Investigation of humoral Investigation of humoral immunityimmunity

Lucie Sedláčková, PhD.

Department of Molecular Biology and Cell

Pathology, Third Faculty of Medicine

lucie.sedlackova@lf3.cuni.cz

Humoral immunity Humoral immunity componentscomponents

• specificantibodies (Ab)

• nonspecificacute-phase proteinscomplement system

• investigation in serum (urine, cerebrospinal fluid, pathological exudates, bronchoalveolar lavage)

IgG 7-19 IgA 0.8-4.8

IgM 0.5-3 IgD 0.01-0.2 IgE 0-0.0002

Physiological levels of Physiological levels of serum immunoglobulins serum immunoglobulins

(g/l)(g/l)

Investigation of humoral Investigation of humoral immunityimmunity

• agglutination

• turbidimetry and nephelometry

• enzyme immunoassay (ELISA)

• electrophoresis, immunoprecipitation,

immunoblotting

• indirect immunofluorescence

Agglutination

• principle: immune complexes form due to

interaction between an antibody (Ab) and

particulate antigen (Ag)

• antigenic particles (bacteria, Ag on

carrier) + patient‘s serum

demonstration of specific Ab

• evaluation: qualitative (y/n),

quantitative (serum titration)

Agglutination – quantitative

determinationserum dilution by geometrical series

of antigen suspension

of diluted serum

final serum dilution

serum titre = reciprocal value of the highest serum dilution, where the reaction is still positive

example: patient XY

last positive reaction in tube with 1:32 serum dilution → titre = 32

Usage of agglutination

• infectious serology (Salmonella typhi,

Listeria monocytogenes,…)

• diagnostics of autoimmune diseases

(autoimmune hemolytic anemia – Coombs

test)

• blood type determination (hemagglutination)

Immunoprecipitation techniques

• principle: immune

complexes form

due to interaction

between an Ab

and soluble Ag

(precipitate)

Immunoprecipitation curve

constant amount of antibody

Radial immunodiffusion

• principle: Ag and Ab react in agarose gel after diffusion (precipitate ring)

• gel with homogenous Ab content + patient‘s serum

• diameter of the ring is proportional to Ag amount

• usage: IgD class determination

Turbidimetry and nephelometry

• principle: measurement of immune complexes Ag-Ab amounts in the Ab excess

• reaction in liquid phase in cuvette

• evaluation: Ag concentration is proportional to

- production speed of complexes (kinetic system)

- turbidity of complexes („end point“)

Turbidimetry

• precipitation in solution

• measurement of light extraction

(precipitate absorption)

• standard curve

Turbidimetry

laser/quartz lampcuvette

extraction measurement

photodetector

Nephelometry

• precipitation in solution

• measurement of scattered light

• standard curve

Nephelometry

Scattered light detector

Scattered light measurement

Usage of turbidimetry and nephelometry

• measurement of serum proteins‘

concentration (immunoglobulins, acute-

phase proteins, complement components

C3, C4, transferrin, albumin,…)

• rapid, fully-automated techniques for

large quantity of samples

Immunoreaction with labelled antibodies

• RIAAb labelled with radioisotope

• EIAAb labelled with enzymedetection of the reaction according to substrate characteristics:

spectrophotometry, fluorometry ELISA

Enzyme-linked immunosorbent assay (ELISA)

colored product

antibodies in tested sample

enzyme-conjugated antibody

substrate for the enzyme

Determination of optical density

• evaluation: photometry

• higher intensity of color reaction, the

higher concentration of Ag/Ab

concentration

OD

ELISA KIT

ELISA usage• determination of serum concentration of

specific antibodies (borreliosis, tetanus,

hepatitis), autoantibodies, cytokines

• high sensitivity

(<1ng/l)

• high specifity

• reproducibility

Photometry

substrate

sample antigen

1st specific antibody (coating)

2nd labelled antibody

washing

washing

ELISA usage

Determination of IgG against Determination of IgG against Clostridium Clostridium

tetani tetani

Indication:

• unclear anamnesis of previous vaccination

• decision about the vaccination way in

traumatized patients with unknown vaccination

status

• humoral immunity investigation -

immunodeficiencies

Screening of IgG levels Screening of IgG levels against Clostridium tetaniagainst Clostridium tetani

basic vaccination: 3x tetanus toxoid (Alteana)

re-vaccination: 1x Alteana

IgG (IU/ml) protection recommendationbelow 0.03 none basic vaccination0.03-0.1 unsure re-vaccination0.1-0.5 present re-vaccination0.6-1.0 sufficient check-up in 2 yrs1.1-5.0 longterm check-up in 5-10 yrsover 5 very high check-up in 10 yrs

ELISA usage

Antibody level determination against Antibody level determination against transglutaminasetransglutaminase

(Anti-tTG = Anti-tissue Transglutaminase Antibodies)

• in class IgA

Occurance: celiac disease

ELISA usageDetermination of allergen-specific IgE Determination of allergen-specific IgE

antibodiesantibodies• ELISA

• UniCAP machine

adsorbed allergen on solid phase (CNBr –cellulose) reacts with IgE in patient‘s serum

detection with enzyme-labelled secondary Ab against IgE, fluorescencefluorescence evaluation

Electrophoresis

• protein separation due to mobility in electric field (according to electric charge and molecular weight)

• gel (agarose, polyacrylamide)

Serum protein electrophoresis

basic orientation in serum protein abundance

cathode

anode

electrophoreogram

globulins

Serum protein electrophoresis

Immunofixation

• serum electrophoresis in several lines

• antibodies application

(anti-IgG, -IgA, -IgM, -κ, -)

• precipitate formation

(e.g. immune complex = IgG+anti-IgG)

• staining

Immunofixation

patient A

healthy control

healthy control: negative

patient A: paraprotein in class IgG ()

Usage of immunofixation

• paraprotein detection

• diagnostics of monoclonal gammapathies

(multiple myelomamultiple myeloma, macroglobulinemia,

CLL, B-lymphoma)

• paraprotein: synthesized by single clones

of myeloma cells

(neoplastic proliferation of B

lymphocytes)

Immunoblotting (Western blot)

1) electrophoresis

2) transfer of proteins from gel to nitrocelullose membrane in electric field

3) immunodetection:+ serum sample+ labelled secondary Ab (HRP)+ substrate – colored reaction

Usage of immunoblotting

• demonstration of Ab presence against particular Ag (bacteria, autoantigens, …)Borrelia burgdorferi

• detection of several Ab against specific Ag at one time

• immunodot: commertial artificially-purified or recombinant antigens fixed on the membrane

Immunodot

Autoantibodies• targeted against body‘s own cells

• physiologically in low concentration, with age

• used in diagnostics of autoimmune diseases

• non-organ-specific (e.g. against nuclei,

mitochondria,…)

• organ-specific (e.g. against pancreatic antigens)

Indirect immunofluorescence

UV light

green light

slide

substrate (healthy tissue)

specific Ig (antibody from the sample)

washing

washing

specific FITC-labelled anti-human Ig

ANA antibodies (anti-nuclear)

• against nuclear antigens (ds-DNA,

histones, nucleolus, mitotic apparatus,

…)

• substrate: HEp-2 cells

nucleolar fluorescence type (autoantibodies against nucleolus)

homogeneous fluorescence type (autoantibodies against ds-DNA

and histones)

Anti-ds DNA antibodies

• substrate: Critidium lucilliae (protozoan with kinetoplast containing ds-DNA)

• positive sample = kinetoplast and nuclei

fluorescence+ -

Occurance of ANA antibodies

• systemic lupus erythematosus, Sjoegren

syndrome, sclerodermia,

dermatopolymyositis, …

patient with SLE

ANCA antibodies (antineutrophil cytoplasmic)

• against neutrophil granula components (e.g.

myeloperoxidase, lactoferrin, proteinase 3,

…)

• substrate:

human neutrophils

• occurance:

vasculitides

ANCA antibodies

human neutrophils stained with antibody against myeloperoxidase

Gastric parietal cell antibodies

• against intrinsic factor (absorbs vitamin B12)

• substrate: rat stomach

• occurance: atrophic gastritis and pernicious anemia

Investigation of complement

• nephelometry – measurement of concentration

C3 and C4indication: suspected genetic deficiency,

pathogenetic role of immune complexes

C1 inhibitorindication: suspected deficiency

(dg. of hereditary angioedema)

Functional assays for complement

• CH50 hemolytic assay

Principle: tested serum + sheep erythrocytes with bound Ab → complement activation, ery lysis

Evaluation:• spectrophotometry –absorbance

measurement of released hemoglobin, proportional to number of lysed ery

• reciprocal value of serum dilution required to produce hemolysis of 50%

Complement-binding assays

• determination of Ab or Ag

1.step – Ag and Ab reaction in presence of known CC amountactivation (fixation) and C C consumption

2. step – hemolytic activity determination of activated (available) CC

Evaluation:• the highest serum dilution with CC activity

(Ab determination)• Ag concentration