• once Ab have been made they can be used for a variety of diagnostic assays in the lab to detect the presence or absence of a particular antigen or bacterium or virus in a sample
  • For example, the use of Ab specific for the myriad of RBC antigens has made routine transfusion possible as well as successful organ transplantation
  • The reaction of antigen with the homologous Ab is a two-stage phenomenon
  1. Initial or primary binding can occur invisibly
  2. Secondary manifestation of that interaction is dependent on several factors like:

a.     isotype of the Ab

b.     valence of Antigen

c.      form of the antigen

  • The type of assay depends on these factors

Primary Interactions between Ab and Ag

  • Ag-Ab interact via multiple weak, non-covalent rxns.
  • Ab-Ag complexes can be readily dissociated by:

1.     low or high pH

2.     High salt concentrations

3.     chaotropic ions

  • Law of Mass Action- can be used because the reaction is noncovalent; measures the strength of the primary interaction between one paratope and its epitope.
  • The binding of a univalent epitope such as a free Hapten (H) to a paratope can be represented by the equation———– Ab + H Û AbH
  • Association constant———— K = [AbH]/[Ab][H]
  • this K value will represent an intrinsic association constant for monoclonal Ab and an average association constant for polyclonal Ab

Affinity and Avidity

  • Affinity- the intrinsic interaction between epitope and paratope
  • Avidity- enhancement due to multivalency

Secondary Interactions between antibody and antigen

Agglutination Reactions

Agglutination- aggregation of insoluble particles

titer- highest dilution of that serum that will agglutinate a standard amount of cells

Prozone

  • high concentrations of Antibodies can totally saturate all epitopes on each cell so that no cross linking occurs
  • Dilution process allows for effective agglutination

Zeta Potential

  • An electrical potential between two like charged particles prevents them from physically associating
  • IgM may be large enough to overcome thus zeta potential; IgG’s fab arms have too short a distance between them

Coomb’s test

  • can overcome zeta potential by using a second layer of Ab to bridge cells
  • if the RBC has some IgG Ab on it, an antiglobulin antiserum can be added (a serum containing antibodies specific for the Fc region of IgG)
  • this can now cross-link the IgG Ab previously bound to the cell thereby agglutinating the red cells

Direct Coomb’s Test

  • Antiglobulin serum is added to a patient’s RBCs that are already suspect to having Ab already bound
  • positive agglutination is diagnostic for the presence of anti-Rh Ab bound to the RBCs.

Indirect Coomb’s test

  • used to detect presence in serum of a non-agglutinating Ab
  • For example, serum from a pregnant patient suspected of having circulating IgG anti-Rh Ab is mixed with Rh+ RBCs.
  • Antiglobulin is then added to this mixture
  • Positive agglutination is then diagnostic for the presence of anti-Rh in patient serum.

Passive Agglutination

  • a way to use agglutination assays for Ab specific for soluble Antigens (Ex. thyroglobulin)
  • The antigen is is attached to something particulate such as micro-latex beads or RBCs
  • The sera containing suspected Ab for the antigen can then be titered in a standard agglutination format
  • Passive hemagglutination- if RBCs are used in the assay as the antigen carrier

Precipitation Reactions

  • Quantitative (precise)
  • constant quantities of Ab in each tube and increasing amounts of Ag.
  • will tell the exact concentration of Ab in a serum
  • Value- to be able to standardize sera that can then be used for standards in other Ag-Ab assays
  • Procedure
  1. Incubation until equilibrium is reached
  2. Centrifugation (precipitate can then be saved for analyzation; supernatants are saved also for further testing)
  3. Each precipitate is analyzed for total protein content which is then plotted against the amounts of added antigen
  4. The supernatants are then each individually split into two equal aliquots
  5. To one aliquot, Ag is added, and to the other aliquot, Ab is added. If there is any free Ab or Ag present it will show further reaction
  6. We can then calculate the concentration of Anitbody in the serum and furthermore if we know which specific Ab it is (Ex. IgG) we can count the molar concentration of Ag-Ab in the precipitate just by simply knowing the molecular weight of the Ab (IgG= 150000 Daltons)

Precipitation Reactions in Gels

  • Qualitative
  • The gel matrix slows down the rate of diffusion of reactants and holds the precipitate in the gel web so that it is effectively immobilized for visual analysis.
  • Several quantitative and qualitative methods are available today for analysis of hormones, enzymes, toxins, and for immune system products
  • Know which of the following are qualitative as well as quantitative

Ouchterlony Double Diffusion Assay

  • Very inexpensive; can compare the relatedness of two antigens
  • QUALITATIVE
  • Double diffusion assay because both the Ag and the antiserum are diffusing
  • Ag and Ab slowly diffuse towards one another
  • A positive result is when there is a thin opaque line or band that forms in the agar at right angles to a line connecting the centers of the two wells
  • The line’s presence is qualitative for the presence of either antibody in the antiserum or for the presence of Antigen
  • COMPARISON OF Similar ANTIGENS
  1. 3 wells equidistant from one another
  2. If the 2 antigens are the same, there would be two lines in a V shape near the Ab
  3. Called a reaction of identity or a reaction of caolescence
  • COMPARISON OF different ANTIGENS
  1. An X-shaped pattern is formed as if there were two individual independent reactions.
  2. This is a reaction of non-identity or a reaction of non-coalescense
  • COMPARISON OF similar but not identical ANTIGENS
  1. The lines that form look like lambda  ( l )
  2. called a reaction of partial identity or a reaction of partial coalescence

Immunoelectrophoresis

  • QUALITATIVE
  • Variation of the Ouchterlony Double Diffusion in gel assay
  • designed to analyze complex protein mixtures containing many different antigens
  • Procedure
  1. Small wells are made on an agar-coated alide and the sample is pipetted into the wells
  2. The slide is the subjected to an electrical field in the long direction
  3. After electrophoresis, a trough is cut at the bottom of the slide and the appropriate antiserum is added. (The serum is whole serum- having about 30 or more proteins)
  4. After incubation the precipitations are read along the slide where the electrical field has pulled the Antigens.
  • Clinical relevance: used for diagnosis of conditions where certain proteins are suspected of being absent or overproduced

Radial Immunodiffusion

  • QUANTITATIVE
  • have to have a standardized anti-serum or standard antigen
  • Usually used as a follow up to immunoelectrophoresis to get exact quantitation of an overproduced enzyme, Ig, hormone, or where an Ag is present but in unusually low concentrations
  • Procedure
  1. The standard antiserum in incorporated into the agar
  2. Small wells are cut and precise quantities of standard antigen are placed into the wells
  3. There are several wells on each slide and different concentrations of standard antigens are added along with the unknown samples to be analyzed into separate wells
  4. Incubation
  5. They are then analyzed by measuring the ring around the wells.
  6. The results of the known antigen standard are then plotted (Ring diameter vs. Ag concentration) on semi-log plots aith Ag conc. on the log scale

Nephelometry

  • accurately measures quantities of the Ig class in serum
  • QUANTITATIVE
  • Procedure
  1. proteins in the sample react with specific antibody
  2. the mixture is placed in a tube and inserted into the Nephelometer
  3. The Nephelometer measures the scattered light that passes through the sample
  • Quantitative method using liquid-phase precipitation principles
Immunoassays
  • A standard curve of inhibition can be generated using precisely known amounts of unlabeled antigen and then test samples containing unknown concentrations of Antigen can be analyzed and simply read of the standard curve to find the concentration of Ag in the unknown
Solid Phase Immunoassays
  • groups of assays in which the antigen or antibody is coated on the surface of a plastic microplate and sensitive indicators such as radioactivity or enzymatic action are used to detect the presence of Ab or Ag
  • The are 5 types in two groups: soluble or cellular

Soluble Antigens

Radioimmunoassay

  • many formats
  • Procedure to detect antigen
  1. Free antigen is coated onto the surface of plastic plates
  2. Excess antigen is rinsed out of the wells with buffer soln
  3. the remaining plastic surface is then blocked by adding an irrevelant protein soln and washing
  4. Antiserum is added to the plate, incubated, then washed out
  5. This leaves the plate with the Ab bound to the Ag (the Ag is noncovalently bound to the plastic)
  6. A radioactive indicator is added which recognized the Ab but not the Ag
  7. The indicator is then read
  • Clinical Relevance: Quantitates the amounts of specified antigens in the body fluids
  • Can measure Renin, Gastrin, Parathyroid Hormone, GH, Urine Microalbumin, Vit B12, and Folate

Enzyme linked Immunoadsorbent Immunoassay (ELISA)

  • Similar to the radioimmunoassay except without the radioactivity
  • Instead the indicator is coupled to an enzyme molecule which converts substrates to a colored product
  • The colored product can then be detected spectrophotometrically due to the color change

Cellular Antigens

Immunofluorescence

  • finds out if a particular antigen is on or in the cells of a particular tissue
  • Uses flourescence methods
  • Direct Immunofluorescence- the antibody specific for the antigen is directly labeled with the fluorophor
  • Indirect Immunofluorescence- Two-step method similar to Coomb’s reaction; First the unlabeled Ab specific for the antigen in question is reacted with the tissue, then the slide is flooded with the fluorophor. This is more sensitive then the Direct method; used to detect anti-DNA Ab in Lupus.

Fluorescence Activated Cell Sorting (FACS) Analysis

  • used to identify, and sometime purify, one cell subset from a mixture of cells
  • used to measure CD4+ cell numbers in AIDS patients to follow disease progression
  • Procedure

1.     Fluorescent labelled Ab is mixed with the cells

2.     The excess is removed and the celss are then passed through a narrow aperture where a laser beam is focused

3.     As a cell passes by the beam, if it has the flourescent Ab bound, it emits a light at a characteristic wavelength for the particular fluorophor used which can be detected by a detector

4.     Each cell is then isolated into a microdroplet and the detector signals an electrode that induces an electrical charge in the droplet

5.     This way droplets with fluorescent cells can be put in one container while those that don’t have the fluorophor bound are placed into another container.

Monoclonal Antibodies

  • Due to cross-reactivity, it is sometimes of great advantage to have a homogeneous antibody preparation that is specific for only a single epitope and with high affinity
  • Two dudes devised a method for making murine antibodies that are monoclonal(all Ab are derived from a single precursor plasma cell) so that all Ab in the preparation are identical and derived from the same original clone
  • Clinical Relevance:
  • Most ELISA and RIA kits used in the clinical labs use monoclonal Ab
  • The method is also used for making T cell hybridomas which are valuable for the large scal production of several T cell-derived lymphokines that are used as Ag in diagnostic kits and therapeutically.