It will be Dr. Diana Fagan's turn to present her research efforts next Friday during our noon seminar. She will be discussing her work with phage display. I look forward to seeing your shiny faces there!
A background reading is available via the Seminar Schedule tab above!
Dr. Fagan discussed her research with phage display for the detection of specific ligands, and for the treatment of disease. She did this through the use of monoclonal antibodies, which are used to detect or identify pathogens, toxins, and hormones, and can also be used to treat infectious disease, organ rejection, cancer, and autoimmune disease. Many different variations of ELISA, including peptide binding ELISA and phage ELISA were employed to detect antibodies. She also used a phage display cycle to isolate antibodies. A very interesting part of her research was her work with the GK-1 thread sensor, which is a detector that is used to detect changes in current. Her aim is to apply these to be able to detect blood and alert that there are casualties during war. I don't have a strong immunology background, so my question is, what is the mechanism of action in a detector like this? And is this a practical/feasible project to pursue?
ReplyDeleteHi Amina. I recently joined the team on this project, I’ll try to give a short summary. Phage display library screening was performed by Bill Reese to identify a variant of interest (BR-1) that selectively binds human serum albumin (HSA - the most abundant protein in blood), much like GK-1. A textile thread is produced using woven single-walled carbon nanotubes (CNTs), which is biologically functionalized with the selected peptide to create a highly specific nanosensor. This functionalization is completed by covalently coupling the peptide variant (BR-1 or GK-1) to the CNTs via carbodiimide bonds, creating a peptide-CNT thread complex. The mechanism is as follows: when HSA is present, it binds to the peptide-CNT complex, changing thread resistivity, thereby creating a signature change in electrical current through the thread, which is measured. This is a pretty simplified explanation -- if you would like to know more please feel free to email me.
ReplyDeleteHi Amina. I recently joined the team on this project, I’ll try to give a short summary. Phage display library screening was performed by Bill Reese to identify a variant of interest (BR-1) that selectively binds human serum albumin (HSA - the most abundant protein in blood), much like GK-1. A textile thread is produced using woven single-walled carbon nanotubes (CNTs), which is biologically functionalized with the selected peptide to create a highly specific nanosensor. This functionalization is completed by covalently coupling the peptide variant (BR-1 or GK-1) to the CNTs via carbodiimide bonds, creating a peptide-CNT thread complex. The mechanism is as follows: when HSA is present, it binds to the peptide-CNT complex, changing thread resistivity, thereby creating a signature change in electrical current through the thread, which is measured. This is a pretty simplified explanation -- if you would like to know more please feel free to email me.
ReplyDeleteAs part of #teamfagan and for clarification purposes, phage display technology does not use monoclonal antibodies. Instead phage display has replaced monoclonal antibodies in the detection and treatment of disease. My project began making monoclonal antibodies to staphylococcus aureus. However it was discovered that monoclonal antibodies that did well in phase two clinical trials in animal models, caused multi organ system failure in humans. This could be due to a variety of reasons. At that point, we decided to use phage display technology, since it is a novel approach for identifying peptide ligands that could potentially bind to a target in my project that target is staphylococcus aureus. In order to do this we began with bio-panning, this is an affinity selection technique to select that peptide from the phage surface that binds most affectively to our target (in my case the target is staph, in Floyd and previous Bill's case the target was HSA). To begin bio-panning we place the target in the bottom of a 96 well plate, following that we add our phage library. Our phage library has many peptides on the surface of the phage particle. Through a series of three washing and binding procedures we are able to identify the peptide that binds most successfully with the target. From there we begin ELISAs to demonstrate the binding of our phage with the target. Although we use a phage library with peptides displayed on the phage surface, there are phage libraries available with antibody fragments on the phage surface.
ReplyDeleteAs far as the feasibility and practicality, absolutely, this is a project worth pursing. A former chemical engineering graduate student, Amy (I apologize I do not know her last name off hand), was successful in created carbon nanotubes that were capable of sending a signal. It is also an amazing opportunity for emergency responders to be able to detect an injured military/police personnel member.
If you have any further questions, or if that did not answer all of your questions, as it is an abridged version, please email me and I can try to clear up any confusion.
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ReplyDeleteI found Dr. Fagan’s lecture to be very interesting as I have worked in the development of ELISA for salivary hormones in the past. She started by explaining how her team has to discard to use of monoclonal antibodies in the case of S. aureas as it stimulates an immune response in humans. They later adopted Phage Display Technology in which is less expensive, less technically difficult, and growing bacteria is easier. Virus is basically nucleic acid material (DNA or RNA) covered in a protein capsid and making changes in virus’s protein coat is easy. The filamentous phage was used as genes (Gene 3) can be inserted in its DNA and it can accommodate the altered DNA. Gene 3 gene will produce gene 3 protein on the capsid of the virus that will bind to pili of bacteria later. Phage will bind to E.coli in phage ELISA , it will be grown on agar plate and later the plaques are extracted. My question for this process is how do make a clone out of this process? Is the clone, E.coli with phage in it and how is it used to identify S.aureas?
ReplyDeleteAlso about the carbon nanotubes that are capable of sending the signals to detect wounded military/police personnel member that detects HSA in blood, I heard Dr. Fagan saying that this apparatus will probably be in the form of a patch. My concern is if the wound is on the leg and the patch is on the chest/ or arm, how would the patch will come in contact with blood, (thus detects HSA) and would be able to send signals?
Phage display is a useful alternative to monoclonal antibodies (mAbs) for a few reasons. For example, as opposed to synthesizing mAbs, phage display has no need to immunize mice against the desired antibody, meaning that no spleen cells are harvested and that no animals are sacrificed. Additionally, mouse mAbs present other problems in terms of detecting and identifying human pathogens, toxins, and hormones. Since they are from mice, they do not effectively interact with human cells and they can stimulate an immune response. It’s a “one and done” situation because once human cells develop their own antibodies against mAbs, they will automatically be targeted and destroyed in subsequent testing. The utility of phage display is that it can generate molecular probes against specific targets for analysis and manipulation of protein/ligand interactions without introducing foreign bodies, and therefore do not induce an immune response. Phage display technology is capable of selecting a single variant that is specific to a particular ligand from a library of 109 variants. S. aureus is not treatable with mAbs, which is why Dr. Fagan initially became interested in phage display, and treatment of staph infections without inducing an immune response may be found using the phage display circle. Since discovering phage display technology, Dr. Fagan’s lab has discovered a peptide that binds to human serum albumin (HSA) much like GK-1, which they named BR-1. HSA behaves as a ligand for BR-1, which I assume means that BR-1 undergoes a conformational change when bound to HSA. This interaction can be utilized for detecting spilt blood in which HSA is a major ingredient. The navy has taken interest in combining BR-1 with nanothread sensor technology because it could be used as an early detector for when troops are injured on the battlefield so that help can rapidly be deployed. I think that the practicality of this technology is apparent, especially if ground troops are not able to communicate by radio.
ReplyDeleteI really enjoyed this presentation. Dr. Fagan's research is extremely interesting. I was not aware of the differences between using phage display and monoclonal antibodies so I walked away actually learning something, which is always a plus.
ReplyDeleteAs far as using the BR-1 attachment to HSA in order to create the nanothread technology that was discussed, I 100% think its worth investing in. This type of technology, if used appropriately, has the potential to save countless lives (especially in battle) so it is something that I would really like to see become a possibility in the future.
Dr. Fagan’s talk on the use of phage display for the detection of specific ligands was both interesting and informative. I found the project regarding HSA most interesting. I personally have very basic knowledge and understanding of ELISA. I was aware Dr. Fagan’s expertise was specified toward immunology, but I did not know of the collaboration across departments, and possibly with the U.S. Navy. From the talk and other comments on this blog by students that work with Dr. Fagan, it seems that the project is very feasible and has very practical applications, thus it is well worth continued investigation.
ReplyDeleteDr Fagan's project can be very demanding and of great help in battle field, not only to the soldiers but also world-wide as a biological alarm to indicate injury. i found that, the use of phage display technology, binds to HSA resulting MWCNT-GK-1 thread sensor- resistance changing in the presence of blood eventually alarming the colleagues.
ReplyDeleteI agree that Dr. Fagan's project could be used world- wide as a biological alarm. phage display technology replaced the use of the monoclonial antibodies, since the Mabs could stimulate immune responses. This technology allows the presentation of foreign proteins at the surface of the phage, which leads to selection of peptides and proteins including the antibodies with high affinity and specificity to a target.
Deletefrom the project, GK-1 peptide binds to HSA, which are then incorporated into the CNTs. CNTs have a sensing ability hence they produce human blood detector. the CNTs- GK-1 biological complex and the HSA has a resistance to the thread. Thus the sensing ability could then be helpful to people working in dangerous environments, since in case of an injury blood could be electrically detected then automatic signal would be send as an emergency, then the res-ponders could be able to reach the injured personel.
Phage display is an efficient way of detecting the presence of diseases or other physiological abnormalities within the body by identifying peptide ligands associated with them. In Dr. Fagan’s lab, they used a certain peptide (BR-1) to bind to HSA (a common protein found in human blood plasma). Similar to an ELISA, a color change will occur if successful binding between the ligand and peptide has occurred, and therefore detecting the presence of any invasions or abnormalities within the body. Due to the advanced technology and efficiency for this detection, this is definitely a project worth pursuing in immunological and biomedical research.
ReplyDeleteI won’t go into too much explanation on the mechanism, as the rest of #teamfagan has already done this in detail. Briefly, the project focused on identifying specific Phage (bacteria virus) that expressed a peptide which would bind to HSA. The team isolated the phage of interest through repeated panning/amplification cycles and then sequenced the peptide. Then, a nanosensor was developed using the peptide which could be attached to soldier/police uniforms to detect blood loss. This means that if a police officer or soldier losses blood during an injury, the information can be quickly relayed to others. This allows them to send help without the injured person having to directly send the message. The project does show a lot of promise but still has much work to be done. Ultimately, this would be a great tool for both military and law enforcement use, but the idea is what interests me the most. If this project works out, where else could the idea be applied? I would speculate that it might be possible to adapt it for aerosolized virus detection. The flu is a virus that changes every year and usually exists in multiple strains as well as have the potential to become severe. In order to combat this, the CDC and other government agencies work together to identify which strain/strains the population should be vaccinated against. I wonder if nanosensors engineered to detect flu virus could help governmental agencies acquire more data on the virus and make more informed decisions.
ReplyDeleteI enjoyed Dr. Fagan’s presentation and found it very interesting. However, I am not much of an expert in immunology. I think that Dr. Fagan’s lab has done a great job explaining the mechanism of GK-1 and exactly what Dr. Fagan is looking at throughout this discussion. From my understanding, phage display is a very useful technique for detecting new proteins and polypeptides. Her research uses protein BR-1 which binds to HSA that is detected in the blood and causes a color change. I think it is pretty amazing that Dr. Fagan and her team are working with the U.S. Navy. I think that this is an excellent project to pursue and it is feasible because if they are able to make a patch to detect wounds on the battlefield then this project could end up saving many lives.
ReplyDeleteFrom what I understand of Dr. Fagan’s research and my extreme interest in immunology, her Phage display replaces the need to develop monoclonal antibodies. Her lab has already isolated and amplified a peptide (expressed on the surface protein of a bacteriophage) that has a high affinity for HSA. After they have amplified that particular peptide, they detach it from the bacteriophage and purify it to be used for treatment and diagnostic purposes. The detector most likely has a sensor that is laced with these peptides that when it comes into contact with HSA (indicative of a person bleeding), the binding of HSA to the peptide completes a circuit and transmits a signal for assistance. There are a few benefits of using phage display instead of monoclonal antibodies. Phage display is simpler to work with and is less expensive than developing monoclonal antibodies (and doesn’t require murine sacrifices). The peptides developed through phage display also would not provoke a host immune response against them, while the mouse constant regions of monoclonal antibodies do (this would allow a patient to receive many injections of phage-derived peptides without developing serum sickness). One possible disadvantage is the difference in size of the peptides versus monoclonal antibodies. Monoclonal antibodies are relatively large proteins with an elimination half life (how long it takes to be cleared from a person’s body) of 10 to 20 days. Peptides are drastically smaller and I’d be concerned that in vivo they would not be able to serve their therapeutic purpose before being excreted by the kidneys. I think that their scope as a therapeutic tool would be limited; they’d serve better as a diagnostic tool.
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