sum imm 2

forces- non cov

electrostatic or ionic bonding- pos to neg

H bonding- 2 electroneg atoms, weak but strengthens ag-ab

hydrophobic binding- between nonpolar groups

van- weak between electron orbital of one atom and nucleus of another

affects of binding

pH, ionic strength, salt

determine how we make tests

forces and factors (specific for each test)

Equilibrium

Constantly associate and dissociate from each other but Amount of free ag and ab and complexes remain constant

law of mass action

proportion of complexes depends on the concentration, immune complex formation depends on the equilibrium between ag, ab, and ag–ab complexes, lower concentration shifts towards disassociation, Higher towards association

Ag-ab concentration ratio affect

Too much ab or ag, binding is weak and undetected in lab

ionic strength affect on ag-ab

changes can cause dissociation, high salt creates inability to bind

inheritance and location affect on ag-ab

diff people have diff ag and loc on cells, how easily ag can find fab, some fab is weak or hidden creating false neg

number of Ag effect on ag-ab

more ag creates stronger rxn with better binding ability, ex- Preg control is very dark, patient less bc less ag (weaker rxn)

pH affect on ag-ab

prefer neutral 7-7.4, 6.5-8 is okay for strong interaction

time affect on ag-ab

some rxns aren’t instant, incubating speeds up, if not enough time before read creates false neg, standardized (preg read 3-4 not after 4)

temp affect on ab-ag

some best at room, some need body (37 C)

affinity

Strength of binding of one Fab (Ab) with one epitope of an Ag, Higher affinity= more Ab-Ag complexed= more sensitive rxn

avidity

strength of all binding sites together, number of binding sites x the affinity

specificity

an ab ability to bind its intended ag, ab should bind one type of epitope or ag, Does pos actually mean disease is present, 100= no false pos, more specific less false pos, Determined by shape, charge, and chem properties of ag and ab

cross reactivity

ab binds structurally similar but non-target ag of greatest affinity, happens bc the ag shares similar epitopes, causes false pos, Issue solved by- screening (screens people for a disease) and if pos confirming with 2nd test (more expen)

chart

pro- excess ab (fix- dilution to get ab out of solution), arch eq zone, post- excess ag (fix- repeat test in a week giving patient more time to make more ab)

pro and post cause false neg by preventing binding

y- precip formed, x- ag added

unlabeled immunoassays rely on

multiple binding sites of ab and ag at appropriate concentrations, soluble ag = precipitate, ag particulate = agglutination

precipitates

Non-covalent soluble ag and soluble ab bind to form insoluble precipitate, Low sensitivity, Ag and Ab to be eq concentrations and have multiple and eq binding sites for one another, semi-quantitative, quantitative- how much, qualitative- present yes or no

agglutination

visible aggregation of particles from combining with specific ab, identify ag or ab, 2 sensitization step= ag-ab, lattice formation step= combine ag-ab to neighbor ag-ab form aggregates), qualitative, dilution to get semi-quantitative

precipitation- immunotrub (trub)

Automated Methods, Ab reagent + patient Ag, Measures reduction in light intensity, Directly corresponds to the amount of ag in a sample

precipitation- nephelometry

automated method, ab reagent + patient Ag, suspension of particles scatters light as immune complexes form, more sesitive than turb, quantitative

precipitation- flocculation

manual method, produces fine suspended flakes in liquid medium, RPR or VDRL test for syphilis or environmental

precipitation- radial immunodiffusion

manual, single diffusion- ab alr in gel, add ag to diffuse out and react with ab forming ring (eq zone), diameter= direct concentration, end point (mancini) method, kinetic (fahey) method- faster, measurmentS taken before rxn ends

precipitation- ouchterlony

manual method, double diffusion- add ag and ab to diffuse into gel wells, line of precip forms at zone of eq, identity (share, arch), partial (1 spur 1 does not share), non (2 spurs no arch)

precipitation- immunofixation

manual method, double diffusion, proteins in patient serum are electrophoresed, ab added to gel, precip forms where ag-ab complexes form

Agglutination- active/direct

ag is naturally on particle (cell or microorganism), diagnosis of disease(bac/fungus, widal test, synoid/ tifle fever), blood typing (hemagglut)

Agglutination- passive/indirect

particles coated with ag not normally found on surfaces, ag is attached to carrier particle (latex, synthetic beads, silicates), is ab it present agglut,

Agglutination- reverse passive

ab is attached to carrier particle, if ag is present agglu

Agglutination- inhibition

looking for ag, comp between particulate and soluble ag for limited ab sites, pos = no agg bc ab alr bound to patient ag, neg = agg bc reagent can bind to unbound ag

Agglutination- hemagglutination inhibition

detect ab to viruses, add rbcs no agg = pos, agglu = neg