LAB FINAL Flashcards
What are the diagnostic methods used for?
identify the pathology of a particular patient
What is an immunoassay?
analytical methods using antigens/antibodies to determine the presence of their corresponding antigen/antibody in different media
What are immunoassays used for?
find the presence of specific antibodies that are produced in the response to a non-self protein (antigen)
Why are immunoassays useful in the diagnostic field?
they are rapid, cost-effective, sensitive and specific for particular antigens
What are the 3 main parts of a Western Blot assay?
separate antigens by weight | transfer (blot) to a membrane | test for antigen via enzyme-substrate reaction
What is the weakness of Western Blot assays?
less sensitive compared to others
What is an ELISA?
enzyme linked immunosorbent assay (ELISA) 96-microtiter plate capture specific antibody/antigen in sample
What are the 3 main steps of ELISA?
absorb protein-interest into wells (solid-phase support system) | use antibody-specific to protein | develop using enzyme-substrate reaction
What are the 3 most common ELISAs?
direct, indirect, sandwich
What is a strength of ELISAs?
fast, cheap, easy to perform, more sensitive
What is a weakness of ELISAs?
false positives
What are ELISAs used to determine?
ability of an antigen to bind to extracellular matrix proteins
What is an agglutination assay?
ability of antibody to attach to specific antigen = create a lattice = clumping of particles results in visible conglomerate (aggluntination)
What is the agglutination assay commonly used to test for?
Syphilis in diagnostic laboratories (VDRL assay)
What is a significant step of the agglutination assay? Why?
dilute antigen and antibody bc agglutination can only occur when the concentration of antibody and antigen is equal = zone of equivalence
What happens if there is an excess of antigen in the agglutination assay?
agglutination will not occur = leads to false positives
What is the precipitation assay?
uses insoluble complex formed between antigen/antibody
How are results observed in a precipitation assay?
aggregate precipitates out of solution = visualization of complex at base of tube
What is a weakness of precipitation assays?
false-negatives
What is an immunofluorescence assay (IFA)?
detect presence of antibody/antigen in serum samples
What does the immunofluorescence assay involve?
binding of a specific antibody to an antigen in the sample
What modification is done to the antibody(ies) used in the IFA assays?
chemically tagged with fluorescent dye = allows pathogen to be detected by fluorescent microscope
What are the 2 types of IFA techniques?
direct and indirect
What is the Direct IFA Antibody assay?
uses 1 antibody | detects antigens in fecal, blood, tissue samples (not serum)
What is Indirect IFA Antibody assay?
uses 2 antibodies, label with fluorochrome | detects antigens in serum samples
Define molecular techniques?
study of molecular basis of physiological processes that occur in microbes - such as translation and transcription
Why are molecular techniques useful?
can detect slow-growing/unculturable pathogens | identify particular bacterial species present in sample via nucleic acid sequence
What are molecular techniques commonly used as by medical professionals?
identify disease prognosis = allows doctors to decide on potential therapies based on specific characteristics og the pathogen
What are the 3 molecular techniques discussed?
PCR | RT-PCR | electrophoretic mobility shift assay
What is PCR?
polymerase chain reaction | identifies particular pathogen in a sample by a targeted nucleic acid sequence
What is the concept behind PCR?
use of one DNA template strand (or many from a sample) and use of polymerase’s high efficiency to make many copies of the targeted nucleic acid
How can you identify a particular pathogen using the PCR technique?
must use primer specific to a particular bacterial species
What is PCR commonly used for?
identify unknown bacterial isolates and uncultivable pathogens
What makes PCR highly usable?
cheap, fast, specific, uses very small sample
What is RT-PCR?
reverse transcriptase-PCR | determine which genes are expressed in a particular pathogen
What are the main steps of RT-PCR?
reverse transcriptase amplifies mRNA »_space;> makes cDNA (complimentary DNA) »_space;> PCR amplifies cDNA
What is RT-PCR useful for?
determine various characteristics of a particular pathogen (ie: toxins)
What is a weakness of RT-PCR?
unstable nature of RNA | RNase can degrade product
What are the results of most immunoassays expressed as?
positive = presence of Ab/antigen | negative = absence of Ab/antigen
