Second seminar-Methods based on antigen-antibody interactions I. Immunoserology Flashcards
immunoassay
An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes).
what is the antigen-antibody interaction?
Antigen-antibody interaction: reversible, non-covalent interaction (vander walls and hydrophoic interactions)
IT IS REVERSIBLE AND THUS USED IN SEVERAL METHODS
Monoclonal polyclonal
Polyclonal antibodies are derived from blood serum of immunized animals; upon immunization with
foreign protein, blood serum of the immunized organization contains large amounts of antibodies
reactive with different (usually conformational) epitopes of the immunizing antigen.
Poduction of polyclonal antibodies is relatively inexpensive and takes 4-8 weeks. The
most frequently selected species is rabbit from which ~250 mg antibodies can be collected at each
venipuncture. Rabbit polyclonal antibodies are often used as secondary antibodies along with primary
mouse monoclonal antibodies.
Monoclonal antibodies are produced in vitro, in tissue culture using hybridoma technology, involving
somatic fusion of immortal tumor (myeloma) cells and antibody producing plasma cells. Daughter cells
of immortal hybridomas formed by the fusion will all produce the same monoclonal antibody.
Commercially available monoclonal antibodies belong to the same immunoglobulin isotype (are almost
exclusively IgGs)
affinity-
Affinity: it is the binding force between an epitope and an antibody antigen binding site, which is a
sum of the attractive and repellent forces
between the antigen and antibody molecules.
Affinity is an equilibrium constant
characteristic of the antigen-antibody reaction.
Most antibodies are characterized by high
affinity.
avidity-
Avidity: it is the sum of binding forces
detected at more than one site (that is the
total binding force between multivalent
antigens and antibodies).
IgG is can bind to one epitope, showing low avidity, but can bind to two and show higher avidity.
but IgM is multivalent avidity (it is a pentamer).
specificity-
Specificity: the feature of an antibody is that it reacts with a single antigen determinant only.
Antibodies distinguish antigens by 1) their primary structure, 2) isomeric forms and 3) secondary and
tertiary structures.
cross reactivity-
Cross-reactivity is a feature of an antibody that it can react with more than a single antigen. The
basis of cross-reactivity is that the antigen has an epitope, a similar epitope that exists in another
antigen.
antibody titer-
Antibody titer: the last dilution at which the antigen-antibody reaction is still detectable
Sensitivity:
Sensitivity: the parameter that shows how many % of the patients is recognized by the antibody used
in a diagnostic test
specificity-
Specificity: the parameter that expresses how many % of healthy individuals is recognized as
„negatives” by the antibody used in a diagnostic test
Methods based on antigen-antibody interaction
QUALITATIVE – SEMIQUANTITATIVE
Immunodiffusion
radial (the antibody, or the antigen is mixed in the gel)
radial double (both components diffuse in the gel)
- Combination of electrophoresis and antigen-antibody reaction
immunoelectrophoresis
Immunfixation
Western blotting
QUANTITATIVE – ULTRASENSITIVE
Detection of immuncomplexes
Turbidimetry
Laser nefelometry
Ligand assays
RIA – IRMA
ELISA
Lateral flow
HAE disease?
Hereditary angioedema (HAE) is a rare autosomal dominant disorder of C1 inhibitor (C1-INH) deficiency, manifested in painless, nonpruritic, nonpitting swelling of the skin.
serum electrophoresis- Combination of electrophoresis and antigen
QUALITATIVE – SEMIQUANTITATIVE
Serum total protein determination is widely used in the daily clinical practice. The total serum protein
level of a healthy adult is 60-80 g/l (35-50 g/l albumin). If the serum protein content is less than 60 g/l,
or more than 80 g/l, serum electrophoresis may help to identify the missing or overproduced proteins.
The serum protein components have five classifications based on size and electrical charge: POSITIVE POLE 1: Albumin 2: Alpha 1 globulin and Alpha 2 globulin (a1-antitrypsin ceruloplasmin, high-densitylipoprotein (HDL), haptoglobin) 3: Beta globulins (transferrin, b2-microglobulin) 4: Gamma globulins (immunoglobulins)
NEGATIVE POLE
Densitometry helps the evaluation by quantification of
the electrophoretogram.
densitometry
Densitometry is the quantitative measurement of optical density in light-sensitive materials, such as photographic paper or photographic film, due to exposure to light.
densitometry fraction
Albumin is the greatest fraction.
α-globulins: α1 antitrypsin, coeruloplasmin
β-globulins: transferrin, β2-microglobulin
-globulins: immunoglobulins
immune complex.. and overproduction diseases related to them.
Immune complex is a soluble antigen-antibody complex
Diseases caused by the overproduction of immune complexes- Endogenous antibodies Hypersensitivity II. ABO incompatibility RH incompatibility Hemolytic anemia Hypersensitivity III. SLE Exogenous antibodies Passive immunization: serum sickness (Hypersensitivity III.)
Turbidimetry and nephelometry
QUANTITATIVE – ULTRASENSITIVE
Detection of immuncomplexes
used in the daily routine practice: measures antigen, antibody immune complexes.
both methods can be used to determine the level of various proteins measures light intensity.
Turbidimetry: (sensitivity 50 µg/ml) to measure protein levels in the serum, liquor Based on the measurement of light intensity.
Nephelometry: (sensitivity: 1 µg/ml) to measure serum, liquor Based on the measurement of light dispersion (turbidity) intensity.
Nephelometry methods are more sensitive than turbidimetric methods.
Detection of antibodies by Precipitation or by Agglutination.
agglutinin
which antibody will you use for agglutination?
agglutination- Antigen is a particle- bacteria or RBC.
agglutinin= antibody.
We should use IGM due to its high valence.
immune complex formation is NOT precipitation
precipitation In laboratory: insoluble - generated from immune complexes through physical, chemical processes,
immune complex- Soluble antigen-antibody complex.
Immune complex formation is a rapid process.
Radial Immunodiffusion
Radial immundiffusion (cheapest, not used recently) based on the precipitation reaction of the antigen and the antibody in agarous gel
- simple (the antibody, or the antigen is mixed in the gel) - radial double (both components diffuse in the gel)
Radial Immunodiffusion construction
Mancini method or single radial immunodiffusion assay) is an immunodiffusion method widely used in
immunology to determine the quantity of an antigen or antibody, by measuring the diameters of circles
of precipitin complexes surrounding samples.
simple- In wells: antigen (from patients serum): During incubation Ags diffuse into gel
In gel mixed in: specific antibody against the appropriate antigen.
When determining the concentration of the antigen by looking at the diameter, remember!!!:
The diameter of the ring is proportional to the log of the concentration of antigen since the amount of antibody is constant.
Double (ouchterlony method)- both antigens and antibodies are diffused freely.
immunofixation
immunofixation facilitates the detection and typing of monoclonal antibodies or immunoglobulins in
serum or urine.
It is used to To detect one antibody isotype (more sensitive then ELFO).
Agglutination
When the antigen is particulate, the reaction of an antibody with the antigen can be detected by agglutination (clumping) of the antigen.
When the antigen is an erythrocyte the term haemagglutination is used.
Types of agglutination, applications
The reaction of antibodies with the antigens of cells (reed blood cells, white blood ells, bacteria) may result agglutination
Types of agglutination
direct: the reactions of antibodies with antigens of cells result cell agglutination
(eg. RBC antigens)
We use IGM AS THE ANTIBODY FOR DIRECT
indirect: the structure of antigens does not allow direct agglutination but the binding of the second antibody (against the Fc part of antigens) induce agglutination (eg Rh antigen)
We use IGG as the primary antibody and a secondary antibody against it.
passive: haptens or antigens bind to RBCs or latex particles, these structures can be agglutinated with specific antibodies
Micro-coloumn test
??
Hemagglutination without antibodies?
viral proteins may induce agglutination
e.g.: mumps, flu viruses
Not an immunological reaction.
Many viruses attach to molecules present on the surface of RBCs. A consequence of this is that at certain concentrations, a viral suspension may bind together (agglutinate) the RBCs, thus preventing them from settling out of suspension.
Addition of anti-viral antibodies may prevent the agglutination reaction.
Passive agglutination
assive Agglutination• An agglutination reaction that employs particles that are coated with antigens not normally found in the cell surfaces•
Particle carriers include: – Red blood cells – Polystyrene latex
Passive Agglutination• Passive agglutination has been used in the detection of : –Rheumatoid factor –Antinuclear antibody in LE –Ab to group A streptococcus antigens –Ab to Trichinella spiralis
rheumatoid factor
RF: rheumatoid factor: serum IgM autoantibodies against self IgG (e.g. in Rheumatoid arthiritis) which is bound to a particle
Erythrocyte sedimentation rate
The erythrocyte sedimentation rate (ESR) is the rate at which red blood cells sediment in a period of one hour. It is a common hematology test, and is a non-specific measure of inflammation. To perform the test, anticoagulated blood was traditionally placed in an upright tube, known as a Westergren tube, and the rate at which the red blood cells fall was measured and reported in mm/h.
Since the introduction of automated analyzers into the clinical laboratory, the ESR test has been automatically performed.
The ESR is governed by the balance between pro-sedimentation factors, mainly fibrinogen, and those factors resisting sedimentation, namely the negative charge of the erythrocytes (zeta potential). When an inflammatory process is present, the high proportion of fibrinogen in the blood causes red blood cells to stick to each other. The red cells form stacks called ‘rouleaux,’ which settle faster, due to their increased density. Rouleaux formation can also occur in association with some lymphoproliferative disorders in which one or more immunoglobulins are secreted in high amounts.
