Lecture 2/26

• • Fractionation/ID/Quantitation - plasma proteins separated based on size
• • A/G ratio: 53-65% of all plasma/serum proteins are albumin
• • “Salting out” - aka salt fractionation; globulins are precipitated out in a sodium sulfate solution (NaSO4)
  • Albumin stays in solution
  • Not commonly used any longer
• common dyes used in dye binding method include methyl orange, HABA, BCG, and GCP (4)
• HABA - 2-4’ hydroxyazobenzene benzoic acid
• BCG is bromocresol green
• BCP is bromocresol purple
• Fractionation method

  • Total globulins - glyoxylic acid in an acid medium can be used
  • Cu2+ ions will produce a purple color
  • Occurs as glyoxylic acid binds to tryptophan in globulins
• • Electrophoresis - Separates proteins on the basis of electric charge densities
  • Gives a percent of total protein rather than specific quantities in grams
• • pH of the buffer must be greater than the pI - therefore protein will have negative charge
• (pI) isoelectric point - pH at which protein has no charge
• • The farther the pH is from the pI, the faster the proteins will move
• • Cations (positive charge) move towards the cathode (negative charge)
• • Anions (negative charge) move towards the anode (positive charge)
• • Electric field strength or the voltage 
  • Increased charge causes increase in movement
  • Too much voltage creates too much heat which denatures the proteins
• electrophoresis

  • smaller molecules move faster through gel
• • Temperature - electrophoresis
  • Heat causes greater movement (to a point)
  • Too much heat denatures proteins 
• • Buffer characteristics - electrophoresis
  • pH 
  • Ionic strength
• • Time - electrophoresis
  • Longer time periods produce more movement
  • If run too long, proteins will travel off of gel and into buffer
• • Electroendosmosis - electrophoresis
  • Interference by a medium with a net charge 
  • Polyacrylamide and agarose do not carry a net charge (from literature)
  • However, because of electroendosmosis, gamma globulins migrate backwards!
• beta-gamma globulins are really fast moving gamma globulins
• • Hi-resolution SPEP
  • high voltage with cooling
  • Uses more concentrated buffer 
  • Can produce up to 12 zones 
  • High res SPEP is ordered to detect unusual bands
  • however, is uncommon - often, unusual proteins we’re looking for are detectable via immunoassays
• Immunoassays

  • Antigen binds to antibody in vitro
  • Known antibodies used to detect or quantify antigens or transport proteins
  • Known antigens used to detect presence of antibodies (e.g. serology - looking for immunity or previous exposure by looking at antibodies present)
• • antigen/antibody interaction - strength of reaction increases as antigen:antibody ratio is 1:1 or close to 1:1
• • Prozone - antibody excess
• • Zone of equivalence - antigen:ab ratio is 1:1
• Postzone - antigen excess
• • Hook effect - ability to manipulate antibody:antigen ratio until it peaks and then starts to weaken (once it starts to weaken, stop diluting)
• types of immunoassay include double diffusion, radial diffusion, immunofixation, rocket electrophoresis, and isoelectric focusing
• • Double diffusion - semisolid agar plate with wells cut out
  • Aka Ouchterlony reaction; found in 1948
• Double Diffusion

  • Pt serum absorbs in middle and radiates outward
  • Antibodies on periphery will radiate outward
  • zone of precipitation will form where Ab-Ag meet
  • occurs at zone of equivalence
• Double diffusion - alternative method

  • known antigen or pathogen in middle; known antibodies on one side, patient serum on other sides of well 
  • used to detect antibodies associated with autoimmune disease
• radial diffusion

  • impregnate the agar with antisera or antibodies
  • patient sample is added after well is cut in agar
  • at zone of equivalence, get reaction ring of precipitate around well
• Two resulting methods for radial diffusion are end point or Mancini method and Fahey-McKelvey method
• Radial diffusion

  • End point or Mancini method
  • Run for 2-3 days
  • Looking for low levels of antigen in patient
• Radial diffusion

  • Fahey-McKelvey method
  • Run fo 18 hours
  • A kinetic methods