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Flashcards in MD Deck (17):
1

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

2

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

3

Steps of Enzyme-Linked Immunosorbent Assay (ELISA)

1. bind sample being test to solid support (plastic microtiter plate)
2. add primary antibody specific for target antigen (from pathogen) to the support and wash off unbound material
3. 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
4. add colorless substrate
5. observe or measure the amount of colored product

4

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

5

Chemiluminescent Detection Procedure –

Nonradiactive DNA Diagnostic Procedure Without Radioisotopes

6

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

7

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

8

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

9

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

10

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)

11

How Antibodies Are Produced

1. purified target molecule (protein) is introduced into an animal (rabbit)
2. rabbit makes antibodies against target molecule – there is a mixture of antibodies that bind to different parts of the target molecule (polyclonal antibodies)
3. 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

12

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

13

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

14

First Human Gene Therapy

ex vivo gene therapy of SCID patient

15

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

16

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

17

3 strategies used to create vaccines

1. subunit vaccines – a portion of pathogen is used as vaccine (genetically engineered)
2. attenuated vaccines – a live virus or bacteria that has been genetically engineered to be weak (can not replicate) is used as vaccine
3. inactivated vaccines – dead or inactive pathogen used as vaccine