PRECIPITATION AND AGGLUTINATION Flashcards
• Involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible.
PRECIPITATION
In this method, the detection device is placed directly in line with the incident light, collecting the light that passes directly through the solution.
TURBIDIMETRY
Measures the reduction in light intensity caused by reflection, absorption, or scatter as a beam of light passes through a solution.
Turbidimetry
The amount of scatter is proportional to the size, shape, and concentration of molecules in the solution and is recorded in absorbance units.
Turbidimetry
Instruments that use 🤨 typically measure light scatter at angles ranging from 10 to 90 degrees.
NEPHELOMETRY
The amount of light scattered is an index of the solution’s concentration, and this measurement can be used to determine the concentration of an analyte in a solution.
NEPHELOMETRY
Measures the light scattered at a particular angle from the incident beam as it passes through a suspension.
NEPHELOMETRY
• In this technique, the rate of increase in light scattering is measured immediately after the reagent antibody is added to the patient sample.
When the antibody concentration is kept constant, this rate change is directly related to the antigen concentration in the sample
RATE NEPHELOMETRY
MEASUREMENT OF PRECIPITATION BY LIGHT SCATTERING
TURBIDIMETRY
NEPHELOMETRY
MEASUREMENT OF PRECIPITATION BY PASSIVE IMMUNODIFFUSION
RADIAL IMMUNODIFFUSION
OUCHTERLONY DOUBLE IMMUNODIFFUSION
These techniques detect antigen-antibody reactions based on the diffusion of reactants through a semisolid medium, such as agarose gel, without the aid of an electrical current.
PASSIVE IMMUNODIFFUSION
• This single-diffusion technique involves uniformly distributing antibodies within the support gel.
The antigen is then introduced into a well cut into the gel.
• As the antigen diffuses radially from the well, it interacts with the antibodies,
forming a precipitate at the zone of equivalence.
The diameter of this precipitation ring directly correlates with the antigen concentration in the sample.
allows for the quantification of antigen concentrations.
RADIAL IMMUNODIFFUSION
• The square of the diameter is directly proportional to the concentration of the antigen.
Mancini method (Endpoint)
• The diameter is proportional to the log of the concentration, and a graph is plotted using semi-log paper.
• Fahey method (Kinetic)
In this classic technique, both antigen and antibody diffuse independently in two dimensions (horizontally and vertically) through the semisolid medium.
• Wells are cut into the gel, with multi-specific antibodies typically placed in the central well and different antigens loaded into the surrounding wells.
• This setup allows for the comparison of
different antigens and determining if they share identical epitopes.
_____form at the zone of equivalence where the diffusing antigen and antibody fronts meet.
OUCHTERLONY DOUBLE IMMUNODIFFUSION
Precipitin lines
• The pattern of these lines reveals the relationship between the tested antigens
OUCHTERLONY DOUBLE IMMUNODIFFUSION
•: When the antigens in two wells are identical, the precipitin lines form a
smooth arc, fusing without crossing
Identity
: If the antigens are unrelated and share no common epitopes, the precipitin lines cross each other.
• Nonidentity
: In cases where antigens share some but not all epitopes, a spur formation is observed, indicating partial cross-reactivity.
• Partial identity
Immunoglobulins, comple-ment, C-reactive protein, other serum proteins
Nephelometry
Light that is scattered at an angle is mea-sured, indicating the amount of antigen or antibody present.
Nephelometry
Immunoglobulins, complement
Antigen diffuses out into gel that is infused with antibody. Measurement of the radius indicates concentration of antigen.
Radial immunodiffusion
Both antigen and antibody diffuse out from wells in a gel. Lines of precipitate formed indicate the relationship of antigens.
Complex antigens such as fungal antigens
Ouchterlony double diffusion
Electrophoresis of serum followed by diffusion of antibody from wells.
Differentiation of serum proteins
Immunoelectrophoresis
Electrophoresis of serum followed by diffusion of antibody from wells.
Differentiation of serum proteins
Immunoelectrophoresis
Electrophoresis of serum followed by direct application of antibody to the gel.
Over-or underproduction of antibody
Immunofixation electrophoresis
• This process is a visible expression of the aggregation of antigens and antibodies through forming a framework.
AGGLUTINATION
• The process by which particulate antigens, such as red blood cells, aggregate to form larger, visible clumps when the corresponding antibody is present in the serum.
AGGLUTINATION
is a two-step process that leads to forming a stable lattice network.
Agglutination
AGGLUTINATION
• The first step,_______, involves the binding of antigens and antibodies.
• This reaction is rapid and reversible.
• The second step,______, is the formation of cross-links that make up the visible aggregates.
• This step stabilizes the antigen-antibody complexes by binding multiple antigenic determinants together.
sensitization
lattice formation
is an antigen antibody reaction that occurs when antigens are found naturally on a particle.
Direct agglutination
DIRECT AGGLUTINATION
• is a specific type of agglutination reaction that uses red blood cells (RBCs) as the indicator particle.
Hemagglutination
• Utilize particles coated with antigens not naturally found on their surfaces.
• Used to detect antibodies
Particles that can be used: erythrocytes, latex, and gelatin.
PASSIVE AGGLUTINATION
Also called indirect agglutination
Passive agglutination
Advantages:
Consistency and uniformity
Reactions are easy to read visually and give quick results.
Passive agglutination
Advantages:
Consistency and uniformity
Reactions are easy to read visually and give quick results.
Used to detect antigens in a patient sample.
In this reaction, it is the antibodies that are attached to the carrier particle.
The antibody attached to the carrier particle must still be reactive and bound so that the active sites face outward.
REVERSE PASSIVE AGGLUTINATION
This method uses Staphylococcus aureus with Protein A on its surface.
Protein A binds to the Fc region of immunoglobulin G (IgG), effectively coating the bacterial cells with the Fc portion of antibodies.
When a sample containing the target antigen is added, the antigen binds to the Fab portion of the antibody, resulting in visible agglutination.
COAGGLUTINATION
Makes use of some viruses that have receptors for red blood cell
(RBC) antigens
• Detects antigens
VIRAL HEMAGGLUTINATION
VIRAL HEMAGGLUTINATION detects
Antigens
A serological technique used to detect the presence of a specific antigen in a patient sample by its ability to inhibit agglutination.
Presence of agglutination =______
Absence of agglutination =______
AGGLUTINATION INHIBITION
Negative
Positive
hinges on the ability of certain viruses to bind to receptors on the surface of red blood cells (RBCs), a process known as hemagglutination.
• The assay leverages this viral characteristic to determine if a patient possesses antibodies against these hemagglutinating viruses.
• Hemagglutination inhibition
The DAT is performed to directly detect in vivo antibody (IgG) or complement coating on RBCs.
COOMB’S TEST
DIRECT COOMB’S TEST
• IAT detects in vitro antibody (IgG) binding to RBCs.
COOMB’S TEST
INDIRECT COOMB’S TEST
TYPES OF AGGLUTINATION REACTIONS
DIRECT AGGLUTINATION
PASSIVE AGGLUTINATION
REVERSE PASSIVE AGGLUTINATION
COAGGLUTINATION
VIRAL HEMAGGLUTINATION
AGGLUTINATION INHIBITION
HEMAGGLUTINATION INHIBITION
COOMB’S TEST (direct and indirect)
TYPES OF AGGLUTINATION REACTIONS
DIRECT AGGLUTINATION
PASSIVE AGGLUTINATION
REVERSE PASSIVE AGGLUTINATION
COAGGLUTINATION
VIRAL HEMAGGLUTINATION
AGGLUTINATION INHIBITION
HEMAGGLUTINATION INHIBITION
COOMB’S TEST (direct and indirect)
