Week 4: Serology agglutination

Serology

Study of components in the blood, especially antibodies

serum

• liquid portion of blood minus coag factors

• most frequently encountered specimen in immunologic testing

• separated from other components of a blood specimen via centrifuge

Serum vs plasma

blood specimen preparation and measuring

• ideally use fresh serum that hasn’t been heated

• however, for certain tests, complement may need to be inactivated

  • heat sample to 56C for 30 min

Blood storage

• between 2-8C for up to 72 hrs

• frozen at -20C or below

dilutions

• solute is the material being diluted

• diluent is the medium making up rest of solution

• relationship between the 2 is expressed as a ratio or fraction

• equations are used to determine:

  • total volume of a solution

  • amt of solute/diluent needed

sensitivity

• proportion of people who have a disease (or condition) and have a + tst

• indicates how small an amount can be measured and still produce a + test result

• sensitivity (%) = (TP/TP + FN) x 100

specificity

• proportion of people who DON’T have a disease and have a - test

• measures the substance that it is designed to measure, not interfering substance

  • specificity (%) = (TN/TN + FP) x 100

Test parameters

Antigen-antibody binding: affinity

• initial attraction force between a Fab site on an antibody molecule and an epitope or determinant site on an antigen

• strength of attraction depends on specificity of antibody for a particular antigen

avidity

• sum of attractive forces between an antigen and an antibody that keeps the molecules tgt

• involves the strength with which a multivalent antibody binds a multivalent antigen

• measure of overall stability of an antigen-antibody complex

law of mass action

• K= [AgAb]/[Ab[[Ag[

• free reactants are in equilibrium with bound reactants

• value of K depends on strength of binding between antibody and antigen

• higher value of K:

  • larger amt of antigen-antibody complex

  • more visible or easily detectable the reaction

Precipitation reactions

• involve combining soluble antigen with soluble antibody to produce insoluble complexes that are visible

• require antigen and antibody to have:

  • multiple binding sites for one another

  • equal relative concentration of each

precipitation curve: zone of equilvalence

• # of multi-valent sites of antigen and antibody are approx equal

• to be detectable, precipitation reactions must be run in th zone of equilvalence

Prozone

• antibody excess

  • antibody combines with only 1 or 2 antibody molecules

  • no cross-linkages are formed

  • false negatives reaction may occur as a result of high antibody concentration

  • if false- rxn is suspected, diluting out the antibody and performing test again may produce a + result

Postzone

• small aggregates are surrounded by excess antigen

• no lattice network is formed

• presence of small amt of antibody may be obscured, causing fn results

  • test is repeated about a week later with specimen to give time for further production of antibody

  • if test is neg again, unlikely that patient has antibody

Precipitation rxn techniques

• nephelometry - light scattered at an angle is measured

• radial immunodiffusion - antigen diffuses out into a gel that is infused with antibody, measurement of radius

• Ouchterlony double diffusion - both antigen and antibody diffuse out from wells and the lines of precipitate formed indicate relationship

• immunoelectrophoresis

• immunofixation electrophoresis

Radia immunodiffusion

• RID is a single diffusion technique

• antibody is in support gel, antigen in a well cut in the gel

• antigen diffuses out until the point of equivalence is reached (end-point method)

• square of the diameter is proportional to antigen concentration

Ouchterlony diffusion

• double-diffusion technique

• wells are cut in a gel, both antigen and antibody diffuse out radially

• line of precipitate forms where antigen and antibody meet

• 3 possible patterns: identity, partial identity, nonidentity

Immunofixation electrophoresis

• double diffusion technique

• unknown antigen is electrophoresed, and then antibody is applied directly to the gel

• precipitates form where antigen-antibody combination has taken place in gel

• technique is used with serum as the antigen to determine over- or underproduction of antibody types

Agglutination

• visible aggregation of particles resulting from combination with specific antibody

• 2 step process

  • sensitization (initial binding)

    • antigen and antibody unite through antigenic determinant sites

  • lattice formation (formation of large aggregates)

    • rearrangement of antigen and antibody bonds to form a stable lattice

• produced by antibodies called agglutinins

direct agglutination

• uses known bacterial antigens to test for presence of unknown antibodies in the patient

• can also occur when antigens are found naturally on a particle

• widal test

  • rapid screening test used to determine the possibility of typhoid fever

  • uses Salmonella O (somatic) and H (flagellar) antigens

• ABO typing

Passive agglutination

• used to detect:

  • RF

  • antibodies to Group A strep

  • antibodies to viruses such as rotavirus, CMV, rubella, varicella-zoster

  • antibodies to hepatitis viruses and HIV

• process

  • antigen is attached to carrier particle

  • agglutination occurs if patient antibody is present

Reverse passive agglutination

• used to detect microbial antibodies

• detecting soluble antigens

• process

  • antibody is attached to carrier particle

  • agglutination occurs if antigen is present in patient sample

Agglutination inhibition

• based on competition between particulate and soluble antigens for limited antibody-combining sites

• lack of agglutination = + rxn

indirect agglutination

• particles are coated with antigens not normally found on their surfaces

• agglutination indicates presence of patient antibody

hemagglutination inhibition

• RBCs spontaneously agglutinate if viral particles are present

• lack of agglutination is + test, indicating presence of patient antibody

Particle-counting immunoassay (PACIA)

• nephelometry

• involves a laser in optical particle counter to measure the # of residual non-agglutinating particles in specimen

• measures serum proteins, therapeutic drugs, tumor markers, certain viral antigens