1050 Unit 3 Flashcards
Describe affinity
-initial attraction force between a single Fab site on an antibody molecule and a single epitope of an antigen
—->strength of attraction depends on specificity of antibody for antigen
—->cross reacting antigens have lower affinity
Describe avidity
-sum of attractive forces between an antigen and antibody
—->strength with which a multivalent antibody binds a multivalent antigen
—->measure of the overall stability of an antigen antibody complex
describe how the law of mass action relates to antigen-antibody binding
-value of K depends on strength of binding between antibody and antigen
-the higher the value of K–>larger the amount of antigen-antibody complex –> the more visible or easily detectable the reaction.
define precipitation
the combination of soluble antigen with soluble antibody to produce insoluble complexes that are visible
define agglutination
process by which particulate antigens (latex beads, RBC, gel particles) react with specific antibody to form large aggregates or clumps
what is the difference between precipitation and agglutination?
agglutination takes place between antibody and particular antigen where as precipitation happens between antibody and soluble antigens
describe antigen/antibody concentration in prozone
-when antibody excess is large, prozone occurs
-antigen combines with only one or two antibody molecule and no cross linkage formed.
-precipitation and agglutination can not be detected
describe antigen/antibody concentration in postzone
-when antigen excess is large, postzone occurs
-small aggregates are surrounded by excess antigen. Every available antibody site is bound to single antigen
describe antigen/antibody concentration in zone of equivalence
-number of multivalent sites of antigen and antibody molecules is approximately equal
describe immunoturbidity
-measures reduction in light intensity (measurement of turbidity)
-spectrophotometer
describe nephelometry
-measures light scatter at particular angles as immune complexes form
-nephelometer
-amount of light is proportional to size, shape, and concentration of molecules. Thus light scatter increases as number of immune complexes increases and is an index of concentration index of antibody or antigen is solution.
what is single diffusion?
involves migration of antigen only.
what is double diffusion?
both antigen and antibody diffuse independently through a semisolid medium in two dimensions, horizontally and vertically
what is the principle of the end point method of radial immunodiffusion (RID)?
-antigen is allowed to diffuse to completion when equivalence is reached
-single diffusion technique
-incorporated into gel
- antigen is placed on gel in wells which diffuse out and react with antibody, forming rings of precipitation around the wells. Diameter of ring is directly related to the amount of antigen in the well.
describe the principle of immunofixation electrophoresis (IFE)
-proteins in patients serum are electrophoresed, then antibody is applied directly to gel (agarose or cellulose acetate gel). Precipitation forms where antigen-antibody combination has taken place
-used to detect monoclonal immunoglobulins produced by patients with immunoproliferative diseases such as multiple myeloma
discuss IgM’s ability to participate in agglutination reactions
-700 times more efficient at agglutination than IgG
-bigger, which contributes to its ability to agglutinate without enhancement
-tested at room temperature
discuss IgG’s ability to participate in agglutination reactions
- usually requires use of enhancement techniques to achieve visible reaction such as ionic strength of solution, pH and temperature
-usually requires the use of second antibody to visualize reaction–> Coombs reagent
-tested at 37C
what are the physiological conditions that can be altered to enhance agglutination
-pH
-Temperature
-ionic strength of solution
describe Agglutination inhibition
- based on competition between antigen coated particles and soluble patient antigen for a limited number of antibody site
-only instance in which agglutination represents a negative test.
define Agglutinins
Antibody, lectin or other substances that causes agglutination
define Coombs reagent
Poly alert and contains species specific anti-IgG, anti-IgM, and anti- c antibodies. If present on patient RBC, cross linking will occur (agglutination)
Define cross reactivity
Reacting of an observed agent which initiates reactions outside the main reaction expected
Define Electrophoresis
Movement of charged particles in a fluid or gel under the influence of an electric field
Define end point method
Measure total amounts of analytes that participate in the reaction
Describe DNA
-made of nucleotides that contain deoxyribose sugar with one of the following: adenine, thymine, cytosine, and guanine
-double stranded and arrange in a double helix
What happens when DNA seperates?
two daughter strands separate; each is a template for a newly synthesized complementary strand
Describe RNA
-nucleotides that contain ribose sugar and the nitrogen bases: adenine, uracil, guanine and cytosine
- single stranded
what is the basis of all molecular diagnostic testing?
the high specificity of detection of nucleic acid sequences through complementary base pairing
what does “central dogma of molecular biology refer to?
-the fact that DNA serves as the template for messenger RNA, which in turn codes for proteins
explain mutations and polymorphisms
-changes in nucleotide sequences that may affect specific protein structure and function
explain technique of gel and capillary electrophoresis
negatively charged DNA fragments are separated by size under the force of an electric current in a semisolid gel or polymer solution
Define Restriction fragment length polymorphisms
-(RFLP)
-change in DNA that result in different size pieces when cleaved by restriction enzymes
What is the CRISPR-Cas9?
gene editing system that can be used to alter DNA at specific location
define hybridization
very specific binding of two complementary DNA strands or a DNA and a RNA strand. Often a probe is used to detect an unknown nucleic acid sequence in sample
what are some hybridization techniques?
1) Southern blot analysis
2) situ hybridization
what is amplification?
involves making many copies of specific nucleic acid sequence to obtain enough material for laboratory identification
describe transcription-mediated amplification
-TMA
-target is RNA instead of DNA. cDNA copy is made of the original RNA and uses to produce millions of RNA copies
describe strand displacement amplification
-SDA
-involves amplification of probes rather than the original DNA
describe Branched DNA
represents a signal amplification method in which multiple probes attach to the original target sequence
-DNA probes used to capture the target nucleic acid
what are types of probe amplifications?
1) strand displacement amplification (SDA)
2) loop-mediated amplification (LAMP)
3) molecular inversion probe (MIP) method
what does DNA sequencing involve?
-determining the order of nucleotides in a DNA chain -the most specific way of detecting polymorphisms and mutations
What is the Sanger chain termination sequencing method?
-involves replicating a single DNA strand in the presence of fluorescent-labeled modified nucleotide bases called dideoxy nucleotide triphosphates
-DNA fragments of various sizes are generated from the original template, and the sequence is determined by detecting the fluorescent labels
-most explicit method for identifying polymorphisms and mutations
what is pyrosequencing?
alternate method that relies on the generation of the light when nucleotides are added to a growing DNA chain
-no gels, dyes or ddNTPs
what is the overall function of Next Generation Sequencing?
-NGS
-allows for rapid sequencing of large number of small DNA templates at one time. The short sequences are then assembled into a complete sequence.
what does targeted gene panels do?
determine the sequence of specific genes
what does whole exome sequencing do?
sequence of only the coding regions within DNA
what does whole genome sequencing do?
sequence of an entire genome to identify mutations associated with a variety of diseases
what is bioinformatics?
uses information technology to analyze the vast amount of data for clinical relevance by comparison with known databases
what is associated only with RNA synthesis?
promoter
the speed at which nucleic acids migrate in gel electrophoresis is determined by which property?
size
what is the function of endonucleases?
they cleave DNA at specific site
what technique is used in RNA-guided enzymes?
CRISPR
To what does situ hybridization refer to?
probes react with the intact cells within tissues
what is the principle of microarrays?
arrays contain multiple unlabeled probes on a solid support
Describe PCR
primers are used to make multiple DNA copies
what happens in the annealing process of PCR?
the primers bind to target DNA
what is the purpose of the amplification control in qPCR?
to avoid false negatives
what technique is based on RNA amplification?
TMA (target mediated)
what is used in Sanger sequencing?
ddNTP
what type of signal is generated in pyrosequencing?
light
what is the NGS sequencing library?
a collection of short templates to be sequences simultaneously
what is the coverage in NGS?
the number of times a region is sequenced
define nucleic acid
carry genetic information that codes protein structure
what binds Guanine to Cytosine
3 hydrogen bonds
what binds adenine and thymine?
2 hydrogen bonds
what are DNA and RNA measured in?
-DNA is measured in base pairs
-RNA is measured in bases
what is a chromosome?
double helix of DNA
how many chromosomes are in the cell nucleus?
46
Define genes
sequences of nucleotides in chromosomes that carry information for either a protein or non-coding RNA molecule
Define diploid
two copies of the each 23 chromosomes per cell or 46 chromosomes total
define genome
entirety of of DNA in cell
what is the cell cycle and what are their phases?
as cells divides, they undergo a series of events in which they increase in size and duplicate their DNA
-phases
—1> G1
—2> S
—3> G2
—4> mitosis/cytokinesis
explain each phase from the cell cycle
- G1–> daughter cell will go/be here
- S –> DNA replication takes place
- G2–> DNA complement of the cell is doubled
-mitosis - one complement of chromosome is divided into two daughter cells
what is the catalyst for DNA replication?
DNA polymerase enzyme
what can DNA synthesis not start without
a preexisting 3’ hydroxyl group
what is a primer?
-begins DNA synthesis in vivo
- a primer of RNA is synthesized by RNA polymerase (primase) enzyme
what does PCR stand for?
polymerase chain reaction
what is the lagging stand of DNA?
copied discontinuously toward replication fork
what is the leading stand of DNA?
copied continuously is direction of replication
what is RNA synthesis catalyzed by?
RNA polymerase
What is RNA polymerase?
begins polymerization of RNA by binding to its binding recognition start site in DNA (promoter)
Define epigenetics
-involves chemical changes is histone proteins
-modification of DNA such as base methylation
-noncoding RNA activities that can influence the expression of genes independent of the nucleotide sequences
define transcription
genetic information flows from DNA to mRNA
define translation
genetic information flows from RNA to proteins
what is a phenotype?
observable properties
what is a codon?
nucleotide sequence of mRNA contains a 3 base recognition sequence
what is the difference between variant and mutation?
-variant is recommened for nucleotide sequence changes that are inherited
-mutation is a more general term for spontaneous changes in DNA
what is a polymorphism?
alterations of DNA or protein sequence shared by at least 2 % of natural population
what are the two types of gels use for nucleic acid testing?
1) agarose (less high resolution but cheaper and less toxic)
2) polyacrylamide
what are 4 approaches to nucleic acid analysts?
1) strand cleavage methods
2) hybridization methods
3) amplification methods
4) sequencing methods
what are restriction enzymes
-endonucleases that will separate the phosphodiester bonds between nucleotides in DNA.
-endonucleases recognize and bind to specific nucleotide sequences in the DNA so that they will only separate the DNA at those locations
what is the Southern Blot test?
-laboratory method used to detect specific DNA molecules from a mixture of DNA molecules.
-informative studies could be performed directly on large and complex genomes by cleaving the DNA into smaller fragments by gel electrophoresis and identifying the region of interest with probes.
define probes
short nucleic acids that bind to complementary sequences
what is the western blot method?
-detection of proteins and protein modifications.
-serum, cell lysate or extracted proteins are separated by gel electrophoresis and blotted to membrane
define genomics
the analysis of hundreds to thousands of target or whole genomes, rather than as single genes.
what are 3 basic types of arrays?
1) comparative genomic arrays (detect amplification or deletion of DNA)
2) RNA expressions arrays
3) high density oligonucleotide (SNPs)
define microarray
-thousands of targets with probes bound to a very small area (example: microscope slide)
-use highly specific unlabeled probes attached directly to a solid support
define in situ hybridization
-(ISH) detection of targets in places as they appear in tissues, cells, and subcellular structures
-labeled probes are used to bind or hybridize to the targets
Define immunohistochemistry
type of in situ hybridization using labeled antibodies to detect the presence of clinically significant protein targets, such as those expressed by tumors
describe Fluorescence in situ hybridization
-(FISH) use fluorescently labeled probes and require specialized microscopes equipped to detect the emitted fluorescent signals
-to detect specific chromosome abnormalities
-can be performed on interphase cells or metaphase chromosome from dividing cells.
what is the ISH method sensitive to?
-buffer
-temperature
what is a false positive and a false negative of the ISH method
false positive -> cause by nonspecific binding of the probes
false negative -> caused by failure of the probe to bind
what is amplification?
-copying of nucleic acids
what is reverse transcriptase PCR?
-method of analysis for cellular RNA or qualitative detection of RNA viruses (HIV and hepatitis C).
what is key component to the specificity of the PCR reaction?
oligonucleotide primers
what is a standard 3 step cycle of PCR
1) denaturation -> separate the double stranded DNA into single strands (94-96 C)
2) annealing -> allow binding of primers (50-70 C)
3) extension -> complementary nucleotides are added to the 3’ end of the primers to complete DNA synthesis (68-72 C)
* each temperature is held for 30-60 seconds
* cycle repeated 20 to 50 times
what are the types of thermal cycler and how do they work?
1) chamber-based -> sample tubes are subjected to the amplification program temperatures through the surrounding air in the chamber
2) block-based -> sample tubes are placed in a metal block that is heated and cooled accordingly
what is sequence specific primer PCR
(SSP-PCR) primers designed so that they will end on a potentially mutated or polymorphic base pair
-designed to detect mutations and polymorphisms
what is qPCR?
target quantification could be achieved by observing the accumulation of PCR products in real time during amplification
-SYBR green is dye used for this test.
what are the 4 types of probes
1) fluorescence resonance energy transfer
2) TaqMan
3) molecular beacon
4) scorpion
what is digital droplet PCR?
-provides absolute quantification
-not affected by variations in PCR efficiencies
-allows detection of small changes in target number that is not possible by qPCR
what is the benefit of amplification methods?
-isothermal –>temperature cycling is not needed.`
what is transcription mediated amplification systems?
-RNA is target cell and primary product
-cDNA is synthesized from the target RNA and transcription of the cDNA produces millions of copies of RNA products
what is loop-mediated isothermal amplification
-(LAMP) has high specificity and sensitivity for target DNA
-shorter run time
-used to detect HIV, cytomegalovirus, staphylococcus aureus and E. coli.
what is signal amplification?
large amounts of signal are bound to the target sequences that are present in the sample
Define Chain termination sequencing
-modification of the DNA replication process.
-uses dideoxynucleotide triphosphates to modify DNA replication
what is in pyrosequencing reaction mix?
1)sequencing primer
2)enzyme
3)substrate
what is ion conductance sequencing ?
nucleotide order is determined through release of hydrogen ions by DNA synthesis.
what reversible dye terminator technology?
carries sequences complementary to immobilized primers on a solid support
when does maximum binding of antigen and antibody?
when aggregate number of multivalent sites on antigen and antibodies are approximately equal
*seen in zone of equivalnce
Describe Ouchterlony method
-double diffusion technique
-both antigen and antibody diffuse from wells and travel toward each other
-Precipitin lines may indicate identity, non-identity, and partial identity, depending on the pattern formed between a known antigen or antibody and an unknown antigen or antibody
what are the two steps in agglutination?
1) sensitization –> initial binding of antigen and antibody, which depends on the nature of the antibody and antigen bearing surface
2) lattice formation –> governed by such factors like pH, ionic strength, and temperature
describe direct agglutination tests
-patient serum is incubated with antigens that are found naturally on the indicator particles
- RESULTs: agglutination indicates the presence of patient antibody to a natural antigen
describe passive agglutination test
-also known as indirect agglutination
-patient serum is reacted with antigens that are artificially attached to the indicator particles.
-RESULTs: agglutination indicates the presence of patient antibody to an artificially attached antigen
describe reverse passive agglutination
-antibody is attached to the indicator, instead of antigen
-RESULTs: agglutination indicates the presence of specific antigen in the patient sample
agglutination reactions are typically used for what?
rapid screening tests. Are sensitive and can yield valuable information when interpreted correctly
describe hemagglutination inhibition
-RBCs spontaneously agglutinate if viral particles are present
-RESULTs: lack of agglutination is a positive test indicating the presence of patient antibody
describe principle of measurement used in particles enhanced turbidimetric (PETINA)
-patient sample incubated with latex beads coated with analyte and reagent antibody
-if amount of analyte is patient sample is high, analyte will prevent antibody from binding to beads and less turbidity results.
-measures small analyte such as therapeutic drugs
identify conditions that must be met for optimal results in agglutination
1) avoid cross reactivity by using monoclonal antibody directed against an antigenic determinant unique to a particular antigen
2) store reagents properly, check expiration dates and follow manufacturers instruction
3) account for sensitivity and specificity of specific test kits used.
4) Be aware that a negative results does not rule out presence of the disease antigen
In a precipitation reaction how can ideal antibody be characterized?
high affinity and high avidity
what are some characteristics of nephelometry?
-readings are taking before equivalence is reached
-more sensitive than immunoturbidity
-measurements are time dependent
a major characteristic of end point method of RID
-the antigen concentration is directly proportional to the square of the ring diameter
what zone might an antibody-screening test be a false negative?
prozone
how does immunoturbidity differ from nephelometry?
nephelometry measures light that is scattered at an angle
what refers to the force of attraction between an antibody and a single antigenic determinant?
affinity
if crossed lines result in an Ouchterlony immunodiffusion reaction with antigens 1 and 2, what does this indicate?
the two antigens are unrelated
agglutination of dyed bacterial cells represent what type of agglutination?
direct
what best describes the law of mass action?
the equilibrium constant is related to the strength of antigen-antibody binding
if a single IgM molecules can bind many more antigens than a molecule of IgG, what is higher?
avidity
agglutination inhibition could best be used for which of the following types of antigens?
soluble haptens
what describes reverse passive agglutination?
-it is used for identification of antigens
reactions involving IgG may need to be enhanced for which reasons?
IgG may be too small to produce a lattice formation
what test is a lack of agglutination for positive reaction?
Agglutination inhibition
typing of RBCs with reagent antiserum represents what type of reaction?
direct hemagglutination
why were labeled immunoassays developed?
measure antigens and antibodies that may be small in size or present in very low concentrations
what is an analyte?
the substance being measured
what are some labeling techniques
?
what are some labeling techniques?
-radioactivity
-enzymes
-fluorescence
-chemiluminescence
-chromatography
what are heterogenous and homogenous immunoassays
-hetero–> physical separation of bound and free components
-homo–> does not require physical separation
what are competitive immunoassays?
-all reactants are added at the same time and labeled antigen competes with patient antigen for a limited number of antibody-binding sites
-amount of bound label is inversely proportional to the concentration of the labeled antigen
-in other words, the greater the amount of labeled antigen detected, the less antigen is present in the patient sample
what are noncompetitive immunoassays?
-reagent antibody is passively absorbed onto a solid-phase material. Excess capture antibody is present to ensure that any patient antigen will be bound. A labeled antibody, directed against a different epitope of the antigen, is added.
- the amount of label detected is directly proportional to the amount of patient antigen present.
what are characteristics of antibodies used in immunoassays?
-must be very specific
-high affinity for the antigen in question
-specificity helps reduce cross reactivity
-affinity determines how stable the binding between antigen and antibody.
-avidity and affinity help determine the sensitivty of immunoassays
what are Radioimmunoassay’s
-(RIA) based on competitive principle using radioisotope label
-extremely high analytical sensitivity
-hazardous to health
what are Enzyme immunoassays?
- (EIA) uses enzymes that catalyze biochemical reactions
-they convert reagent substrates to produce chemically modified products can be detected
what are competitive enzyme-linked immunosorbent assays?
-(ELISAs) antigen in the sample competes with enzyme-conjugated antigen for a limited number of binding sites on antibody molecules attached to a solid phase
-enzyme activity present in the final reaction catalyzes the conversion of the substrate to a detectable product
what are indirect ELISAs?
-noncompetitive immunoassays
-enzyme labeled reagent does not participate in the initial antigen-antibody reaction
-associated antigen is bound to solid phase
-patient with unknown antibody concentration is added
-a second antibody reacts with any patient antibody that is bound to the solid phase
what are capture immunoassays?
-detect antigen
-antibody is bound to solid phase, and any patient antigen is allowed to binded or captured
-second enzyme-labeled antibody is added
-final complex formed with sample antigen in between the two reagent antibodies creates the sandwich.
-label amount is directly proportional to the amount of of patient antigen present.
what are homogenous enzyme immunoassay
-require no separation steps
-based on principle that enzyme activity changes as specific antigen-antibody binding occurs
-when antibody binds to enzyme-labeled antigen, steric hindrance results in a loss of enzyme activity
how can increased sensitivity of labeled immunoassay be achieved?
complexing biotin to the capture antibody and streptavidin to the solid-phase material.
-bioton-SAv labeling can be used in both indirect ELISAs and capture immiunoassays
what is the most common limitation of capture immunoasay?
high dose hook effect–> excess patient antigen causes falsely decreased detection, leading to an analyte concentration that appears to be low or normal
how do antibodies interfere with immunoassays?
-whenever the antibody is produced is similar to that of a test reagent
-heterogenous interference occurs when the individual produces antibodies similar to those used in the test reagent kit
-test kits using a biotin-SAv complex can cause unpredictable interferences
what is cross reactivity?
describes detection of a substance other than analyte of interest. It is typically observed as a false-positive results
what homogenous immunoassays are used for?
detection of low-molecular-weight analytes, such as hormones, therapeutic drugs, and drugs of abuse. Example –> CEDIA
what are rapid immunochromatographic test
-are able to capture antigen or antibody in specific location on membrane
describe immunometric assays
-based on chemiluminescence label detection
-chemiluminescence is produced by certain compounds when oxidized. Substances that do this can be used as markers in reactions that are similar to RIA and EIAs
what are fluorochromes?
fluorescent compounds that absorb energy from an incident light source and convert that energy to light of a longer wavelength
describe direct immunofluorescence assays
-involve antigen detection through a specific antibody that is labeled with a fluorescent tag.
- the presence of fluorescent microscope that utilizes UV lights
-fluorescence on cell surface indicates that the cell is positive for antigen
describe indirect immunofluorescence assays
-the original antibody is unlabeled.
-incubation with antigen is followed by addition of a second fluorescent-labeled anti-immunoglobulin that detects antigen-antibody complexes
-if fluorescence is detected, antibody or antigen is present in the sample, and the test is positive
describe multiplex immunoassay
-involves using polystyrene beads is as the solid phase
-to detect antibodies in patient serum, beads conjugated to different antigen are used
-allows for multiple antibodies or antigens to be detected simultaneously
describe fluorescence polarization immunoassay
-(FPIA) was a type of homogenous fluorescent immunoassays
-it was based on the principle that when an antigens is bound to to antibody, polarization of light increases
-competes with patient antigen for a limited number of soluble antibody-binding sites
-when patient antigen binds to antibody, less reagent antigen binds and less polarized light will be detected.
describe newer single use immunoassays
-rapid tests based on immunochromatography that are designed to detect analytes such as hCG and drugs in urine
what best describes heterogenous competitive binding immunoassays?
the concentration of patient analyte is inversely proportional to bound label
how do heterogenous immunoassays differ from homogenous immunoassays?
heterogenous immunoassays require a separation step.
what best characterizes a capture/sandwich immunoassays?
excess number of antibody sites on solid-phase material
what is an advantage of enzyme immunoassay over radioimmunoassay?
decrease in hazardous waste
what is the best characteristic of direct fluorescence immunoassays?
this method can be used for rapid identification of microbial antigens
what is TRUE about enzyme-multiplied immunoassay?
a)it is classified as a heterogenous method
b) nicotinamide adenine dinucleotide (NAD+) is a substrate use in the test reaction
c)when patient sample antigen concentration is high, the final test signal will be low
d)patient sample antigen blocks the enzyme active site in the test reaction
B
a fluorescent substance is best described as one in which
light energy is absorbed and converted to a longer wavelength
In a noncompetitive ELISA, if a negative control shows the presence of signal. what is a possible explanation?
washing steps were incomplete
what best characterizes chemiluminescent immunoassays?
a chemical is oxidized to produce light
Immunofluorescence assays may be difficult to interpret for what reason?
-autofluorescence of substances in serum
-nonspecific binding to serum proteins
-subjectively in reading results
what best describes rapid immunochromatography assays?
they are designed primarily for point-of-care testing
what is a characteristic of an indirect enzyme immunoassays?
color is directly proportional to the amount of patient antibody present
in a indirect ELISA, what would be the outcome of an improper wash after the antibody-enzyme conjugate is added?
results will be falsely increased
in a heterogenous enzyme immunoassay, if the patient sample produces more signal than the highest positive control, what action should be taken?
repeat the immunoassay using one-half the volume of the patient sample
what can uniquely cause interferences in immunoassays designed with streptavidin bound to the solid phase surface?
elevated concentrations of biotin in the patient test sample can cause falsely increased results
what are two noncompetitive immunoassays?
1) capture
2) immunometric
what order does analytic sensitivity appear in?
(most sensitive)
1) chemiluminescence
2) fluorescence
3) colorimetric
4) radioactive
(least sensitive)
what are common enzymes found in enzyme immunoassays?
1) alkaline phosphates
2) horseradish peroxidase
what are common enzymes found in enzyme immunoassays?
1) alkaline phosphates
2) horseradish peroxidase
3) glucose 6 phosphate dehydrogenase (G6PDH)
4) Beta-galactosidase
what vitamin is biotin?
-vitamin B7 (vitamin H)
what are the steps of noncompetitive in-direct enzyme immunosorbent assays (ELISAs) to detect an antibody in a patient sample?
1) reagent antigen immobilized on solid phase binds to antibody in patient sample
2) wash to remove unbound material.
3) add enzyme-labeled (detection) antibody that binds to human immunoglobulin
4) wash to remove unbound materials
5)add substrate and measure signal. Signal is directly proportional to concentration of ANTIBODY in patient sample
what are the steps of the capture immunoassay to detect antigen in patient sample?
1)reagent antibodies immobilized on solid phase capture antigen/analyte in patient sample
2) wash to remove unbound material
3) add enzyme labeled (detection) antibody that binds to different epitopes on the antigen
4)wash to remove unbound materials
5) add substrate and measure signal (color intensity) signal is directly proportional to concentration of ANTIGEN in the patient sample
what are two clinical applications for homogenous (EIAs)
1) enzyme-multiplied immunoassay technique (EMIT) –> based on principle of change in enzyme activity as specific antigen-antibody interaction occurs in solution
2) cloned-enzyme donor immunoassay (CEDIA)–> developed using genetic engineering techniques to produce the Beta-galactosidae enzyme in two parts: acceptors and donors
describe the difference between direct and in direct immunofluorescence technique
-direct –> labeled antibody
-indirect –> unlabeled antibody
what are pros and cons of radioimmunoassays?
pro–> detectors are able to measure very low quantities of radioactivity
con–> health hazards involved in working with radioactive substances
what are pros of enzyme immunoassays?
-eliminates concern for health hazards
-enzyme improve analytical sensitivity
what is pro of non competitive immunoassay?
most frequently used because of its specificity, sensitivity, and simplicity
what is a pro of capture immunoassay?
use of monoclonal antibodies has made this a very sensitive test system
what is a pro of CEDIA
reaction occurs without the need for a solid phase
what are pros of chemiluminescence immunoassay?
-can be applied to heterogenous and homogenous assays
-has superior analytical sensitivity
-has wide dynamic range while also reducing the need to dilute the sample
what is a pro of fluorescent immunoassay?
rapid results
what is a pro of direct immunofluorescent assay?
antigens can be detected with high specificity and sensitivity
what is a pro and con of in direct immunofluorescent assay?
pro–> one antibody conjugate can be used for many different types of reactions, eliminating need for numerous purified labeled reagent antibodies
con–> reading slides is partly a subjective interpretations
what are pros of Multiplex immunoassay?
-easier
-allows for multiple antibodies to be detected simultaneously
-also can be used to simultaneously detect multiple antigens in test sample
what are pros of rapid immunoassay?
-easy to perform
-give reproducible results
-faster turn around
-doesn’t take up a lot of space
-disposable
