MODULE 2: PRECIPITATION AND AGGLUTINATION

OUTLINE
I Precipitation Tests and Assays
A Fluid-Phase Precipitation Tests
i Nephelometry
ii Turbidimetry
B Methods in Measuring Precipitation
C Precipitation by Passive Immunodiffusion Tests
i Radial Immunodiffusion (RID)
ii Oudin Test
iii Ouchterlony
ii. TURBIDIMETRY
iv Double Linear Diffusion • Detect the presence of bacterial growth
D Precipitation by Electrophoretic Techniques • Measures the cloudiness / turbidity of a solution
i Immunoelectrophoresis (IEP) • Measures the decrease in light intensity in a solution of
ii Counterimmunoelectrophoresis (CIE)
antibody-antigen complexes
E Precipitation Reactions/Precipitation Curve
II Agglutination Test o The lower the light intensity, the higher the
A Agglutination Tests concentration of complexes
i Hemagglutination Test • Uses transmitted light and the measurement is
ii Direct Agglutination Test (DAT) spectrophotometer
iii Indirect Agglutination Test or Passive Agglutination • Measurements are made at 180 degree from the incident
B Antiglobulin Technique light beam
C Viral Neutralization
i Neutralization
ii Opsonization
D Complement Fixation

• In microbiology — when preparing bacterial suspension —

I. PRECIPITATION TESTS AND ASSAYS once there is a presence of turbidity, it is suggestive that it
• Precipitation: Soluble antigen + antibody in equal and has bacterial growth.
optimal proportions • In antibiotic susceptibility testing, you compare the
o Process of forming insoluble complexes/ solid masses bacterial suspension to McFarland standard through
o Simplest method for the detection of antigen-antibody cloudiness/turbidity.
• Measurement of Precipitation in Fluid Medium
o Principle: B. METHODS IN MEASURING PRECIPITATION
▪ Soluble antigen + antibody (in proper proportions) 1. In Liquid: Ring Test
→ Visible precipitate o Ascoli test
▪ Lattice Formation (antigen binds with Fab sites of 2 ▪ For Bacillus anthracis
antibodies) o Lancefield flocculation
o Soluble antigens interact with antibodies and form a ▪ Slide flocculation: VDRL
lattice that eventually develops into a visible precipitate ▪ Tube flocculation: Kahn test for syphilis; not
o Precipitin ring test is performed in a small tube. performed anymore
2. Gel Diffusion/Immunodiffusion
A. FLUID-PHASE PRECIPITATION TESTS o Oudin
• Measurement of Precipitation by Light Scattering o Oakley-Fulthorpe
(Optical Methods) o Radial
a. Turbidimetry o Ouchterlony
b. Nephelometry o Immunoelectrophoresis
o Electroimmunodiffusion
▪ Rocket
▪ CIE
▪ Laurell’s
o Immunodiffusion is affected by:
▪ Size of particle
- The bigger the particle, the slower it moves
▪ Temperature: 40-45°C
▪ Gel viscosity and hydration
- Gel (Agar): Agarose gel

i. NEPHELOMETRY
• Scattered (reflected) light is proportional to number of
insoluble complexes
• Light is passed through suspension
o More light refracted = more sensitive
• Deals with measurement of intensity of scattered light;
usually measured at the right angles to the incident light
beam
o Example:
▪ Complement component concentration
▪ Antibody concentration
• Quantitative determination of complement factors and
immunoglobulins
DARIO, GRANADA, LAGUD, PRIETO, TRESIANA
C. PRECIPITATION BY PASSIVE
IMMUNODIFFUSION TESTS
• Immunodiffusion procedures are carried out in an agar gel
medium
• The precipitate is easily seen in gels yield visible precipitin
lines
• But no visible precipitate forms in regions of Ab or Ag
excess.
i. RADIAL IMMUNODIFFUSION (RID)
• Single diffusion, double dimension
• Gel containing known antibodies with small holes cut as
wells.
o Sample containing the antigen is placed in the well
(labeled) with the plate in the normal position.
o Afterwards, the plate is placed in the incubator in its
normal position as well.
▪ If turned upside down, the antigen will spill.
3
 MODULE 2: PRECIPITATION AND AGGLUTINATION

o After 24 hours or depending on what method was • Antibody in the agarose gel and the antigen solution is
used, there will be a zone of inhibition or also known layered over it.
as ring which is the one measured. • If there is an antigen-antibody reaction, the antigen
• Antibody is uniformly distributed in the gel - Agarose gel diffuses towards the agar gel and will form a precipitin
• Line of precipitation forms in a circle around the well band.
(Precipitin line)
• Used to determine the concentration of an antigen in a iii. OUCHTERLONY
biological fluid
• Double diffusion, double dimension
o Diameter (size) of the ring/zone of inhibition is directly
proportional to the concentration of the antigen in the • Also called Double Radial Diffusion
sample • Most widely used
o Example: • Used for comparing antigens
▪ Salmonella typhi test (Widal) • Usually, this is the first generation that was used for
▪ IgM HbsAg.
▪ IgG • Qualitative test
▪ C4 • Wells are cut in gel with multivalent antibody is placed in a
▪ C3 center well and antigen is remaining wells; both antigen
▪ Transferrin and antibody is moving
• Two methods: • Precipitation lines that form will identify relationship
1. Fahey and McKelvey/Kinetic method between antigens
▪ Diameter is proportional to log of the • Example:
concentration; measures diameter before the o Elek’s gel precipitation
completion of reaction o Test for Corynebacterium diphtheriae
▪ Reading time: 18 hours • Diffusion patterns: after 24 hours
▪ Can be performed in multiple measurements o If you are only comparing a few antigens, then you can
- To monitor the activity of the whole test use a small plate. But if there are many, it is better to
2. Mancini/Endpoint method use a big plate.
▪ IgG: 24-48 hours o Agarose gel is always used.
▪ IgM: 50-72 hours a. Identity:
▪ Square of diameter is proportional to the - precipitation lines meet but do not cross, no
concentration/measures diameter after spurs, indicating no relationship between the
completion of reaction (endpoint method) antigens; smooth curve; antigen in the sample
▪ Only one measurement is performed/ measured is same as known antigen
usually at the end of the test - Positive reaction: fusion of two lines
forming an arch meaning the antigen present
have the same epitopes/antigenic determinants
b. Non- Identity
- precipitation lines formed will crisscross, as
an X, indicating no identical epitopes and no
identity between antigens; antigen in the
sample is different from known antigen
- Demonstrates two separate reactions, that’s
why they crossed; different antigens, different
antigenic determinants
• Like Mueller Hinton Agar (sensitivity testing) - Positive Reaction: no formation of arch;
• Can use small or big plate where we will put a hole antigen do not have the same determinant
• Sources of errors: c. Partial Identity
o Overfilling and underfilling the well - precipitation lines formed will appear partially
o Spilling sample outside of the well crossed, as a line with a spur formation on the
o Nicking the well end, indicating cross reactivity between the
▪ there are scratches or the agar is cut antigens
o Improper incubation time or temperature - antigen in the sample has some similarities with
known antigen
ii. OUDIN TEST - Positive result: fusion of lines but with a
• Single diffusion, single dimension spur; meaning the antigen in the sample have
• Most simple immunodiffusion test similarities with the known antigen
- Example: heterophile antigens
• No electrical current used
• Result: forms a precipitin line or band (semi-
quantitative) iv. DOUBLE LINEAR DIFFUSION
• Advantage: • Oakely and Fulthorpe
o Simple and cheaper • Double diffusion – single dimension
• Disadvantage • Agar on slide or plate; ab incorporated in gel
o Long turnaround time • Antigen and antibody in opposing wells
• Incubation time: 24-48 hours • Reactions:
• Example: Quantification of antigens (Factor VII antigen) o react equidistantly and at equivalence
o contamination or impurity present in antigen reacting
with antibody
• Agarose gel with antibody and antigen on top but there is
an intervening layer in between which is a plain agar.
• Positive result: If there is antigen-antibody reaction, the
antigen and antibody will move toward each other through
the plain agar layer, forming a precipitin band.

DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 4

 MODULE 2: PRECIPITATION AND AGGLUTINATION

D. PRECIPITATION BY ELECTROPHORETIC o Ab migrates to electrode (-)

TECHNIQUES o Ag migrates to electrode (+)

i. IMMUNOELECTROPHORESIS (IEP) E. PRECIPITATION REACTIONS/PRECIPITATION

• Combination of electrophoresis and immunodiffusion CURVE
1. Zone of antibody excess
• precipitation in agar under an electric field
o Prozone Phenomenon
• Expensive and procedure is more complex
o precipitation is inhibited and antibodies not bound to
• analyzes serum proteins antigen can be detected in the supernatant
• Excellent screening test to differentiate serum proteins o Many antibodies (Y-shaped) scattered, few antigens
and to detect abnormalities o Indicates late infection
• Since it is electrophoresis, the antibody is positively 2. Zone of equivalence
charged (+) and the antigen is negative (-). o Window Period
• two-step double diffusion technique which first involves the o maximal precipitation in which antibody and antigen
electrophoretic separation of proteins form large insoluble complexes and neither antibody
• Result: separate precipitation lines between each protein nor antigen can be detected in the supernatant
and its antibody o Area where antigen and antibody are present in
• useful procedures for the ID of monoclonal proteins optimal concentrations or proportion.
(Bence Jones proteins) 3. Zone of antigen excess
• double diffusion + electrophoresis o Post Zone Phenomenon
• Enhances the semiquantitative of antigens o precipitation is inhibited & Ag. not bound to Ab. can be
• Used to treat myelomas and malignant lymphomas detected in the supernatant
o Many antigens (round-shaped), few antibodies
a. ROCKET ELECTROPHORESIS o This is suggestive of early infection
• Also known as: Laurell’s
• One dimension, Electroimmunodiffusion Note: Both prozone and post zone can cause false negative
• Combination of RID + electrophoresis serologic reactions and erroneous titer results.
• The height of the rocket is proportional to the
concentration of the antigen II. AGGLUTINATION TEST
o The higher the rocket, the higher the antigen • Agglutination occurs due to the cross-linking of
• Total distance of antigen migration and precipitation is particulate antigens by antibody molecules.
directly proportional to antigen concentration • visible reaction of antibody with the particulate antigen
• Antibody in gel and antigen pipetted into a cut out well • Disadvantage: mostly qualitative examination (positive or
• Electrical current is applied and resulting precipitation lines negative) only; actual value cannot be obtained
form narrow triangles, like a rocket • Advantages:
• The rocket lines are formed as antigen concentration o Easy to perform
decreases o No need for any complicated equipment
• A quantitative method for measuring serum proteins • Example: Widal test: for typhoid fever
antibody • Principle
• Advantage: The result can be obtained in a few hours o Particulate antigen + antibody –> clumping
o Lattice formation (antigen binds with Fab sites of 2
b. IMMUNOFIXATION ELECTROPHORESIS antibodies forming bridges between antigens)
• Combination of Immunoprecipitation + electrophoresis • Two steps to agglutination:
o Sensitization
• Most sensitive method used to detect, confirm and
▪ occurs when the antibody and specific antigen (on
characterize monoclonal gammopathies, such as multiple
the surface of the particulate) combine thru single
myeloma, Waldenstrom’s macroglobulinemia, monoclonal
epitope
gammopathy of unknown significance
▪ Factors affecting sensitization:
• To replace IEP; similar to IEP except that after
- Size of the antigen and antibody
electrophoresis is performed the antiserum is applied
- Class of antibody
directly to the surface of the gel
- Nature of antigen
• This method is used to detect the M-band. o Lattice formation
o M stands for monoclonal. ▪ involves interactions between antibody and
o Consists of monoclonal proteins which are abnormally multiple antigenic determinants
present in plasma cells
• Types of Agglutination Reactions
• In this test, you are finding out what particular a. Direct agglutination reactions
immunoglobulins (IgG, IgA, IgM, kappa, and lambda) are ▪ Patient is tested immediately
the M proteins present. ▪ For example, in blood typing, you prick, get the
o Kappa and lambda: parts of the heavy and light chain blood, and put antisera.
of the immunoglobulins - If it agglutinates, then it is positive.
• Example: Western blot b. Indirect or passive agglutination tests
o Confirms the HIV proteins ▪ antigen must be bound first to an inert particle
o Also confirms the ELISA before you can detect the antibody
Note: electrophoretic errors can occur when current is too ▪ Inert particles can be:
strong or too weak, current is applied backward, buffer is not - Charcoal
at correct pH, or time to run is too long or too short. - Sheep’s red blood cells
• Capillary tube precipitation (Ring Test) - Red blood cells
- Latex
ii. COUNTERIMMUNOELECTROPHORESIS (CIE) c. Hemagglutination reactions
• Modification of ouchterlony ▪ Type of agglutination reaction wherein the RBCs
• antigen and ab are placed on the well directly opposite to are the carrier.
each other
• Advantage: speeds up the migration of antigen and A. AGGLUTINATION TESTS
antibody

DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 5

 MODULE 2: PRECIPITATION AND AGGLUTINATION

i. HEMAGGLUTINATION TEST iii. INDIRECT AGGLUTINATION TEST OR PASSIVE

• Assays that use RBCs as the antigen particulate reacting
with antibody
ii. DIRECT AGGLUTINATION TEST (DAT)
• Particulate antigen reacts directly with antibodies
• Antigen found naturally on the surface of the particles
(cells).
• Ex. Blood typing, Widal, Weil-Felix, Cold Agglutinin test
AGGLUTINATION
• Soluble antigen is artificially attached to particulate
carriers (inert particles)
• Antibodies cause visible agglutination of soluble antigens
affixed to latex spheres.
• This can be used to test the antibodies against viruses
such as Rubella, Herpes zoster (shingles), CMV.

B. ANTIGLOBULIN TECHNIQUE
• Antiglobulin
o Used to demonstrate incomplete antibodies
• Identifies reactions of reagent RBC antigen with specific antibody when the reaction occurs in vitro • Applications:
Indirect
Antiglobulin • Detects antibodies that are floating in the serum o Detection of anti-Rh Ab
(Coombs) Test • Detects in vitro sensitization of cells o Autoimmune hemolytic
anemia
•Qualitative agglutination test o Blood group typing
Direct •Detects in vivo sensitization of cells o The identification of viruses.
Antiglobulin Test •Detects antibodies that are stuck or attached to the RBCs o The diagnosis of certain diseases
•Applications: • Practical considerations: Easy & Semi-quantitative test
• Assays based on the principle of no agglutination as a (+) result o Example: GRAVINDEX
Agglutination o If samples are reactive, it will not produce a visible result. If there is no visible result, then it is positive. ▪ Pregnancy test
Inhibition • Serum (containing antigen) is mixed with beads or latex coated with antigen ▪ Uses urine as specimen
• If the agglutination occurs, the antigen is not present - it is negative. to detect HCG
Reverse Passive • Antibodies (known) are bound or attached to • Used for detection of microbial antigens such as Group A and B streptococcus, S.
Agglutination particulate carriers instead of antigen aureus, N. meningitidis, H. influenza, C. neoformans, M. pneumonia and C. albicans
Coagglutination • Carrier: Bacteria • Used to identify Streptococci, Neisseria meningitidis, Neisseria
(Coat) • Most commonly used carrier: Staphylococcus aureus gonorrhoeae and Haemophilus influenzae.
• Form floccules
Flocculation • Seen in VDRL and Kahn test
• Agglutination of colloidal particles (non-treponemal tests)

C. VIRAL NEUTRALIZATION o consist of known target antigen reagents (beef heart

• This test can also be used to test toxins extract, bacterial antigen) and complement.
• Advantages: Sensitive and specific enough to identify • Indicator system
whether an individual has been exposed to a particular o consist of sheep’s rbc sensitized with hemolysin
virus or viral strain • Positive result: no hemolysis
o Wasserman test for syphilis (in the past)
i. NEUTRALIZATION o Certain viral & fungal diseases
• If complement is fixed by specific antigen-antibody
• Neutralization Reactions reaction, it will be unable to combine with indicator system
• After binding, antibody is not available to react in indicator o Complement binds to the antigen-antibody complex
system and is used up.
• Like agglutination inhibition o Complement-fixation can be used to detect very small
• Results: amounts of antibody
o No agglutination/ hemolysis = positive reaction
o Agglutination or hemolysis = negative reaction
• Antibody did not bind to the origin
• Generally, positive control samples used in inhibition or REFERENCES
neutralization tests show no reaction and negative control
samples show a reaction Notes from the discussion by Mrs. Maria Redora R. Esteban, RMT
• Example of inhibition: Hemagglutination inhibition test
for rubella carrier: bacteria (S. aureus) seen in VDRL CANVAS Notes
• Example of neutralization: antistreptolysin O test (ASO)
1. Toxin Neutralization
▪ Toxin-Antitoxin reaction
▪ ASO
2. Virus Neutralization

ii. OPSONIZATION
• Antigen is covered with antibodies that enhance its
ingestion and lysis by phagocytic cells
• Coating of pathogens (antigen) with antibodies to increase
phagocytosis (the ingestion of phagocytes)
• Opsonins may be
o Specific antibody alone (IgG1, IgG3)
o Specific antibody plus complement, via the classical
pathway
o Complement alone, via the alternative pathway

D. COMPLEMENT FIXATION
• First, the body needs to produce complement fixing
antibodies to bind to the antigen before forming
complement fixation.
• Test system

DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 6