Serology Methods

prozone vs postzone

xs antibody // xs antigen

agglutination principle

lattice formation = Ag binds w Fab sites of 2 Ab forming bridges

agglutination direct vs passive

direct ie blood bank, Ag is naturally there

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passive = treating cells or latex agglutination (unnatural)

if competitive assay, what makes a pos?

pos if result is less than the cutoff

if non-competitive assay, what makes a pos?

pos if result is more than cutoff value

agglutination inhibition

indicator = latex bead w target Ag

pos: no agglutination

neg: agglutination

complement fixation (CF)

indicator = srbc coated w hemolysin

pos: no hemolysis

neg: hemolysis

radial immunodiffusion (aka single immunodiffusion) (RID) steps

a. unlimited Ab incorporated into agar

b. serum + standards are in circular wells precut in agar

c. incubate

d. diffusion occurs & rings of ppt forms

e. meas diameter of ring

RID methods

Fahey (kinetic)

• read before ring reaches max size (6-12 hr)

• logarithmic relationship b/t d of ppt ring & Ag conc → read from plotted stnd curve

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Mancini (end-point)

• read at max size 24-48h

• linear relationship b/t area of ppt ring (d²) and Ag conc→ ““

double diffusion (aka Ouchterlony)

used to determine relationship b/t Ag & Ab

1. AB added to wells in center of agar

2. pt sera & stnds are alternated in wells around center

3. incubate

4. diffusion → band of ppt

5. pt wells are read in relation to stnd in adjacent wells

6. location of bands dep on conc & rate of diffusion

immunoelectrophoresis

m/c used to determine heavy & light chains involved

serum IEP: monoclonal (sharp peak) gammopathies or polyclonal (inc but wider peak)

urine IEP: Bence Jones protein

immunofixation

PEL + immunoppt

a. apply spcm to 6 positions on agar

b. electrophorese to sep proteins

c. apply monospecific antisera to 5 lanes

d. if Ag present, Ag-Ab complexes form & ppt; wash, stain

v sensitive, used to classify monoclonal gammopathies

rocket IEL (Laurel)

similar to RID but EL is used to speed formation of ppt

cone-shaped area of ppt forms, meas height & compare to stnd curve

countercurrent IEL

a. 2 rows of wells in gel

b. add Ag in one row & Ab in other

c. elec current

d. migrate towards each other

e. ppt forms if specific

radioimmunoassay (RIA)

detects Ag or Ab

ie RIST (total IgE) or RAST (IgE to specific allergens)

EIA/ELISA

sandwich technique

a. mono or polyclonal Ab adsorbed on solid surface (bead or well)

b. add pt serum; if Ag is present, binds to Ab-bead

c. add xs enzyme-labeled Ab (Ab conjugate) > forms Ag-Ab-label Ab sandwich

d. add enzyme substrate, incubated & read absorbance

e. **wash req b/t each step

f. absorbance = direct proportional to Ag conc

enzyme multiplied immunoassay (EMIT)

used to measure conc of small molecules ie drugs & hormones

add pt serum to enzyme-drug conjugate; also add anti-drug Ab

add enzyme substrate & incubate

pos: color produced

neg: no color

nephelometry

a. serum substance reacts w specific antisera & forms insoluble complexes

b. light is passed through suspension

c. scattered (reflected) light is proportional to number of insoluble complexes; comp to stnd

immunofluorescence

direct: add fluorescein-labeled Ab to pt tissue, wash & examine under fluor microscope

indirect (IIF) - add pt serum to tissue w known Ag, wash, add fluorescein label antiglobulin, wash, examine under fluor microscope

• ie testing for antinuclear Ab (ANA) or fluorescent treponemal Ab test (FTA-Abs)

fluoresence polarization immunoassay (FPIA)

add rgt Ab & fluor-tagged Ag to pt serum

pos: unbound tagged Ag rotate quickly → reduce amount of polarized light

neg: tagged Ag bind to rgt Ab → tagged Ag rotates slowly → inc polarized light

serial dilutions

testing for inf dz on acute & convalescent (recovering) spcms coll abt 2 weeks apart

must see 4-fold or 2 tube rise in titer to be clin sig

sensitivity vs specificity

detect v small amounts, gives pos result if pt has dz (no false neg)

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detect substance w/o x-reacting; gives neg result if pt doesn’t have dz (no false pos)

test sensitivites

most sensi = immunoassays

• RIA, EIA, FIA, nephelometry

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less sensi = lattice

• CIE, CF, agglutination, flocculation (pptn), rocket elp, RID, ouchterlony, IFE< IEP

titers for current infn

2 weeks apart titers, need at least 4 fold inc (or 2 tubes)

ie 320 → 1280

if not may be past infn

if given 1:20 titer, what should you do next

rpt titer in 10 days - 2 weeks

1:10 neg (why neg?)

1:20 +

1:40 +

1:80 +

1:160 +

1:320 neg

what is the titer to report?

160 bc last tube w agglutination

1:10 is showing prozone (xs ab)