Serology Principles

humoral vs cell-mediated immunity

humoral= substances found in humors (body fluids) ie serum ie Ab, complement, antimicrobial peptides

cell-mediated = phagocyte activation, cytokines, T-cell activation

IgG

most prevalent in serum

longest half-life

opsonization of Ag, fixing complement (but IgG4 doesn’t fix complement!)

can cross placenta!

agglutination

IgM

pentamer → 10 binding sites

1st Ab to be produced

predominant in primary Ab response

complement fixation, agglutination, neutralize toxin, primary Ab in neonates

IgA

monomer in serum BUT dimer in secretion

passive immunity from mom -> newborn

can activate complement via alternate pw

IgD

part of B-cell Ag recognition unit

no protective fxn

IgE

important for parasitic infns

attaches to basophils & tissue mast cells

allergic hypersensitivity rxns

direct action or neutralization

Ab may bind directly to foreign bacteria, virus, poison, or enzymes to remove them from circulation

agglutination/aggregation

all Ab have at least 2 binding sites to attach to foreign organisms to remove from circulation

aggregated organisms are less free to move & can be engulfed more readily by macrophages = less mobile

opsonization

opsonin=prepare for eating

macrophages/PMNs recognize IgG1 and IgG3

lysis (complement activation)

refers to series of 9 serum protein that are normal present → mediate inflammation

complement activation → lysis of animal cells & Gram neg bacteria

inflammation

IgE stimulates an inflammatory response which is an important host defense

it is only w prolonged or extensive inflammation that the destructive effects outweigh benefits

enzyme linked immunosorbent assay: non-competitive

if reactive → +

ELISA: competitive

add unlabeled (unmarked) Ab to test Ag → add labeled Ab → if color changed → neg bc that means not enough binding sites for unlabeled so labeled will bind to Ag

chemiluminescent assay

measures light emission

precipitation assays

prozone vs postzone

can get zone of equivalence (optimum precipitation) = lattice formations

prozone: xs Ab present

postzone: xs Ag is present

—> both lead to false NEG (bc doesn’t lattice correctly?)

3 ways to measure precipitation

turbidimetry

nephlelometry

immunodiffusion (visually)

rocket electrophoresis

antisera incorporated into agar → Ag added to wells → electric current to drive Ag

Ag ppt when concentration is equal to that of antisera in agar

height of rocket = conc of Ag in well → compare unknowns to standards

countercurrent electrophoresis

pH of Ab soln to Ag of interest & unknown Ag are adjusted so have opposite charges → placed in wells in agar + current → ppt line at equivalence point = Ag from pathogen

→ when provider needs rapid dx → replaced w latex agglut or lateral flow assays

immunoelectrophoresis

used to see components of serum for abnormalities

Ag are separated by current

plate developed by placing Ab into trough cut in agar b/t 2 wells → ppt lines form as arcs in agar

passive immunodiffusion

line of ppt on agar plate from Ab & Ag w/o electric current

radial immunodiffusion

Ab incorp into agar

wells cut out of agar → filled w pt serum

Ab diffuses out & makes ppt bands

Ouchertorlony

double diffusion bands

fluorescent Ab: direct vs indirect?

direct fluor Ab → looking for Ag

indirect fluor Ab: Ab from rabbit + anti-rabbit labeled Ig → looks for Ab

IFA principle

slide w Ag

Ab pt sample

Ab+Ag complex

incubate 30 min & rinse

add conjugate w/fluorescent tag, incubate, rinse

read w fluorescent microscope

western blot

proteins

used as confirmation for HIV Ab/Ag = gold standard

agglutination

sensitization = initial binding

lattice formation = enhance lattice formation via low ionic strength saline, inc viscosity, agitation, temp, pH

direct agglut = natural Ag-Ab

passive/indirect agglut= latex particles Ag

prozone vs postzone

prozone = xs Ab

postzone = xs Ag

acute hepatitis panel includes

HAV IgM

HBV core IgM

HBSAG

HCV IgG

Diasorin Liaison XL runs

MMR

Varicella IgG

CMV IgG & IgM

HSV1/2 IgG

EBV panel

Quantiferon TB Gold plus

syphilus (Trepsure)

Syphilis work up

1. screen w/EIA test for IgG and IgM

2. rapid plasma reagin (RPR) - non treponemal test - can be false +

3. Treponema pallidum - Particle Agglutination (TP-PA) (Treponemal test)

RPR = rapid plasma reagin

antibody in pt = reagin=anti-cardiolipin (Ab to tissue lipids)

Ab produced as the body responds to lipid material released from damaged cell → can have false +

Ag - cardiolipin w/choline chloride, cholesterol, lecithin; charcoal added for macro viewing

50 uL serum + 1 drop of Ag

calibrated needle (20 gauge)

100 RPM at 8 min

Ag made fresh daily

microscopic flocculation 100x

VDRL: venereal disease research laboratory

performed on CSF or serum

Ag-cardiolipin, cholesterol, and lecithin

50 uL + 1 drop Ag

calibrated needle: 22 gauge, CSF

180 RPM rotation at 8 min

Ag made fresh daily

microscopic flocculation 100x (like agglutination)

Treponema pallidum particle agglutination

tanned sheep rbc coated w Nichols strain of T. pallidum

pt sera incubated w sensitized & unsensitized gelatin particles

pt sera w specific Ab will bind w Ag → smooth mat of agglutinated particles

compact button results from the absence of specific Ab

agglutination in w both cells results from non-specific Ab

titers =

the last positive dilution

lateral flow assay (LFA)

Cryptococcus neoformans/gattii

complement fixation

Ag mixed w/ test serum to be assayed for Ab

standard amount of complement is added to bind to any Ag-Ab complexes

sensitized erythrocytes are added

amount of rbc lysis is determined

no lysis = reactive (no complement available to lyse the cells)

lysis = non reactive (complement is free to lyse the cells)

IgG and IgM interpretations

G+/M+ = recent infn

G+/M- = past infn

G-/M+ = acute infn or false pos IgM

Herpes simplex virus type 1 (HSV-1)

generally assoc’d w oral, ocular, pharyngeal lesions ie cold sores, watery blisters, keratitis

HSV-2

accounts for 2/3 of genital/anal infns; oral can occur

recurrent genital herpes

HSV-1 and 2 latency

virus remains latent indefinitely → reactivation via different factors → cutaneous outbreaks

HSV 1 and 2 lab ID

• viral culture

• direct Ag test by immunofluor

• ELISA

• cytology - Tzank Smear

  • real time PCR - DiaSorin

Herpes ELISA tesets

microtiter plates coated w/ recombinant glycoprotein 1 Ag/glycoprotein 2 Ag

proxidase-conjugated antihuman IgG (HSV1/HSV2)

…

read optical densities

Varicella-Zoster virus (HHV-3)

chickenpox (primary)

• most freq in children

• starts w rash → maculopapules, vesicles, scabs → highly contagious until scab falls off

Zosters or Shingles

• occurs in adults w painful eruption of vesicular lesions w inflammation of dorsal root or cranial nerve sensory ganglia

Varicella-Zoster virus pathology

transmits via direct contact, resp secretions, airborne

complication: neonatal varicella → VZV infn in early pregnancy may result into congenital Varicella syndrome

VSV lab ID

• culture: shell vial - mouse monoclonal Ab specific for VZV glycoprotein conjugated to FITC

• direct immunofluor

• cytology

• PCR

• ELISA

Epstein-Barr Virus (HHV-4)

aka “kissing disease”/infectious mononucleosis

Ab secretion due to inf mono = EBV specific Ab, heterophile Ab, auto-Ab

EBV assoc’d Ab

heterophile Ab =

= Ab that are capable of reacting w similar Ag from 2 or more unrelated species

detected using “monospot test”

in IM formed due to Ag on inf’d cells

Forssman: formed after exposure to certain bacteria (Salmonella, Shigella, Strep pneumo)

serum sickness: formed in resp to injections of horse serum (in some vaccines)

auto Ab

auto anti-i, common cold agglutinin

uhhhhh

heterophile Ab’s

inf mono Ab → absorbed by beef rbcs

Forssman Ab → ““ guinea pig rbcs

serum sickness by both beef & guinea pig (rxn to protein in antiserum of non-animal source)

rapid latex test “monospot” : purified bovine rbc extract as Ag

Paul-Bunnel test: used to titer pt’s serum (EBV-mono)

CMV IgM and IgG

in newborns → IgM Ab is dx significant for congenital infn bc IgM does not cross placenta

IgG persists for life (can cross placenta)

CMV lab ID

• shell vial

• FITC

• …

• PCR = useful in i-compromised pt (bc don’t have Ab → false NEG)

• ELISA = determine pt’s immune status

why is fungal serology important?

bc fungi grows slow → can rapid detect Ag

titer of 1:32 or a 4-fold or greater rise in titer are significant in making dx

Coccidiomycosis tube precipitin (TP)

• detects IgM

• POS as early as 1-3 weeks

• rarely in CSF (IgM can’t cross BBB)

• highly specific

  • disappears w/in 4-6 mo’s → little prognostic value

Cocci complement fixation

detects IgG in serum, pleural fluid, peritoneal fld, joint fluid

serum titer 1:2-1:4 = presumptive early infn

> 1:16 = disseminated infn

CSF titer 1:2 = meningitis

cross-reactivity w/ Histoplasma

immunodiffusion

m/c method for screening Ab

single bands = chronic infn

2 or more bands = dissemination or active dz

Aspergillosis

opportunistic pathogen: A. fumigatus, flavus, niger, terreus

lab dx: immunodiffusion, ELISA, skin test

Blastomycosis

B. dermatitidis

broad based budding yeast

dx: immunodiffusion, ELISA, complement fixation

Candidiasis

Candida albicans & other Candida sp

opportunistic pathogen

Cryptococcosis

pigeons = chief vector

Histoplasmosis

tuberculate macroconidia

Histo immunodiffusion

qualititative test for serum, plasma, CSF, pleural fluid

Ag H&M proteins of histoplasmin

M band = early dz, inactive dz, or skin testing

H & M band = active progression of dz & chronic pulmonary dz

Histo complement fixation

titers of 1:64 or higher = presumptive Histo

yeast form = primary Histoplasmosis

mycelial form = chronic dz

x-reactivity occurs

Name the markers in AHP panel

HAV IgM

HBV core IgM

HBSAG

HCV IgG

What is the confirmatory test for reactive Hepatitis B surface antigen result?

neutralization?

What is confirmatory test for HIV infn (old gold standard)

Western blot

Name different methodologies used in serology.

complement fixation

immunodiffusion

lateral flow

What is the principle for EIA & what does ELISA stand for?

ELISA = enzyme linked immunosorbent assay

Name the tests used to test for H. pylori in the lab & name which one is the best test and why.

Name differences b/t VDRL & RPR in testing for syphilis (especially the Ag components).

Which agglutination tests are used at UCI and what do they test for?

Name the purpose of the IgM diluent.

Explain the principle of complement fixation & what the results mean if there is a button vs no button of rbcs.

button = no rbc’s lyse → Ab-Ag complex formed = POS

no button = rbc’s lysed bc complement protein didn’t bind to Ag-Ab complex → binds/lyse rbc = NEG