binding events of antibodies to proteins in our setup. With this promising result we were one step further along our diagnostic application.  
 
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             <strong>Figure 4: Relative light intensity at a spot related to the background</strong>  
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             <strong>Figure 4: Change of relative light intensity at a spot related to the background during an iRIf measurement</strong>  
 
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             <strong>Figure 6. Western Blot of HIV multi-epitopic antigen.</strong> In this Western Blot the HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. The anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.
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             <strong>Figure 6. Western Blot of HIV multi-epitopic antigen.</strong> The HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. Anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.
 
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     As we suppose a new diagnostic device for the detection of several diseases, we expressed several antigenic peptides in <i>E.coli</i>. Due to time constraints we could only overexpress some antigens and verify them by Western Blot or SDS-PAGE (<a href="https://2015.igem.org/Team:Freiburg/Labjournals">see labjournal protein purification</a>). In addition to the expressed <i>S.</i> Typhimurium and <a href="http://parts.igem.org/Part:BBa_K1621003"target="_blank"><i>C. tetani</i></a> antigen, we were able to overexpress the HIV multi-epitopic antigen as well as a HCV antigen. Figure 6 shows the Western Blot of the <a href="http://parts.igem.org/Part:BBa_K1621006"target="_blank">HIV multi-epitopic antigen</a>. For this verifcation we used the anti-HIV-1 P24 polyclonal antibody.
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     As we suppose a new diagnostic device for the detection of several diseases, we expressed several antigenic peptides in <i>E.coli</i>. Some of these antigens were successfully overexpressed and verified by Western Blot or SDS-PAGE (<a href="https://2015.igem.org/Team:Freiburg/Labjournals">see labjournal protein purification</a>). In addition to the expressed <i>S.</i> Typhimurium and <a href="http://parts.igem.org/Part:BBa_K1621003"target="_blank"><i>C. tetani</i></a> antigen, we were able to overexpress the HIV multi-epitopic antigen as well as a HCV antigen. Figure 6 shows the Western Blot for verifcation of the <a href="http://parts.igem.org/Part:BBa_K1621006"target="_blank">HIV multi-epitopic antigen</a> using anti-HIV-1 P24 polyclonal antibody.
 
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Revision as of 20:21, 15 September 2015

""

Julia: Bin grad dabei das hier komplett neu zu machen, nach Plan vom Website-Team.
find ich gut, bin gespannt (NG)

Diagnostics

The following section summarizes the most interesting results we obtained this summer establishing our diagnostic tool. On our way to detecting anti-Tetanus antibodies in human blood serum we achieved many other results in the field of diagnosis. In addition to the detection of anti-Tetanus antibodies, we could identify anti-GFP antibodies with our own cell-free expressed GFP immobilized on our specific surface (Essential Results). Before we achieved these major results we demonstrated that our device is indeed capable of detecting specific antigen-antibody binding.

Detection of Salmonella Typhimurium Single Chain Antibodies

We obtained the sequence for an immunogenic Salmonella Typhimurium antigen and a corresponding anti-S. Typhimurium antibody from Prof. Dr. Hust's laboratory. Both His-tagged proteins were successfully expressed in E. coli and spotted on a PDITC surface. In an iRIf measurement we analyzed the binding between S. Typhimurium antigen and antibody (figure 1). The measurement was a great success. When the S. Typhimurium antibody was flushed over the chip a distinct shift in the binding curve for the S. Typhimurium antigen spot was detectable, whereas the negative control showed no binding event (figure 2). Moreover, the obtained iRIF result was validated with a standardized method. The purified antigen was analyzed by a 12.5% SDS-PAGE. Afterwards a Western Blot was performed using the self-purified antibody. This antibody is genetically fused to a c-Myc tag that is not present in the antigen. Therefore, we used an anti-c-Myc antibody derived from goat as secondary antibody. For detection via chemiluminescence an anti-goat HRP was used. The conventional method confirmed the binding of the anti-S. Typhimurium antibody to the corresponding antigen (figure 3 (B)). The presence of both proteins was additionally validated by Western Blot with an anti-His conjugated antibody (figure 3 (A)). With this measurement we demonstrated that our system is able to detect a specific antigen-antibody binding.

Figure 1: Quotient picture indicating changes of layer thickness caused by antibody binding at the Salmonella Typhimurium antigen spot. Positive control: GFP-His; negative control: biotinylated BSA.

Figure 2: Change of relative light intensity at a spot related to the background while flushing the slide with anti-S. Typhimurium

Figure 3: Western Blot of S. Typhimurium antigen (DHAD) and anti-S. Typhimurium antibody (anti-DHAD, scFv). (A) Western Blot of His-tagged S. Typhimurium antigen as well as the corresponding scFv using anti-His HRP conjugate. The expected molecular weights of the antigen and the scFv are 63 kDa and 30 kDa, respectively. (B) Western Blot of S. Typhimurium antigen using the purified S. Typhimurium scFv; the purified antibody was used in a 1:100 dilution. The scFv is c-Myc tagged. Anti-c-Myc antibody (1:1000; rabbit) for the detection of c-Myc tagged scFv was used in a second step. For detection via chemiluminecence anti-rabbit HRP antibody (1:5000) was used.

Specific Detection of Multiple Binding Events

Another important experiment on our way to the establishment of the DiaCHIP was the immobilization of three proteins on one single slide (GFP, a rabbit- and a mouse-derived antibody). In this experiment the slide was sequentially flushed with three different antibodies, each specifically binding to one of the immobilized proteins. In three different outputs, dependent on the antibody, we received a highly specific binding at each spot. The corresponding binding curve shows the changes in relative light intensity at each spot. The occuring binding events are specific, except a slight cross-reactivity of the anti-mouse antibody (figure 4). Quotient pictures additionally visualize distinct binding of the respective antibodies to the corresponding protein (figure 5). This confirmed specific binding events of antibodies to proteins in our setup. With this promising result we were one step further along our diagnostic application.

Figure 4: Change of relative light intensity at a spot related to the background during an iRIf measurement

Figure 5: Quotient pictures indicating changes of layer thickness caused by binding of the indicated antibody

Expression of several antigens

Figure 6. Western Blot of HIV multi-epitopic antigen. The HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. Anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.

As we suppose a new diagnostic device for the detection of several diseases, we expressed several antigenic peptides in E.coli. Some of these antigens were successfully overexpressed and verified by Western Blot or SDS-PAGE (see labjournal protein purification). In addition to the expressed S. Typhimurium and C. tetani antigen, we were able to overexpress the HIV multi-epitopic antigen as well as a HCV antigen. Figure 6 shows the Western Blot for verifcation of the HIV multi-epitopic antigen using anti-HIV-1 P24 polyclonal antibody.

As ‘Health and Medicine’ is one of the most popular tracks chosen in the iGEM competition, we want to share the sequences encoding for these antigenic peptides with the iGEM community. Thus, future iGEM teams have the opportunity to take advantage of our research, if they are planning to work in the field of diagnostics. BioBricks iGEM Team Freiburg 2015