1. Antigen-antibody reactions

2. Let’s start
The immune system is a system of biological structures and processes within an organism that protects against disease.
To function properly, an immune system must detect a wide variety of agents, from viruses to parasitic worms, and distinguish them from the organism’s own healthy tissue.

3. Antigens
Antigens are any substances that are capable, under appropriate conditions, of inducing the formation of antibodies and reacting specifically with the antibodies so produced.
These antigenic molecules may have several antigenic determinants, called epitopes, and each epitope can bind with a specific antibody. Thus, a single antigen can bind to many different antibodies with different binding sites

4. Chemical Nature of Antigens ((Immunogens
A. Proteins The vast majority of immunogens are proteins. These may be pure proteins or they may be glycoproteins or lipoproteins. In general, proteins are usually very good immunogens. B. Polysaccharides Pure polysaccharides and lipopolysaccharides are good immunogens. C. Nucleic Acids Nucleic acids are usually poorly immunogenic. However, they may become immunogenic when single stranded or when complexed with proteins. D. Lipids In general lipids are non-immunogenic, although they may be haptens.

5. Antibodies
An antibody is a protein produced by the body’s immune cells “ activated B-lymphocytes” when it detects a foreign antigen.
Classes of antibodies:
IgG: The main antibody in blood(70%), made in 2ry immune response and has a long half life”up to 20 years”.
IgM: accounts for(10%) of Igs, has a key role in 1ry immune response.
IgA: (20%) of Igs present in serum and secretions.
IgE: (0.001%), involved in allergy and parasitic infections.
IgD: (1%) present on the surface of B cells, so that it plays a role in induction of Ab production

7. Nature of antigen-antibody reactions
A. Lock and Key Concept  The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains
B. Non-covalent Bonds  The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds.
C. Reversibility Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible.

8. Affinity and avidity
Affinity  Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.
Avidity Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies.

10. Specificity and cross reactivity
Specificity refers to the ability of an individual antibody combining site to react with only one antigenic
Cross reactivity 
Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant
Sensitivity
Cross reactivity refers to the ability of an individual antibody to locate antigen even when it is greatly diluted.

11. Factors affecting measurement of antigen-antibody reactions
   The only way that one knows that an antigen-antibody reaction has occurred is to have some means of directly or indirectly detecting the complexes formed between the antigen and antibody.
The ease with which one can detect antigen-antibody reactions will depend on a number of factors.
1. Affinity  The higher the affinity of the antibody for the antigen, the more stable will be the interaction. Thus, the ease with which one can detect the interaction is enhanced.
2. Avidity  Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.

12. Factors affecting measurement of antigen-antibody reactions
3. Antigen to antibody ratio  The ratio between the antigen and antibody influences the detection of antigen-antibody complexes because the size of the complexes formed is related to the concentration of the antigen and antibody.
4. Physical form of the antigen  The physical form of the antigen influences how one detects its reaction with an antibody. If the antigen is a particulate, one generally looks for agglutination of the antigen by the antibody. If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble antigen-antibody complexes.  

13. Types of Antigen – Antibody Reaction
The types of antigen – antibody reactions are:
Precipitation Reaction.
Agglutination Reaction.
Complement Fixation.
ELISA – Enzyme Linked ImmunoSorbent Assay
Immunofluorescence.

14. Agglutination Agglutination is the clumping of particles.
Agglutination occurs if an antigen is mixed with its corresponding antibody called isoagglutinin.
This term is commonly used in blood grouping.

15. Agglutination This occurs in biology in several examples:
The clumping of cells such as bacteria or red blood cells in the presence of an antibody or complement. The antibody or other molecule binds multiple particles and joins them, creating a large complex. This increases the efficacy of microbial elimination by phagocytosis as large clumps of bacteria can be eliminated in one pass, versus the elimination of single microbial antigens.
Another example occurs when people are given blood transfusions of the wrong blood group.

16. Characteristics
Is the aggregation of particulate matter due to combination with specific antibody.
Takes place on the surface of the particle and thus antigen must be exposed and able to bind with antibody
Antigens may be:
On a cell (direct agglutination)
Attached to latex spheres (indirect or passive agglutination)
Agglutination reaction is aided by elevated temperature (37-56°C) and by movement which increases the contact between antigen and antibody.
Clear supernatant.
Clumps aggregate and settle as large visible clumps.

17. Steps in Agglutination
1- Sensitization Involves antigen-antibody combination through single antigenic determinants on the particle surface
Antibody molecules attach to their corresponding Antigenic site (epitope) on the red blood cell membrane. There is no visible clumping.

18. 2- Aggregative Stage (lattice formation)
Represents the sum of interaction between antibody and multiple antigenic determinants on a particle
Dependent on environmental conditions as well as the relative concentrations of antigen and antibody
Antibody molecules crosslink RBCs forming a lattice that results in visible clumping or agglutination.

19. Uses of Agglutination Reactions
Aid in the identification, by means of known antisera (serum containing antibodies specific for a given antigen), microorganisms cultured from clinical specimens.
Help estimate the titer of antibacterial agglutinins in the serum of patients with unknown diseases.

20. Types of Agglutination Reactions
Direct Agglutination
Passive Agglutination
Reverse Passive Agglutination
Agglutination inhibition
Hemagglutination-inhibition
Coagglutination/Conglutination

21. Direct agglutination
Occurs when antigens are found naturally on a particle (Serotyping of Salmonella)
e.g. identification of bacterial types , O antigen (somatic) - compact, fine and granular agglutination , H antigen (flagellar) - form a loosely woven network of clumped cells (coarse and floccular), called snowflake agglutination

22. Hemagglutination
Hemagglutination, is agglutination that involves red blood cells (RBCs). It has two common uses in the laboratory:
Blood typing
Quantification of virus dilutions in a Haemagglutination assay.

23. Blood typing
Blood type can be determined by using antibodies that bind to the A or B blood group antigens in a sample of blood.
For example, if antibodies that bind the A blood group are added and agglutination occurs, the blood is either type A or type AB.
To determine between type A or type AB, antibodies that bind the B group are added and if agglutination does not occur, the blood is type A.
If agglutination does not occur with either antibodies that bind to type A or type B antigens, then neither antigen is present on the blood cells, which means the blood is type O.

24. Blood typing

25. Viral hemagglutination assay
Hemagglutination phenomenon is almost commonly used for diagnosis of infection produced by some viruses.
The presence of virus in infected cell cultures can be detected by hemagglutination
The identity of the virus or of antibodies in a patient’s serum can be determined by specific inhibition of that hemagglutination.

26. Viral hemagglutination assay
The basis of the HAI assay is that antibodies to that particular virus (for example-influenza virus) will prevent attachment of the virus to RBC. Therefore hemagglutination is inhibited when antibodies are present.

27. Hemagglutination and hemagglutination inhibition

28. Passive agglutination
Employs particles that are coated with antigens not normally found on their surfaces.
Inert materials commonly used:
Bentonite
Colloidion
Latex particles
Colloidal charcoal
Passive agglutination tests have been used to detect rheumatoid factor and antinuclear antibody.

29. Reverse Passive agglutination
Reverse Passive agglutination - antibody rather than antigen is attached to a carrier particle.
Several kits are available today for rapid identification of such antigens from such infectious agents as group A and B streptococci, Staph. , Neisseria, and others.

30. Passive and reverse passive

31. Agglutination inhibition
Agglutination inhibition - based on competition between particulate and soluble antigens for limited antibody combining sites, and a lack of agglutination is an indicator of a positive reaction.

32. The classic example of agglutination inhibition is the early types of home pregnancy test kits included latex particles coated with human chorionic gonadotropin (HCG) and antibody to HCG
The addition of urine from a pregnant woman, which contained HCG, inhibited agglutination of the latex particles when the anti-HCG antibody was added; thus the absence of agglutination indicated pregnancy.

34. Coagglutination/Conglutination
Coagglutination/Conglutination - name given to systems using bacteria as inert particles to which antibody is attached (S. aureus).
The Fc region of antibody attaches to protein A of staphylococcal cell leaving the Fab region to combine with the antigen
Killed staphylococcal cells coated with antibody can be used to identify bacteria and detect soluble extracellular bacterial antigens in specimens and body fluids.

35. Antiglobulin mediated agglutination
The antiglobulin test (Coomb’s test) can be used to detect red cells sensitized with IgG antibodies
In order for agglutination to occur an additional of anti- antibody , which reacts with the Fc portion of the IgG antibody
This will form a "bridge" between the antibodies or complement coating the red cells, causing agglutination.

36. Instrumentation
Several systems were developed to increase sensitivity of results reading, many of these utilize turbidimetry
Particle Enhanced Turbidimetric Inhibition Assay (PETINA)
Particle counting immunoassay (PACIA)

37. Particle Enhanced Turbidimetric Inhibition Assay (PETINA)
This method uses the creation of light scattering particles to measure drug levels.
The latex particle-bound drug binds to the drug-specific antibody, forming insoluble light- scattering aggregates.
This causes an increase in the turbidity of the reaction mixture.
Non-particle- bound drug in the patient sample competes with the particle-bound drug for antibody binding sites, inhibiting the formation of insoluble aggregates.
Therefore the rate of increase of absorbance (hence the rate of the increase in turbidity,) is inversely proportional to the concentration of the drug.

39. Particle counting immunoassay (PACIA)
Involves measurement of the number of residual non-agglutinating particles in a specimen.
Latex particles are coated with whole antibody molecule, if antigen is present complexes will form and will screened out by counter .
An inverse relationship exists between the number of unagglutinated particles counted and the amount of unknown in the patient specimen.

40. Qualitative Agglutination Test
Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody.
The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen.

41. Quantitative Agglutination Test
Agglutination tests can also be used to quantitate the level of antibodies to particulate antigens.
In this test
one makes serial dilutions of a sample to be tested for antibody
and then adds a fixed number of red blood cells or bacteria or other such particulate antigen
and determines the maximum dilution, which gives agglutination.
The maximum dilution that gives visible agglutination is called the titer.
The results are reported as the reciprocal of the maximal dilution that gives visible agglutination. This can be done using a microtiter plate.

42. Quantitative Agglutination Test
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Pos.
Neg.
Titer 64 8
512
<2 32
128 4
Patient 1 2 3 5 6 7

43. Determining Antibody titer
Titer is the quantity of a substance required to produce a reaction with a given volume of another substance.
Antibody titer is the highest dilution of the biological sample that still results in agglutination, with no agglutination being observed at any higher dilution.
The term is used in serological reactions and is determined by preparing serial dilutions of antibody to which a constant amount of antigen is added.

44. Determining Antibody titer
Prozone
Equivalence Zone
Post
Zone
Serum Dilution
1:10
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
1:2560
1:5120
Antigen Conc. X
Agglutination – + ++
+++
++++

45. Advantages and disadvantages
Advantages of agglutination tests:
Low individual test cost.
Ability to obtain semi quantitative results.
Short time to obtain result.
Don’t need expensive instrument.
Agglutination of insoluble native antigens or antigen- coated particles simple to read with or without the aid of a microscope
Increased degree of sensitivity
Great variety of detectable substances
If the sample contain micro-organisms, it does not need to be viable

46. Agglutination Requirements
Availability of stable cell or particle suspension
Presence of one or more antigens close to the surface
Knowledge that ‘incomplete’ or nonagglutinating antibodies are not detectable without modifications, e.g. antiglobulin (Coomb’s) technic

47. Advantages and disadvantages
Disadvantages of agglutination tests:
It must kept in mind that agglutination reaction are screening tests only, and negative result doesn't rule out disease; the quantity of antigen or antibody may be below the sensitivity of the test system
May give false positive or negative results

48. False positive results in agglutination reactions
Overcentrifugation
Button is packed too tight and is difficult to resuspend.
Contaminated Equipment
Dust, dirt or fingerprints may cause cells to clump.
Autoagglutination
Test cells clump without specific AB present, mainly problematic with RBCs.
Delay in Reading Tests
Dried out Ag may look like agglutination
Saline Stored in Glass Bottles
Colloidal Silica may leach out and cause agglutination
Using plasma instead of serum, hemolysed or lipemic sample.
Cross reaction

49. False negative Undercentrifugation Inactive Reagents
Cells may not be close enough to interact
Inactive Reagents
May cause improper storage
Incorrect Incubation Temperature
May result in the lack of association for AGs and ABs
Insufficient Incubation Time
AGs and ABs may not have time for association
Prozone Phenomenon
Too much patient AB for amount of testing
Delay in Testing Procedure
AB may be eluted from RBCs

50. Determination of the titer of blood group cold antibody
Experiment 1
Determination of the titer of blood group cold antibody

51. Materials Isotonic saline.
Volumetric pipettes, or equivalent: 0.1- to 0.5- mL delivery, with disposable tips.
Red cells with the antigen to be tested.
The tested serum.

52. Procedure
Using 0.5-mL volumes, prepare serial twofold dilutions of serum in saline. The initial tube should contain undiluted serum and the doubling dilution range should be from 1 in 2 to 1 in 2048 (total of 12 tubes).
Place 0.1 mL of each dilution into appropriately labeled test tubes.
Add 0.1 mL of the 2% suspension of red cells to each dilution.
Gently agitate the contents of each tube; incubate for 15 min.
Centrifuge for 15 sec.
Examine the red cells macroscopically; grade and record the reactions.

53. Preparation of serial dilution8 7 6 5 4 3 2 1
Tube #
1/256
1/128
1/64
1/32
1/16
1/8
1/4
1/2
Dilution
500
Serum
Saline
Results

54. Interpretation
The titer is reported as the reciprocal of the highest dilution of serum at which 1+ agglutination is observed.

55. Preparation of serial dilution
Exercise
Preparation of serial dilution

56. Volumes used in dilutions mustn't be below 10ul or more than 1000ul .
Dilution is decreasing the concentration of a solution by a calculated factor using an approved diluent.
As well, dilution is used to prepare samples, buffers, and controls.
In serology tests it is Used to detect the titer of a specific Ab.
When a strong positive reaction is encountered, dilution should be made to detect the titer.
Volumes used in dilutions mustn't be below 10ul or more than 1000ul .

57. 1 express the volume of sample
for example: (1:4)
1 express the volume of sample
4 express the total volume( sample + reagent)
This is done by mixing 100 ul of sample with 300 ul of reagent.
Serial dilution :
100ul
sample
100ul
From 1
100ul
From 2
sample 1 2 3
300ul
saline
300ul
saline
300ul
saline
Total Dillution
1:4
1:16
=1/4 * 1/4
1:64
=1/4 * 1/16
Dillution of tube 1
Dillution of tube 2

58. Another example on serial dillution :
100ul
sample
100ul
From 1
100ul
From 2 1 2 3
sample
900ul
saline
900ul
saline
900ul
saline
Total Dillution
1:10
1:100
= 1/10 * 1/10
1:1000
=1/10 * 1/100
Dillution of tube 1
Dillution of tube 2

59. 9 : 250 9:25 * 1:10 9:250?? It can be done only by serial dilution…
9:25 * 1:10
90ul
100ul
From 1 1 2
sample
160ul
900ul
Total Dillution
9:25
9:250
1:10