SEROLOGY (PT2) (page 4-7)

direct agglutination

• antigen is found naturally on the surface of the particle

• example: hemagglutination, widal test

passive/indirect agglutination

• antigen is artificially attached to carrier particle (bentonite, charcoal, rbc, latex, gelatin, and silicates)

• agglutination occurs if patient antibody is present

reverse passive agglutination

• antibody is artificially attached to carrier particle

• agglutination occurs if patient antigen is present

agglutination inhibition

• competition between particulate and soluble antigens for limited antibody-binding sites

• positive result: lack of agglutination

• example: hcg test, hemagglutination inhibition (rbc as indicator)

coagglutination

• uses bacteria as inert particles to which antibody is attached

• staphylococcus aureus is most frequently used because of its protein a which is naturally adsorbs the fc portion of igg 1,2,4

antiglobulin-mediated agglutination

• detects non-agglutinating antibody by means of coupling with a second antibody (ahg)

direct antiglobulin test

• specimen: red cell

• in vivo sensitization

• investigation of hdn, htr, aiha, diha

indirect antiglobulin test

• specimen: serum

• in vitro sensitization

• cross matching (serum)

• antibody determination and identification (serum)

• red cell antigen phenotype (patient rbc)

polyspecific ahg

• contains anti-igg and anti-c3d obtained from rabbits

monospecific ahg

• contains anti-igg or anti-c3d obtained from mice

particle counting immunoassay

• measurement of the number of residual non-agglutinating particles in a specimen using a laser beam in an optical particle counter similar to one that is designed to count blood cells

homogenous assay

• no separation step; enzyme activity diminishes when antigen-antibody binding occurs

heterogenous assay

• requires a separation step

solid-phase

• medium to which an antigen or antibody may be attached

• glass beads

• cellulose membranes

• polystyrene test tubes

• microtiter plater

enzyme immunoassay

• label: enzymes

• horseradish peroxide - most common

• alkaline phosphatase

• glucose oxidase

• b galactosidase

• g6pd

• change in absorbance is measured using spectrophotometry

competitive enzyme immunoassay

• enzyme-labelled antigen competes with unlabelled patient antigen for a limited number of antibody-binding sites

• enzyme activity is inversely proportional to analyte concentration

non-competitive/indirect enzyme immunoassay

• indirect because enzyme labelled secondary antibody does not participate in the initial antigen-antibody binding

• enzyme activity is directly proportional to the analyte concentration

capture assay/sandwich

• antigen captured must have multiple epitopes

• enzyme activity is directly proportional to antigen concentration

• prone to hook effect

enzyme multiplied immunoassay technique

• homogenous assay

• antigen is labeled with an enzyme tag

• when antibody, the active site on the enzyme is blocked, resulting to loss of enzyme activity

hook effect

• in case pf ag excess, majority of binding sites are filled, and the remainder of the patient ag has no place to bind

• this result in unexpected fall in amount of measured analyte when an extremely high concentration is present

fluorescent immunoassay

• label: fluorophore/fluorochrome

• fluorescence can be measured using fluorometer, fluorescent microscope, flow cytometer, or spectrofluorometer

• atoms emit light of longer wavelength and lower energy during transition to the ground from excited state

• extremely specific and sensitive

• disadvantages: autofluorescence, quenching

direct immunofluorescent assay

• unknown antigen is fixed to a microscope slide

• fluorescent labelled antibody added directly to unknown antigen

• slide is read using a fluorescence microscope

indirect immunofluorescent assay

• same principle as indirect elisa

inhibition immunofluorescent assay

• blocking test in which antigen is first exposed to unlabelled antigen, then to labelled antibody, and is finally washed and examined

• if the unlabelled and labelled antibodies are both homologous to the antigen, there should be no fluorescence

fluorescence polarization immunoassay

• based on the change in polarization of fluorescent tag emitted from a labelled molecule when it is bound by antibody

• degree of fluorescence polarization is inversely proportional to analyte concentration

radioimmunoassay

• first type of immunoassay developed

• label: radioisotopes

• radioactivity is measured by a scintillation counter

  • crystal: gamma

  • liquid: beta

competitive binding assays

• radiolabelled antigen competes with unlabelled patient antigen for a limited number of antibody-binding sites

• radioactivity is inversely proportional to the analyte concentration

non-competitive immunoradiometric assays

• analyte measured is sandwiched between two antibodies

• radioactivity is directly proportional to the analyte concentration

polymerase chain reaction

• in vitro replication procedure; amplification of the target dna

denaturation

• dsdna is heated to 95 deg celsius to separate dna into single strands

annealing

• dna is cooled to 52 deg celsius to allow primers to bind/anneal to complimentary sequences on the separate dna strands

elongation/extension

• 72 deg celsius, the heat-stable dna polymerase (taq polymerase) binds to the 3’ end of each primer and synthesizes a new strand of dna

amplicon

• is a piece of genetic material that can be formed as the product of a natural event or artificial amplification

primers

• single-stranded nucleic acids

• 18-30 bases in length

• complementary to the 3’ ends of the sequence to be amplified

reverse transcriptase pcr

• is an rna-dependent dna polymerase that converts rna to dna

• detection of rna viruses

quantitative pcr

• originally called real time pcr

• uses fluorescence0-resonance energy transfer to quantitate specific dna sequences

• results can be seen at the end of each cycle

taq polymerase

• is a thermostable polymerase isolated from the bacterium thermus aquaticus