MD Flashcards
(17 cards)
Molecular Diagnostics
prevention, control and treatment of infectious diseases require identification of the pathogen causing the disease
• traditional diagnostic methods required growth in culture to ID specific properties (costly and slow)
• molecular diagnostic methods designed to use either immunological or DNA detection procedures – specific, sensitive, and simple
Immunological Diagnostic Procedures
- antibodies bind to specific target sites called antigens
- diagnostic assays based on identifying a specific antibody-antigen complex are ideal standard method (antigen is produced by pathogen)
- detecting antibody-antigen complex – most common method is Enzyme-Linked Immunosorbent Assay (ELISA)
- applications – drug testing, monitoring/assessing cancers, detection of specific metabolites, pathogen identification/monitoring
Steps of Enzyme-Linked Immunosorbent Assay (ELISA)
- bind sample being test to solid support (plastic microtiter plate)
- add primary antibody specific for target antigen (from pathogen) to the support and wash off unbound material
- add a secondary antibody that binds specifically to the primary antibody (it does not bind to target antigen). Secondary antibody has enzyme attached to it that can convert a colorless substrate into a colored product. Wash to remove unbound material
- add colorless substrate
- observe or measure the amount of colored product
DNA Diagnostic Systems
• nucleotide sequence is used as a diagnostic determinant
• benefits – extremely specific and highly sensitive
• nucleic acid hybridization is the basis of most of these assays
• basic steps
1. bind single-stranded DNA from target (patient) to a membrane support
2. add single-stranded labeled DNA probe (probe DNA is isolated from pathogen)
3. wash the support to remove unbound labeled probe DNA
4. detect hybridization of probe DNA and target DNA (using X-ray film)
• limitations – phosphorus-32 is short-lived, potentially dangerous, and requires special lab equipment for handling and safe disposal
Chemiluminescent Detection Procedure –
Nonradiactive DNA Diagnostic Procedure Without Radioisotopes
Applications of DNA Diagnostic Procedures
- probes designed to detect tiny quantities of the plasmodium that causes Malaria
- also probes that detect Salmonella and other bacteria
Another DNA Diagnostic System – DNA Fingerprinting
• DNA fingerprinting is based on the presence of restriction fragment length polymorphisms (RFLPs) in the human genome
• RFLPs result from variations in nucleotide sequence that create or destroy restriction enzyme cutting sites
• one region in the human genome known to have variations in sequence from individual to individual is minisatellite DNA
• minisatellite DNA occur throughout the genome and consists of tandemly repeated sequences (repeats vary from 10 to 30)
• basic steps
1. DNA is isolated from subject or obtained from crime scene and restricted with a specific enzyme
2. DNA fragments separated by gel electrophoresis
2. DNA is transferred to a nylon membrane and specific radioactive probes are added to membrane
4. Probes bind to fragments making up specific minisatellite sequence
5. visualize banding pattern
• applications - id paternity/maternity testing, kinship, ID human remains, study/diagnosis of diseases, criminal forensics
Another Molecular Diagnostic System – Bacterial Biosensors
- need for a system that easily and rapidly detects toxic compounds in the environment
- bacteria have been genetically engineered to detect certain pollutants – used as a first screen to detect the presence of pollution
- followed by more sophisticated, analytical methods that indentify and quantify pollutants
Therapeutic Agents
- a wide variety of biotech-based substances are now in use as therapeutic agents
- we will focus on antibodies, DNA (gene therapy), and whole cells
Antibodies
- antibodies are proteins that immune system cells called B cells produce in response to the presence of foreign substances
- antibodies bind to a specific part of the foreign material (antigen)
- after antibodies bind to the foreign material it is then eliminated
- using biotech-based procedures antibodies can be produced in the lab – they have specificity for certain disease-related substances (proteins on cancer cells)
How Antibodies Are Produced
- purified target molecule (protein) is introduced into an animal (rabbit)
- rabbit makes antibodies against target molecule – there is a mixture of antibodies that bind to different parts of the target molecule (polyclonal antibodies)
- antibodies are purified
• usually polyclonal antibodies are used in molecular diagnostic procedures, not as therapeutic agents
• problem with using polyclonal antibodies as therapeutic agents – variation in the effectiveness of each batch
• monoclonal antibodies (bind to only one region on antigen) are used are therapeutic agents
Chemically-Linked Monoclonal Antibodies
- another way that monoclonal antibodies can be used therapeutically
- mechanism for delivery drugs directly to the site where they need to work
Gene Therapy
• involves the delivery of therapeutic genes into the human body to correct disease conditions created by a faulty gene or genes
• 2 primary strategies
1. ex vivo gene therapy – cells are removed from patient, genes are put into cells, then cells returned to patient (more effective so far)
2. in vivo gene therapy – introduce genes directly into tissues and organs in the body without removing body cells
• a variety of vectors are used for gene delivery for in vivo therapy – viruses, liposomes, and DNA guns
First Human Gene Therapy
ex vivo gene therapy of SCID patient
Whole Cell Therapy
- involves the use of whole cells that produce and secrete a protein to alleviate a disorder
- tissue specific cells that grow well in culture are used – fibroblasts, skin cells, certain brain cells, liver cells, or genetically modified cells with therapeutic gene
- these cells are encapsulated in artificial semipermeable membrane – membrane prevents patient’s immune system to detect cells
- encapsulated cells are implanted into the patient in specific locations where therapy is needed
- use of this therapy is still being tested
Vaccines
- biotechnology can help our immune systems by boosting our immunity through the use of vaccines
- vaccines are parts of a pathogen or whole organisms that can be given to humans or animals by mouth or injection to stimulate the immune system against infection by those pathogens
- when people are vaccinated their immune system recognizes the vaccine as an antigen and responds by making antibodies and B memory cells
- if exposure to the real pathogen occurs the person would have a stockpile of antibodies and memory cells to react immediately
3 strategies used to create vaccines
- subunit vaccines – a portion of pathogen is used as vaccine (genetically engineered)
- attenuated vaccines – a live virus or bacteria that has been genetically engineered to be weak (can not replicate) is used as vaccine
- inactivated vaccines – dead or inactive pathogen used as vaccine
