Precipitation reaction

1. Precipitation reaction

2. Precipitation Serological Tests • One of the easiest of serological tests • Soluble Ag & Ab interact and form a lattice that develops into a visible precipitate. • Occur best when antigen and antibody are present in optimal proportions (Equivelance). • Antibodies that aggregate soluble antigens are called precipitins.

3. no precipitate is formed if an Ag contains only a single copy of each epitope Lattices or large aggregates Ag / Ab Precipitation reactions • Polyclonal antibodies can form lattices, or large aggregates. • However, monoclonal antibody can link only two molecules of antigen and no precipitate is formed. • Precipitation occurs in two media; liquid or gel

4. Precipitation in Liquid

5. In liquid: • Antigen – Antibody reaction perform by placing a constant amount of antibody in a series of tubes and adding increased amount of antigen. • Antigen –Antibody reacts together resulting in precipitation. • Plotting the amount of precipitate against increasing antigen conc. Yeilds a precipitation curve.

6. Precipitation curve Plots of the amount Ag/Ab complexes precipitated when increasing Ag concentrations are added to constant concentration of Ab. It reveals 3 zones: Zone of antibody excessProzone precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant Zone equivalence Maximal precipitation in which antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant; Zone of antigen excess Postzone Precipitation is inhibited & Ag. not bound to Ab. can be detected in the supernatant

7. Precipitation Curve Equivalence – Lattice formation Ab excess Ag excess

8. Precipitation in Solution Precipitation reactions in fluids follow the same roles of precipitation curve zones.

9. Precipitation in liquid a)Bottom Precipitate Occurs when Soluble Ag interact with soluble Ab and form a visible precipitate that give bottom ppt after centrifugation.(Widal test)

10. Precipitation in Solution (cont.) • b)Ring Precipitate (Ring Test) (Tube Precipitation test) • Involve soluble antigens with antibodies in tubes (test or Capillary tubes). • Layer Ag over Ab • Precipitate occurs at the interface of the two reagents, forming a ring. • Simplest test • Qualitative Test Tube reaction Capillary Tube reaction Figure 18.4

11. Measurement of precipitation by light scattering 1- Turbidometry 2- Nephlometry

12. Immunoturbidimetry • Turbidimetry is an analytical technique that uses light scattering, a physical phenomenon resulting from the interaction of light with particles in solution, to measure the concentration of particles. • The photometric signal is generated by a decrease in light intensity as a direct consequence of increasing turbidity in the reaction well. • Immunoturbidimetry uses the classical antigen-antibody reaction. The antigen-antibody complexes are particles which can be optically detected by an photometer.

13. Nephelometry • Is a technique used in  immunology to determine the levels of several blood plasma proteins. • For example the total levels of antibodies isotypes or classes:IgM, IgG, and IgA. •  It is important in quantification of free light chains in diseases such as multiple myeloma. • Quantification is important for disease classification and for disease monitoring once a patient has been treated.

14. Nephelometry Principle: • It is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. • The amount of scatter is determined by collecting the light at an angle (usually at 30 and 90 degrees).

15. Principle: • Antibody and the antigen are mixed in concentrations such that only small aggregates are formed that do not quickly settle to the bottom. • The amount of light scatter is measured and compared to the amount of scatter from known mixtures. • The amount of the unknown is determined from a standard curve. • Nephelometry can be used to detect either antigen or antibody, but it is usually run with antibody as the reagent and the patient antigen as the unknown.

16. Types of nephelometry • In the Immunology Medical Lab, two types of tests can be run: • End point nephelometry • Kinetic (rate) nephelometry".

17. End point nephelometry • End point nephelometry tests are run by allowing the antibody/antigen reaction to run through to completion (until all of the present reagent antibodies and the present patient sample antigens that can aggregate have done so and no more complexes can form). • Unfortunately, the large particles will fall out of the solution and cause a false scatter reading, thus kinetic nephelometry was devised.

18. kineticnephelometry • In kinetic nephelometry, the rate of scatter is measured right after the reagent is added. • As long as the reagent is constant the rate of change can be seen as directly related to the amount of antigen present.

19.  Immunologic Assays Performed by Nephelometry • Acid α1-glycoprotein • Albumin • α1-Antitrypsin • α2-Macroglobulin • C1 esterase inhibitor (C1 inhibitor) • C3 • C3b inhibitor (C3b inactivator) • C3PA (C3 proactivator, properdin factor B) • C4 • C6 • C7 • Transferrin • C8 • Ceruloplasmin • Complement components (C1r, C1s, C2, C3, C4, C5, C6, C7, C8) • C-reactive protein (CRP) • Cryofibrinogen • Cryoglobulins • Haptoglobin • Hemopexin • Immunoglobulins • Properdin factor B

20. Turbidimetry Nephelometry • precipitation in solution • measurement of scattered light (proportional to number of insoluble complexes • standard curve • precipitation in solution • measurement of light extraction (precipitate absorption) • standard curve

21. Advantages Of Turbidimetry And Nephelometry • Measurement of serum proteins‘ concentration (immunoglobulins, acute-phase proteins, complement components C3, C4, transferrin, albumin,…) • Rapid • Fully-automated techniques • For large quantity of samples

22. Precipitation in gel

23. Precipitation in gel • Immune precipitates can form not only in solution but also in an agar matrix. • When antigen and antibody diffuse toward one another in agar, or when antibody is incorporated into the agar and antigen diffuses into the antibody-containing matrix, a visible line of precipitation will form. • As in a precipitation reaction in fluid, visible precipitation occurs in the region of equivalence, whereas no visible precipitate forms in regions of antibody or antigen excess.

24. Passive Immunodiffusion • Reactions of antigens and antibodies in agar gel. • Migrate towards each other and where they meet in optimal proportions form a precipitate. • “Passive” because they are allowed to react to completion with no enhancements such as an electrical charge applied. • Factors Affecting Rate of Diffusion: • Size of the particles. • Temperature • Gel viscosity and hydration • Interaction of reactants with gel

25. Types of immunodiffusion • Two types of immunodiffusion reactions can be used to determine relative concentrations of antibodies or antigens • Radial immunodiffusion (the Mancini method) • Double immunodiffusion(the Ouchterlony method) Both are carried out in a semisolid medium such as agar.

26. Radial immunodiffusion • In radial immunodiffusion, an antigen sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of an antiserum. • As the antigen diffuses into the agar, the region of equivalence is established and a ring of precipitation, a precipitin ring, forms around the well The area of the precipitin ring is proportional to the concentration of antigen.

27. Two methods • Endpoint – allows reaction to go to completion. • Kinetic – measurements taken at a specific time before zone of equivalence is reached. • This is a QUANTITATIVE technique which will give the actual concentration.

28. Radial Immunodiffusion

29. Radial Immunodiffusion Precipitin Rings By comparing the area of the precipitin ring with a standard curve (obtained by measuring the precipitin areas of known concentrations of the antigen),the concentration of the antigen sample can be determined. A B C a b c Standards Samples Standard Curve

30. RID Sources of Error • Over or under filling the well. • Spilling sample onto the outside of the well. • Nicking the well with the pipette tip. • Improper incubation time or temperature. • Incorrect measuring of the wells.

31. Ouchterlony Gel Diffusion • In the Ouchterlony method, both antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration gradient. • As equivalence is reached, a visible line of precipitation, a precipitin line,forms.

32. Ouchterlony Gel Diffusion • Holes punched in agar. • Known antibody or antigen added to center well. • Known sample added to outer well. • Unknown sample added to outer well next to unknown sample. • Wait for bands to form. • This is a QUALITATIVE technique, simply determines the presence NOT the concentration.

33. Ouchterlony Immunodiffusion

34. Ouchterlony - Identity • Precipitation appears as a continuous line in the form of an arc between the two outer wells and the center well. • There are no spurs at the angle and this type of reaction is termed a band of identity.

35. Ouchterlony – Partial Identity • If a solution with antigens X and Y is placed in well 1, a solution with antigen X only is placed in well 2, and antiserum containing antibodies specific for both X and Y is placed in well 3, a reaction similar to that appearing in Fig. 2 will occur. Notice that there is a spur reaction towards the XY well. • This indicates that the two antigenic materials in wells 1 and 2 are related, but that the material in well 1 possesses an antigenic specificity not possessed by the material in well 2. • Such a reaction with spur formation indicates partial identity

36. Ouchterlony – Non-Identity • If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines as in Fig.

38. Electro-Immnodiffusion Techniques • Immunodiffusion can be combined with electrical current to speed things up. • Electrophoresisseparates antigen molecules according to differences in their electrical charges and molecular weight then specific antibodies diffuse and react with separated antigen forming precipitin bands. • Electrophoresis is a technique which separates molecules using electrical current. • For immunolectrophoresis antigen and antibody migrate through gel faster. • Can be single or double diffusion.

39. Electro-Immnodiffusion • Immunoelectrophoresis (IEP) • Rocket Electroimmunodiffusion (EID) • Counterimmunoelectrophoresis (CIEP) • Immunofixation

40. Immunoelectrophoresis • Two-dimensional immunoelectrophoresis. • Antigens are separated on the basis of electrophoretic mobility. • Ab is placed in channel cut in the agar • The second separation is run at right angles to the first which drives the antigens into the antiserum-containing gel to form precipitin peaks; the area under the peak is related to the concentration of antigen.

41. - + Ag Ag Ab Ag Ab • Interpretation- Precipitin arc represent individual antigens • Qualitative - Relative concentration

43. Imunoelectrphoresis (cont.)

44. Rocket Immunoelectrophoresis • Adaptation of radial immunodiffusion (RID). • Antibody incorporated (mixed) into the gel. • Antigen added to wells. • Apply electrical current and antigen will move forward and will bind to antigen. • Height of precipitin band related to concentration of antigen. • Much faster than RID.

45. Rocket Immunoelectrophoresis Antigen is electrophoresed into gel containing antibody. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

46. Reactions occurrbetween migrating Ag’s and Ab’s during electrophoresis (Ag and Ab migrate toward each other by electrophoresis) Used only when Ag and Ab have opposite charges Pairs of wells are punched in agarose plates in which Ag is placed in one well of each pair and Ab in the other. Following electrophoresis, precipitin lines will be visible between the wells of a pari of wells of matching specificity. Qualitative Its major advantage is its speed. - + Ab Ag Counter immunoelectrophoresis

47. Immunofixation Electrophoresis • Immunofixation Electrophoresis (IFE) combines electrophoresis with immunoprecipitation. • This technique may be used to identify and characterize serum or urine proteins. • In IFE, proteins of sample are first separated by electrophoresis on a support (agarose) according to their charge and after that the medium is overlaid with monospecific antisera reactive with specific protein - antigen. • If the antigen is present a characteristic immunoprecipitin band will be formed.

48. patient A healthy control patient A: paraprotein in class IgG (k) healthy control: negative

49. Immunofixation Electrophoresis • Test is frequently ordered to identify moncolonal proteins. • May be done on urine or serum depending upon what doctor suspects. • Multiple myeloma • Production of large amount of specific protein. • Will be excreted in urine

50. Electrophoresis Sources of Error • Applying current in wrong direction. • Incorrect buffer pH • Incorrect timing • Incorrect current applied. • Concentration of reactants must be appropriate.