Exam 3 - Lecture 14 Precipitation and Agglutination Reactions

Antibody-antigen interactions are

critical to many diagnostic tests

Immunoassays used to detect specific antigen in patient sample

Direct evidence of infection

Immunoassays to detect specific antibodies in patient sample

Antibodies: indirect evidence, although strong evidence of infection

• so as long as detected antibodies are specific to pathogen 

• Often: reagent antibodies are used to detect patient antibodies

Precipitation 

• Soluble Ag + soluble Ab → insoluble immune complexes

• Antigens must possess multiple antibody-binding sites

• Complexes can be visualized directly

Agglutination

• Aggregation of larger molecules in presence of specific Ab

  • Cells; Antigen on cells

  • Cross-linking of the larger molecules

  • Aggregations can be visualized directly

Affinity

attraction force between one Fab site and a single epitope on an antigen

Affinity Interaction

• Relatively weak bond

• An Antibody can initially attract different antigens

• Good structural fit between Ag & Ab:

  • More stable binding

  • Perfect fit = Maximum binding

Cross-reactivity 

antibodies binding to antigens that closely resemble the inducing antigen 

Avidity

overall antibody-antigen binding strength

• Measure of overall stability of Ab-Ag binding = Force that keeps molecules bound

• Sum of individual affinities

  • More Bonds = Higher Avidity

  • IgM vs IgG: 10 binding sites vs. 2

• Can have high avidity with low affinities

  • Avidity compensates

Higher Affinity =

More Sensitive test results

More bonds =

Higher Avidity

Precipitation Curves

Relative proportions of Antibody and Antigen determine ability to precipitate complexes

Zone of Equivalence

Number of binding sites on Antibody and Antigen are about equal

Precipitation

• Each antibody binds to more than one antigen

• Each antigen binds to more than one antibody

• Stable structure = lattice

Precipitation decreases when

either antigen or antibody are in excess

• Size of complexes, if any are smaller

Prozone

Antibody excess, relatively less antigen = No precipitation

• Both antibody binding sites may bind one antigen = No cross-linking of antigen

• Antibody “competition” for antigen binding sites prevents cross-linking

Prozone =

too much antibody, not enough antigen (No precipitation)

• Your a “Pro” everyBODY likes you

Postzone

Antigen excess, relatively less antibody; no precipitation

• Not enough antibodies for extensive cross-linking

• Each antibody binding site is bound to a single antigen

Postzone =

too much antigen, not enough antibody (No Precipitation)

False Negatives in Prozone

• Specific Antibodies may exist, but may be too concentrated to form complexes

• Solution: dilute Antibody to see if complexes form

  • Reduce Antibody-antigen ratio to reach zone of equivalence

False Negatives in Postzone

• Specific Antibodies may exist, but in small amounts

• Obscured (concealed) by larger amount of Antigen

• Solution: retest at a later date

  • Allow more Antibodies to develop in patient

Light Scatter

measure of light passage through suspension

Turbidimetry

• Detection in line with light source

• Ratio: incident light/transmitted light

• Spectrophotometry

  • A light source shines directly through the sample (in line with the detector).

  • The detector measures the light that continues straight through (transmitted light)

Nephelometry

Measure of scatter at angle

• More complexes = More scatter

• Compared to scatter of known concentrations of complexes

Passive Immunodiffusion (PID)

a technique used to detect and quantify antigen or antibody (or whatever is being measured) in a sample using agarose gel medium.

• Binding leads to precipitation, which appears as visible bands or rings.

• Factors affecting diffusion rate

  • Particle size, temperature, composition of agarose

Radial Diffusion 

1. Agarose gel contains a uniform concentration of known specific antibody of interest throughout

2. Wells are cut into the gel and filled with patient serum (the analyte, usually antigen).

3. Antigen diffuses radially (in all directions) from the well.

4. Where antigen meets antibody at optimal ratio (zone of equivalence) → precipitation occurs.

5. Precipitation forms a ring around the well 

6. The diameter of the ring = measure of antigen concentration.

7. Compare to a standard curve made from rings obtained with known antigen concentrations.

Radial Diffusion Errors

• Too much or too little serum (analyte/antigen) added

• Improper temperature, time allowed

• Damage to the gel

• Not as sensitive as ELISA

Electrophoretic Diffusion

Electrophoresis to speed up diffusion, uses electric field to separate molecules on basis of electrical charge

Immunoelectrophoresis

1. Separation of proteins by electrophoresis

2. Often Serum is used as source of antigen/analyte

3. Antibody of known specificity is placed in trough adjacent to antigen lane

4. Antigen/analyte and Antibody both diffuse

5. Precipitation bands form with complexes

6. Takes 18 hours to complete 

Immunofixation Electrophoresis

1. Separation of antigen/analyte by electrophoresis

2. Antibody placed on top of gel

3. Shorter diffusion distance = higher resolution 

4. Short time = 1 hour 

Example of Immunofixation Electrophoresis - Detection of patient Antibodies in serum

1. Here, patient Abs act as analyte to detect

2. Detect using Abs of known specificities

3. Serum loaded in 6 different lanes

4. Components separated by electrophoresis

5. Different known Ab added atop each lane

6. Ab to heavy chains for IgM, IgA, and IgG

7. Ab to κ and λ light chains

8. Ab to all serum proteins

9. Can detect under-, over-production of Abs

Agglutination

visible clumping of particles caused by combining with specific antibody

• Multiple antibody-binding sites; cross-linking to form lattice

Agglutinins

antibodies causing agglutination 

Aggultination reactions commonly involves

• larger antibody-binding components than precipitation such as

  • Red blood cells (RBCs)

  • Latex Beads

  • Bacteria

    • Ex. ABO blood group testing

Agglutination Steps

1. Sensitization

2. Lattice Formation

Sensitization

Antibody-antigen binding on one binding site

• This binding is reversible.

• No visible clumping yet.

Lattice Formation 

Cross-linking to form aggregates (visable)

• Stronger antibody-antigen binding (larger clumps)

IgM

stronger agglutinins than IgG

• Larger, more flexible, more antigen binding sites

• Agglutinate between 4-27 degrees C

THINK MOMS ARE STRONGER AND HAVE FLEXIBILITY TO MAKE TIME FOR YOU

IgG

Smaller - harder to cross-link larger antigen

• Less flexibility

• Agglutinate best between 30-37 degrees C

GRANDMAS ARE SMALLER AND LESS FLEXIBLE

IgG based reaction often require

additional antibody

• Smaller - harder to cross-link larger antigen 

• Additional antibody: Coombs reagent

• Binds Fc region of IgG to help make cross-linkages

Essentially:

• Used in IgG-based agglutination reactions, especially when IgG alone cannot cross-link antigens effectively.

• Binds to the Fc portion of IgG molecules, helping form lattices and produce visible agglutination.

Direct Agglutination

• When Antigens are naturally on the particle (usually a cell)

• E.g., testing patient Antibodies for known Antigen on bacterial cell

  • Dilutions of patient serum (Abs) used

  • Helpful when infectious agent is difficult to culture

  • Test for increases in Antibody titer over time

Hemagglutination

agglutination (clumping) of red blood cells (RBCs) by specific antibodies

ABO Typing used in Hemagglutination

Detect the presence or absence of A and B antigens on a patient’s RBCs.

How it works

1. Take patient RBCs.

2. Add:

   • Anti-A antibody

   • Anti-B antibody

3. Observe for visible clumping (agglutination).

Interpretation

• If anti-A antibody causes clumping → A antigen is present on RBC

• If anti-B antibody causes clumping → B antigen is present on RBC

• If no clumping occurs → that antigen is absent on RBC

Semiquantitative Methods

Test Tube and Slide Method

Test Tube Method

• Mix antibody (Ab) + red blood cells (RBCs) in a test tube

• Centrifuge to form a pellet at the bottom.

• Shake to see if/how much pellet is resuspended

Observations:

• One larger clump = Strong Positive

• Uniform resuspension, No clumps = Negative

• Gradations between these possible

Slide Method

Place Antibody + RBC on a slide and mix

• Larger clumps with clear fluid = Strong Positive

• Smaller clumps with cloudy fluid = Weak Positive

• No clumps with cloudy fluid = Negative

Passive Agglutination

• Uses particles (like latex beads) coated with antigen that is not naturally on the particle surface.

• Detects antibodies in patient samples

• Several infectious, autoimmune disease tests

Passive Agglutination (Steps)

1. Some kind of particle is coated with a specific antigen thats not naturally on surface

2. Mix particles with patient serum.

3. If patient contains antibodies to that antigen, visible agglutination occurs

Reverse Passive Agglutination

• Particles are coated with antibody, instead of antigen

  • Also uses Monoclonal Antibodies = Higher Specificity

• Detects antigens in patient samples

• Several infectious diseases tests

Reverse Passive Agglutination (Steps)

1. Particles are coated with specific antibody

1. Antibody bind beads via its Fc region, leaving Fab to capture antigen

2. Mix with patient sample

3. If antigen is present, visible agglutination occurs.

Both Passive and Reverse Passive Agglutination are

rapid and quicker treatments

Agglutination Inhibition - Basis

Competition for limited antibody-binding sites

• Uses Antigen-coated particles (ex. coated latex beads)

• Positive Reaction = No agglutination

• Some drug testing

Agglutination Inhibition - Patient Sample

May or may not have antigen of interest 

1. Sample is mixed with reagent antibodies specific to the antigen 

2. Antigen-coated particles added

3. No agglutination at end = Patient (+) for Agglutination

Sample is mixed with reagent antibodies specific to the antigen 

• Ag (+) Patient = Reagent antibodies bind to antigen

• Ag (-) Patient = Unbound antibodies

Antigen-coated particles added

Ag (+) Patient = No agglutination

• Antibodies already bound

• Can’t cross-link antigen-coated particles

Ag (-) Patient = Agglutination

• Free antibodies

• Can cross-link antigen-coated particles

Hemaagultination Inhibition - Basis

• Similar concept; RBCs are the particles

• Detection of Antibodies to some viruses

• RBCs have virus receptors (bind to viruses)

Hemaagultination Inhibition - Patient Sample

May or may not have antibodies to virus

1. Sample mixed with reagent viral antigen

2. RBCs added

3. Performed on dilution of patient serum

Sample Mixed with reagent viral antigen

• Infected patient = Antibodies bind to viral Antigen

• Non-infected patient = Free viral Antigen

RBCs added

• Infected patient = No hemagglutination

  • Viral Antigens already bound to patient Antibodies

  • Too few viral Antigens to link RBCs

• Non-infected patient = Hemagglutination

  • Free viral Antigens; were no Antibodies to bind them

  • Antigens link RBCs