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The ability of a particular antibody to combine with one antigen instead of another.
specificity
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fit between the antigenic determinant and the combining site of the antibody.
non-covalent force
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some of the determinants of an antigen are shared by apparently unrelated molecules.
cross-reactivity
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strength of a multivalent antibody to bind to a multivalent antigen
avidity
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the bond between a single antigenic determinant and an individual combining site of an antibody.
affinity
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excessive amount of antibody that can give a false negative result.
prozine
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decreased amount of antibodyt caused by an excess of antigen
postzone
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point of equal antigen and antibody amounts which yield the greatest reaction.
zone of equivalence
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deviance from the optimum pH of ___ will alter reactivity of the antibody.
7.0
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different classes of antibodies react at different __________.
temperatures
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what is the antibody that reacts at warm temperatures?
IGG
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what is the antibody that reacts at cold temperatures?
IgM
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incubatin time varies with the _____ of antibody.
class
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which types of antibodies will be all the same class and have only one type of heavy and light chain?
monoclonal antibodies
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these are uniform, highly specific antibodies that caon be produced in large quantitites, cloned from a single cell to bind to a single specific antigen.
Monoclonal antibodies
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what are the three uses of monoclonal antibodies?
- typing erythrocyte antigens
- typing leukocyte antigens
- as a reagent
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serological samples should be centrifuged for __ minutes at moderate speed.
10
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if testing cannot be done promptly the serum should be frozen at what temperature?
20oC
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how is complement inactivated?
heat serum to 56oC for 30min
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if serum is not used within four hours, reheat serum at 56oC for ___ minutes.
10
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the highest dilution in which a reaction occurs.
titer
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what is the purpose of a titer?
to determine the concentration of antibody
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this is used to make serum less concentrated.
diluent
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each succeeding dilution is the same as the preceding dilution
fold
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calculations of any given dilution can be determined by using this formula:
D1(V1)=D2(V2)
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in the formula D1(V1)=D2(V2) what is the starting dilution.
D1
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in the formula D1(V1)=D2(V2) what is the starting volume?
V1
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in the formula D1(V1)=D2(V2) what is the final dilution
D2
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in the formula D1(V1)=D2(V2) what is the final total volume?
V2
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pipettes should be periodically calibrated for what three things?
- accuracy
- precision
- reproducibility
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Red Blood cell Suspensions should be used in serological tests as an ________.
indicator
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in an RBC suspension what should be used as the diluent?
2-5% NaCl
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RBC's contain _______; serve as receptor sites for antibodies or complement.
antigens
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what are the four carriers used in serology?
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what is the calucation that can be used to determine the minimum packed cell volume needed to make a solution?
PCV= (total volume X % desired)/100
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in an RBC suspension this represents the packed RBCs.
PCV
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true or false
Hemolyzed RBC's can be used in an RBC suspension.
False
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Mono and ASO use what carrier?
Latex
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Floculation tests use what carrier?
Charcoal
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what carrier is used in hemaglutination?
RBC's
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in what testing method, if antigens are present in serum, they will bind with antibodies forming visible cross-linked aggregates.
latex agglutination
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in latex agglutination antibody molecules bind to latex _____.
beads
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what are the five latex agglutination immunologic assays?
- antistreptolysin O
- Mono Test
- C-reactive protein
- IgG and IgM rheumatoid factors
- Rubella antibody
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in what test are latex particles coated with strptolysin O antigen?
Antistreptolysin O
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in Antistreptolysin O visible agglutination will be seen mixed with patient's serum containing what?
ASO antibody
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titers begin to rise at __ days after the onset of infection and peaks at ____ weeks after infection.
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In ASO, a four fold increase between acute and convalescent tube indicates a recent infection with what?
Group A strep
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ASO antibodies are elevated in acute/convalescent sera with what disease?
acute rheumatic fever
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serological testing should compare acute and convalescent sera collected ______ weeks apart.
three
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how long are ASO samples stable at 2-8oC?
eight days
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how long are ASO specimens stable at -25oC?
3 months
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this is based on agglutination of horse RBC's by hetrophil antibody present in infectious mononucleosis.
Monotest
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in monotest horse RBC's exhibit antigens against both __________ and ______________ threfore a differential absorption of patient serum is necessary.
- forssman
- infectious mononucleosis
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the monotest immunologic theory is based on __________ principle.
Davidshon principle
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in the monotest immunologic theory, what contains only forssman antigen which will absorb forssman heterophil antibody?
guinea pig kidney
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in monotest immunologic theory what will absorb only no-forssman heterophil antibody of infectious mononucleosis?
beef RBC's
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in a monotest immunologic theory this is tested with both the guniea pig-serum specimen and the beef RBC-serum specimen.
horse RBC's
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to be positive for IM heterophil antibody, the guinea pig should be _________ for agglutination and the beef RBC should be _________ for agglutination.
- Guinea pig: positive
- Beef RBC: negative
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what is the sample for Monotest?
Serum or plasma mixed with EDTA
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what is the confirmatory test for infectious mononucleosis?
Epstein-Barr virus antibody
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this is the causitive agent of infectious mononucleosis?
ebstein-barr virus
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this is a DNA herpes-type virus that infects the B-lymphocytes?
EBV
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this is tested to confirm infectious mononucleosis on immune suppressed patients.
EBV antibodies
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this test is beneficial in defining immune status and stage of infectious mononucleosis.
EBVA
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antigen-antibody __________ when combined in proportions at or near equivalence.
precipitate
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in what type of test is interaction of soluble antigen with antibody that results in the formation of a precipitate of fine particles?
flocculation
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in flocculation test particles are ____________ or _____________ visible.
- macroscopically
- microscopically
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what are two examples of flocculation tests?
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this test is based on agglutination of RBC's with antigens that are natural or bound to RBC's
hemagglutination
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what are three examples of hemagglutination immunologic assays?
- ABO antibodies
- Rh antigens
- Cold Agglutinins
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Precipitation methods are performed in what two mediums?
- semi solid media (agar or agarose)
- non-gel support medium (cellulose acetate)
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what are the two types of precipitation methods?
- double immunodifusion
- radial immunodifusion
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in this test antibody dilutions and specific antigens are placed in adjacent wells. Antigen and antibody diffuse out, bind together to form a visible precipitate.
Double immunodiffusion
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what are the three basic reaction patterns in double immunodiffusion?
- Identity
- Non-identity
- partial identity
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In which double immunodiffusion result in precipitin band forms a single smooth arch?
Identity
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in which double immunodiffusion resuld do precipitin lines merge in a spur formation?
partial identity
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in which double immunodiffusion reslut do precipitin lines cross each other?
non-identity
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what are the acceptable specimens for double immunodiffusion?
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in double immunodiffusion why are urine specimens tested in concentrated and unconcentrated forms?
wide range of light cahin concentrations
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what are the two double immunodiffusion immunologic assays?
- Rheumatoid arthritis
- systemic lupus erythrematosus
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Radial immunodiffusion (RID) is used to identify and quantitate the number of _________ found in human serum.
proteins
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in radial immuno diffusion (RID) the antigen will diffuse throughout the agan and form what?
a precipitin ring
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in radial immunodiffusion specific antibody is added to a buffered agarose medium, test ________ is added to a well in the agarose.
antigen
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in radial immunodiffusion (RID) diameter of the precipitin rig is related to what?
antigen concentration
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what are the four clinical applications of radial immunodiffusion (RID)?
- Alpha-1-antitrypsin
- Transferrin
- C3
- Immunoglobulin
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what are the two electro immunodiffusion methods?
- Countercurrent immunoelectrophoresis (CIE)
- rocket immunoelectrophoresis
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in what test do antigens and antibodies move more quickly toward each other with addition of electrical current?
countercurrent immunoelectrophoresis (CIE)
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in CIE agarose gel is used. pH is such that antibodies are ________ charged and antigens are ________ charged.
- antibodies: positively
- antigen: negatively
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in countercurrent immiunoelectrophoresis how is the zone of equivalence represented?
antigen-antibody complex forsm a visible line of precipitin
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what is the clinical application of countercurrent immunoelectrophoresis (CIE)?
- detection of anti-nuclear ribonucleoprotein (nRNP):
- associated to sharp's syndrome (mixed connective tissue disease)
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what test is associated with sharp's syndrome (mixed conective tissue disease)?
detection of anti-nuclear ribonucleoprotein (nRNP)
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in rocket electrophoresis is based on antigen ______________ in an antibody containing gel.
electrophoresis
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in rocket electrophoresis what is adjusted to inhibit antibody migration?
pH
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in rocket electrophoresis the height of the rocket is __________ to the antigen concentration.
proportional
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what ar the four types of labeling techniques?
- radio immuno assay (RIA)
- chemiluminescence
- enzyme immunoassay (EIA)
- immunofluorescent techniques
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what are the two assays that involve chemiluminescence?
- competitive assay
- sandwich assay
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what are the two types of immunofluorescent techniques?
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in what test are radioisotopes used to measure the concentration of antigens or antibodies
radioimmunoassay (RIA)
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in what test are radiolabled ligands bound to antigen-antibody complex?
radioimmunoassay
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in RIA radiactivity is counted on what?
gamma counter
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this test is extremly sensitive and able to detect trace amounts of antigen or antibody.
radioimmunoassay (RIA)
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what are the two clinical applications of radioimmunoassay (RIA)
- hepatitis A IgM antibody
- hepatitis A antigen
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in this test lables can be attached to antigen or antibody.
chemiluminescence
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in this chemiluminescence assay, a fixed amount of labeled antigen competes with unlabeled antigen (patient sample) for a limited numberr of antibody-binding sites.
competitive assay
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in a competitive assay the amount of light emitted is _________ proportional to the amount of analyte (antigen) being tested.
inversely
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in this chemiluminescene test sample antigen binds to a fixed antibody on a solid phase, then a second antibody labled with chemiluminescent binds to the antigen-Ab complex on the solid phase.
sandwich assay
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in a sadwich assay the amount of light emitted is __________ proportional to the amount of the analyte (antigen) being tested.
directly
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what are the two clinical applications of chemiluminescence?
- hepatitis A IgM antibody
- hepatitis A antigen
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this test is similar to RIA but used a non-isotopic label.
enzyme immunoassay
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this test is safer than RIA yet has the same specificity and sensitivity as RIA.
enzyme immunoassay (EIA)
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in Enzyme immunoassay (EIA) what is used instead of a radioisotope?
an enzyme labeled antibody or antigen conjugate
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what ar the most commonly used enzymes to detect the presence and quantity of antigen or antibody in patient serum?
- peroxidase
- alkaline phosphatase
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in EIA substrate causes color production and is measured how?
spectrophotometry
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what are the three clinical applications of Enzyme Immunoassay (EIA)?
- hepatitis B
- HIV antibody
- Cytomegalovirus antigen/antibody
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in this extremely specific and sensitive test, fluorescent molecules are used instead of radioisotopes or enzyme labels.
immunofluorescent techniques
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what are the three clinical applications of immunofluorescent techniques?
- CMV antigen
- CMV antibodies
- HIV antibodies
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in this immunofluorescene technique conjugated reagent antibody is used to detect antigen-antibody complex at microscope level.
Direct Immunofluorescence
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in direct immunofluorescence what is used ot visualized many bacteria in direct smears?
fuorescein-conjugated antibodies boud to fuorochrome
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direct immunofluorescence uses the immunological/physiological theory of:
intracellular antigen detection
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what are the two sepcimens used for direct immunofluorescence?
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in this test, antigen source such as toxoplasma microorganism or virus of infected tissue culture cells to the specific antibody being tested is affixed to a slide.
indirect immunofluorescence
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in indirect immunofluorescence what reacts with antigen forming an antigen-antibody complex?
unkown antibody (patient's Ab)
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in indirect immunofluorescence what is added to the Ab-Ag complex to bind to the antibody as a marker?
labeled anti-immunoglobulin (fluorescent Ab)
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indirect immunofluorescence measures what?
antibody
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direct immunofluorescene measures what?
antigen
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what is the specimen collected for indirect immunofluorescence?
serum
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what are three indications for performing indirect immunofluorescene on serum?
- toxoplasma microorganism
- virus infected tissue culture cells
- ANA antibodies
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what is the classic method for demonstrating presence of a complement fixing antibody in serum?
complement fixation
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what is the indicator system for complement fixation?
- sheep RBC's
- anti-sheep antibody
- complement from guinea pig serum
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in complement fixation patient serum is added to _______ antigen, and complement is added to the solution.
known
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in complement fixation, if serum contains the antibody for the antigen, then an antigen-antibody complex will bind with what?
complement
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in complement fixation sheep RBC's with anti-sheep antibody are added to the solution of antibodies, antigen, and complement. if complement has not been bound, it is available to bind with what?
coated sheep RBC's
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hemolysis of indicator sheep cells indicates a lack of antibody in patient serum and a _________ complement fixation test.
negative
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how is a positive complement fixation test demonstrated.
lack of hemolysis
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why is a positive complement fixation test demonstrated by the lack of hemolysis in indicator sheeps RBC's?
patients serum does contain a complement fixing antibody
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what are the three clinical applications of complement fixation?
- Varicella-zoster virus
- Rubella virus
- Herpes simplex infection
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this is an in vitro method that amplifies low level of specific DNA sequences in a sample to higher quantities.
polymerase Chain Reaction
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in PCR ___ short DNA primers are used as templates that follow the enzymatic process.
two
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in PCR the enzymatic prcess is carried out in cycles and repeated up to ___ times.
30
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what are the four steps of enzymatic process in PCR?
- DNA denaturing
- Primer anealing
- Extension of primed DNA sequenced
- The amount specific DNA sequence double and examined in various ways
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polymerase chain reaction is used in the detection of ____.
DNA
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what are the two clinical applications for PCR?
- human papilloman virus (HPV)
- coronary artery disease
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If the nucleic acid of interest is RNA rather than DNA, the PCR procedures can be modified to include the conversion of RNA to DNA using what?
Reverse Transcriptase-Polymerase (RT-PCR)
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the clinical application for RT-PCR is to test for what?
RNA
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what are the two clinical applications of RNA?
- Human Immunodeficiency Virus
- Hepatitis C Virus
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