Prostate specific antigen (PSA) is produced in the prostate gland. It is produced by the epithelial cells of the prostate gland. It is a member of the tissue kallikrein which is a family of proteases.[2] PSA is a major protein of the seminal fluid. The function of PSA is to liquefy the seminal fluid by a process of proteolysis of gel-forming proteins. This allows sperm free movement in the female genital tract. An increase in PSA can be a result of prostate cancer as a tumour may be growing. It may also be the result of a benign disorder.[2] By monitoring the PSA levels of the blood it is possible to detect the presence of prostate cancer. Prostate cancer is a slow growing cancer; a marker is required for it to be detected.[2] By using the PSA level in the body as the tumour marker it is possible to detect and treat prostate cancer.
In this report I will be discussing the process from start to finish of how a Point of Care (POC) device can be used to detect the PSA of a sample. An immunoassay point of care device is the chosen type of device. The immunoassay format has become the standard format for point of care devices in the last ten years.[3] An immunoassay takes advantage of the specific binding of antibodies to the antigen; in this case the antigen is PSA. By using an immunoassay POC device a number of advantages are apparent[3]:
It produces quicker results than a biopsy.
It is cost effective and easy to manufacture.
The results obtained are reliable.
Can be easily regulated.
As a result immunoassay POC devices can hold a lot of benefits for the use of detecting prostate cancer. Biosensors can then be used to show the results. A biosensor is a transducer that uses biological components as the key element in the transducer sequence. Biomolecular interaction analysis (Biacore) is an example of a biosensor.[4]
Prostate Specific Antigen (PSA) as target Antigen
As an increase of prostate specific antigen (PSA) can be a sign of prostate cancer it is chosen as the target antigen. It is produced in the prostate gland and can be found in semen. PSA is a glycoprotein enzyme.[2] A blood sample can be used to extract PSA from the body. In order for an immunoassay to be used an antibody must bind to the PSA. Firstly the antibody must be chosen.
Antibody Type
There are 3 different types of obtaining antibodies which can be used for this process. These include
Polyclonal
Monoclonal
Recombinant
Polyclonal
Polyclonal antibodies are obtained from multiple plasma cells.[8] They are made up of a variation of different immunoglobulin molecules.[7] They have varying specifics and epitopes. Polyclonal antibodies are generally purified by serum analysis from immunised animals such as a mouse or rabbit which can then be purified.[6] As a result high titre and high affinity antibodies are produced.[7]
Monoclonal
Monoclonal antibodies are monospecific they represent a single B lymphocyte which are specific to one epitote. They are produced from a clone of a parent cell. They have one specific affinity.[7] Like polyclonal antibodies they are extracted from immunised animals. Monoclonal antibodies are derived from a single clonal hybridoma.[8]
Recombinant
A recombinant antibody is produced in a laboratory. It is not natural occurring in the body. By using genetic engineering a recombinant antibody can be produced. The specifics, stability, size and function can be customised. Using DNA technology the antibody can be tailored.[5]
Production
For the production of polyclonal and monoclonal antibodies animals such as mice, rabbits and chickens are used. The antigen required and the serum produced should reflect the choice of the immunisation protocol.[9] Once the animal is immunised a serum sample is extracted from the animal. For polyclonal antibodies the serum is purified and the antibodies are produced. For the monoclonal antibodies the specific B-cells are extracted from the serum.[8] From this the hybridoma is produced which produces the monoclonal cells.
For recombinant antibodies genes are extracted from the spleen or bone marrow of a mouse. The RNA is extracted. A reverse transcription of the mRNA is changed to cDNA. The Vh and Vl genes are amplified by PCR. The SOE PCR anneals the Vh and Vl. Sfil produces a digest of the SOE PCR product.[5]
Selection
As it may take an extensive amount of time to select the correct antibody using various methods such as ELISA or cloning a different method is required to make a selection. In order for a solution to this problem phage display is required in order to make a selection. By using this method the protein peptides are exposed on the surface of the bacteriophage.[5] Antibody engineering can also be used to customise aspects of the antigen such as specificity and strength.
Immunoassay
An immunoassay is a diagnostic test used to diagnose diseases. By detecting the level of reaction of the binding of an antigen to an antibody a diagnosis can be made. There is a large number of immunoassay techniques developed which have specific recognition to the antigen. Immunoassays make it possible to have highly specific and high affinity antibodies to use against any antigen. Monoclonal antibody technology allows for an endless supply of identical antibodies. For the process of an immunoassay to undergo an antigen must be chosen. This will be the antigen that will be detected. Once the antigen is found, the specific antibody that will bind to it has to be recognised. Finally a method of detecting the level of binding must be produced. Immunoassay tests may be qualitative or quantitative. Tests with different complexity’s and different formats have been developed.[14]
The choice of using an immunoassay to detect the presence of disease depends on the specific disease. An immunoassay test holds a number of advantages over other methods. Tests which detect the presence of antibodies can only measure the one time existence of the infection. It is not clear whether the infection is still present or is clearing. Therefore antibody detection in blood indicates the person has suffered from the infection once in their lives.[13]
Tests that indicate the number of antigens in a patient can often be misleading. Antigen capture test can sometimes return false positives. This may occur if the patient is a disease carrier or has a latent infection. It may also return false result’s if there is only a low level of infection.[13]
After the diagnosis of the sample shows the presence or the lack of antibody/antigen binding, a signal or read out must be produced in order for a nurse of clinician to interpret. In general immunoassays use a reagent dye which acts as a label. This label can amplify the signal which in turn can be visualised by eye or quantified using a plate reader or other detection methods.[13]
ELISA
ELISA is an example of a heterogeneous assay.[14] The antibody us labelled with an enzyme which then converts a colourless substrate into a coloured product. The change in colour allows for detection. The main purpose of ELISA is to detect if there is protein or antigens present in a blood sample. There are several different methods of ELISA. These methods vary in degrees of complexity.[14]
ELISAs are performed in 96 well plates. This permits higher throughput results. A coating of protein is located at the bottom of each well. This protein binds to the antibody which is being measured. To obtain the clear serum from the blood sample, the blood is allowed to clot and is centrifuged. The serum is put into a well and each serum contains a different serum.[15]
When the serum is removed it is washed in a rinse buffer in order to the weak antibodies. In order to detect the bound antibodies a secondary antibody is added to each well. An enzyme is also attached to tis secondary enzyme. These enzymes may metabolise and will turn the colourless substrates into coloured products. A plate reader may be used to analyse the results. The deeper the colour change, the bigger the quantity of antibodies present in the well.[15]
Immunoassay POC Device
Immunoassay point of care devices are immunoassay tests which are done at the point of care. Immunoassay testing lends itself to point of care use. For diseases where the antigen/antibody binding level is high an immunoassay test can visibly produce a colour change to the eye. Point of care can be used outside of a lab environment. There are a number of reasons for this. Firstly the user of the device doesn’t need specialised training to use the device so nurses and lab technicians can use it. Point of care devices produce quick results compared to the time of sending a biopsy away to a lab. The point of care devices have easy to read visuals which show the results. [13]
Figure
Mediwatch: Bioscan PSAwatch
The Mediwatch PSA watch is an immunoassay point of care device. It is designed as a low cost device which can be used in a clinical setting. It is small in size and can produce test results in 10 minutes. A blood sample or plasma can be used for use with the device.[10]
Principal
The PSA watch uses a lateral flow chromatographic membrane based palette assay. It is timed and read on the bioscan analyser. An algorithm is used in the bioscanner as well as information from a barcode on the front of the palette to convert the measured intensity of the test line into a PSA concentration.[11] For analysing plasma the front half of the test palette is inserted into the machine. The plasma sample is then dropped into the sample window on the palette. When ready the palette is fully inserted into the device and the timing begins. For a blood sample a wash buffer solution is required.
Components
The Bioscan PSAwatch consists of a palette array which takes the blood sample. The bioscan then analyses the sample. Finally the touch screen shows the results.
A lateral flow device is composed a number of standard components [3]:
A backing card
Absorbent pad
Sample pad
Membrane
A diagram of how these components are configured:
Figure [3]
Example of Commercial Devices
FastPack IP Toatal PSA immunoassay is a chemiluminescent immunoassay for the in-vitro quantitative determination of PSA in humans as shown in figure 2.
Figure
http://www.qualigeninc.com/products/fastpackipsystem.html
Vedalab produces the easy reader Quantitative PSA device as shown in figure 3.
Figure
http://www.vedalab.com/
Lateral Flow Immunoassay
The device uses a lateral flow immunoassay. [11] It uses a quantitative method. It is important that the devices assay has an appropriate analytical range. By using a chromatography process the sample will separate from complex mixtures which rely on differential affinities of substances. In order to have an effective assay there must be some requirements. The ligand must be specific enough; there must be suitable conditions for binding. A suitable matrix must be found as well. Lateral flow immunoassays take advantage of how antibodies bind to specific antigens. The main feature of using lateral flow immunoassay is the ability to visualise to label of the antigen to the antibody. As a result this label can be visualised and analysed in order to produce results. In this way the stronger the reaction of the antigen to the antibody the greater the intensity of the colour change. In lateral flow immunoassays the reaction is shown chromatographically as coloured lines. [16]
There are a number of advantages of using this format [16]:
It has an adequate analytical sensitivity
Reader technology can be used
It’s convenient to use
It produces rapid results
It is cheap and easy to manufacture
For these reasons lateral flow immunoassay is a useful tool in the use of a point of care device.
Validation and Quality Issues
In order for the device to meet regulations and be fully reliable a validation of the device must occur. The validation of an analytical procedure is to demonstrate that the procedure is suitable for its intended purpose.[12] It involves in depth planning by conducting investigations. Only when the assay is proven reliable will it be used commercially. A list of expectations must be laid out in order to design the device to these specifications. By planning what antibody is going to be used and creating an immunoassay plan the chances of success is greater. The assay must be appropriately sensitive in order to diagnose the correct results.
Ethical Issues
As this device is a medical device for humans there are strict regulations and standards that must be met before the device is mass manufactured. If the product were to be a cause of harm to a user there would be a big problem for the company. As the device will be involved in the detection of a potentially life threatening illness such as prostate cancer it is required to be fully reliable. If the device misdiagnoses a patient there is a chance the misdiagnosis may cause a death. For this reason it is vital the device works in the way that it should. As in the process of selection there are animals involved it is important that a code of ethics is followed for the animals. The animals must live in the appropriate conditions that are required by law.
The specificity is the ability to measure the macromolecular therapeutic unequivocally in the presence of other components in the assay matrix.[17] It is a requirement of the device to meet a certain level of specificity which adhere to the rules and regulations of the governing body. The device must meet the reference standard of purity of the required reagents which are being used in the device. If there are no reference standard materials available they must be researched and developed. The precision and accuracy of the device is extremely important. Rigorous planning and experimenting should be done before the device is developed in order to use antibody which is ideal for the antigen. If any of these mishaps would occur it may impact on the diagnosis of a life threatening disease. The impact of this may be a person dying. It may also impact the quality of life of a person suffering the symptoms of prostate cancer while not being diagnosed with it. It also may have emotional and traumatic effects on the person misdiagnosed as well as family and friends of the person.
