Types of agglutination reactions and Prozone phenomenon

Agglutination is one of the antigen and specific antibody reactions that occur when the two are mixed in vitro in the laboratory in the presence of electrolytes at a suitable temperature and pH. Agglutination is from the Latin word ” agglutinare,” meaning ” to glue,” also clumping of particles.Clumping of cells such as bacteria( Widal test) or red blood cells( blood grouping) in the presence of specific antibodies are examples of agglutination in biology. The antibody binds and joins multiple antigen particles, creating a large, complex lattice that we can see with a naked eye.

The agglutination reaction used to diagnose diseases in the laboratory either uses particulate or soluble antigens.For example, Salmonella typhi bacteria are used to detect specific antibodies in serum in patients with typhoid fever( Widal test).The latex agglutination and other particle agglutination tests are examples of agglutination reactions.Soluble antigens are first produced by coating them on inert particles such as red cells, latex particles, gelatin particles and microbeads.These particles support or carry soluble antigens to make the naked eye visible.

Agglutination tests are sensitive, do not require sophisticated equipment, are easy to perform, do not require washing procedures and are cost- effective.The lattice network formed during the agglutination reaction can be visualized macroscopically or microscopically depending on  the manufacturer’s instructions.

What is Agglutination

Large antigens carrying many epitopes, easily sedimented particles such as animal cells, erythrocytes or bacteria when mixed with specific antibodies lead to a cross- linking of the particles at appropriate temperature and ionic strength, forming a lattice- like structure seen as clumps with a naked eye.This sensitive and specific reaction is referred to as agglutination.

Agglutination is a serological reaction such as precipitation; the only difference is that antigen is large and particulate when agglutinated.Blood group testing is the most common example of agglutination.Agglutination clumps together in cells with antigen( epitopes)/antigen bearing microorganisms or particles in the presence of specific antibodies called agglutinins.We can imagine a lock and key concept to understand the characteristics of the agglutination response.

What is the Principle of Agglutination

An antibody is a molecule with a ” Y” shape.The two ” Y” arms are the Fab portion, which has the combination site and consists of the hypervariable regions of the heavy and light chains.The antigenic determinant nestles in a clamp formed by the combination site of the antibody as shown below.The antigenic determinant is therefore the ” key” that fits the cleft formed by the lock ” Fab”.If the fit is right, agglutination occurs.This concept applies to all reactions with antigen( Ag) antibody( Ab).The agglutination process involves two steps.Sensitization is the first step and lattice formation is the second one.

What is Sensitization

It is an antigens attachment of specific antibodies. The reaction is influenced by pH, temperature and incubation time. IgM antibodies at 4 to 22 degrees C react best, and IgG antibodies at 37 degrees C react best. Incubation time can be 15 to 60 minutes.

What is Lattice formation

Like a ” Jaal,” Lattice consists of a cross link between sensitized particles. It takes longer than sensitization and we can see the result with naked eyes. IgM best in this type of reaction because of its large size, but IgG antibodies may need to be improved.

How to enhance agglutination reaction

Agglutination reactions can be enhance by :

1. Centrifugation( Bridges distance)
2. enzyme treatment( Reduces Zeta Potential)
3. colloids( Albumin reduces zeta potential) and anti- human globulin use.
4. Electrokinetic potential is referred to as Zeta potential in colloidal systems.In fact, it is the degree of repulsion in a solution between adjacent, similarly charged particles.A high zeta potential confers so that agglutination can be resisted.Zeta reduction will favor agglutination.

How to grade agglutination reaction

Grading is either macroscopic or microscopic. Follow the criteria set out in the commercial kit package insert. The following is an example of blood grouping used in the blood banks.

Macroscopic grading

Blood grouping for example:

Microscopic grading

What are the Methods of agglutination reaction

The agglutination test can be performed with three different techniques. This includes: rapid agglutination tests; slow agglutination tests in tubes; slow agglutination tests in microtitration plates.

The rapid agglutination tests

This method involves mixing undiluted patient / client serum and antigen on a glass slide or plate, rotation or agitation of the plate in accordance with the instructions in the literature of the kit, and macroscopic examination, usually after 2 minutes for agglutination.In general, the antigen and serum are mixed in a fixed proportion.The agglutination intensity indicates the concentration of the antibodies in the serum.Sometimes the sera has to be heated to confirm strong agglutination reactions( 56 C for 30 minutes) to destroy non- specific agglutinins or repeat the test with various serum dilutions.

Slow agglutination in tubes/tube agglutination

This involves serum dilution and mixing with a fixed amount of unstained antigen.The tubes are kept at temperature and for a while( usually overnight) as indicated in the manufacture kit.The positive results are shown by the presence of a precipitate in the bottom of the tube and by the clearing of the supernatant( compared to antigen without a serum).

Micro-agglutination

This test is carried out using small amounts of antigen and patient serum in a microtitration plate.A large number of samples / different dilutions can be tested on a single plate at a time.The positive reaction is indicated by the formation of a ragged blanket of colored antigen that covers the base of the U- shaped microtiter well.The negative result appears as an unreacted antigen button in the well.

Note that : “Qualitative agglutination tests” which is an agglutination test used to detect the presence of an antigen or an antibody.The antibody is mixed with the particulate antigen and the agglutination of the particulate antigen indicates a positive test.For example Red blood cells of a patient mixed with a blood group antigen antibody to determine the blood types of a person.

” Quantitative agglutination test” that is  agglutination tests used to quantify particulate antigens in the level of antibodies.Serial dilutions of an antibody- tested sample are mixed with a fixed number of red blood cells or bacteria or other particulate antigens, and the last / highest dilution with agglutination is the amount of antibody in the sample and is expressed as the titer.The results are reported as a reciprocation of the maximum dilution which gives visible agglutination.

What is Prozone and postzone effect

False negative antibody reactions, whether agglutinated or precipitated, can occur if antigen and antibody are not mixed in the correct proportions. This can happen if the antibody is either in excess( prozone) or if the antigen is in excess( post zone).

Prozone effect (prozone antibody)

Some sera show no agglutination when tested undiluted.The same sera shows a positive agglutination / precipitation reaction when tested after dilution.This phenomenon is referred to as the ” Prozone phenomenon” or hook effect in which agglutination or precipitation occurs at higher serum dilution ranges, but is not visible at lower dilutions or when not.Excessive antibody levels lead to false negative reactions as excess antibodies lead to the formation of very small complexes that do not clump to visible agglutination.The reaction of the prozone is the likely cause of false negative results.The reaction of the prozone may also result from the presence of blocking antibody or serum inhibitors.If different antigens are near each other, the antibodies corresponding to each antigen can block binding and compete with each other.

Postzone effect

This refers to the reaction in which excess antigens do not form a lattice and cause a false negative agglutination. Antigen excess is also the probable cause of the false- negative agglutination / precipitation reaction of antigen- antibodies.

What are some applications of agglutination reaction

Agglutination tests help you find antigens or antibodies in a sample.The sample can be any body fluids such as urine, blood, saliva and cerebrospinal fluid( CSF), in which either antigen or antibody is detected, the sample can also be a bacterial culture that we want to type to determine whether it is pathogenic.The first step is to have the sample collected.Blood is collected by venipuncture following standard working precautions; the clinician collects CSF by lumbar puncture and the patient collects urine using the clean catch method.

Applications of agglutination in clinical diagnosis include:

1. To identify and type bacterial cultures (Widal test)
2. Typing blood cells of the recipient and donor for blood transfusion (Blood grouping)
3. To detect the presence of specific antibody and quantitate the amount of antibody in patient’s serum ‘(Widal test)
4. Haemagglutination
5. Latex agglutination  (Crag test) cryptococcal antigen

What is Haemagglutination

As the name suggests, this is agglutination of RBCs by RBC antigen- specific antibodies( e.g. ABO blood grouping or agglutination of RBCs coated with some antigen to detect specific antibodies in the serum of the patient( e.g. Treponema Pallidum Haemagglutination).

Materials and reagent required for Haemagglutination

• Adjustable multi-channel micropipette (50-300 µl)
• Adjustable single-channel micropipette (50-200µl)
• Adjustable single-channel micropipette (5-50µl)
• Disposable tips
• Reagent troughs
• Waste discarders
• Micro plates (U bottom)
• Sample diluents
• Control cells (uncoated)
• Test cells (sensitized with T.pallidum antigen)
• Kit controls

Haemagglutination procedure

1. About 30 minutes before the start of the test procedure; bring kit components to room temp. 15- 30 degrees C). Gently mix the liquid reagents. Determine the total number of test specimens and the number of plates needed for the test.
2. Place a unique plate identifier on the upper middle side of the plate, if necessary
3. Label the plate with the last three digits of the donation I d at the bottom left of the well to identify the positions. Include one negative and one positive control per sample batch
4. Arrange the samples in a sampling rack according to the plate map.
5. Use the first extra well by number of specimens and add 190 μl of diluent sample.
6. Use a fresh pipette tip for each addition, take 10 μl of specimen and mix it in the extra well / plate with 190 μl sample diluent in the same position.
7. Transfer 25 ml of the diluted sample to both test and control wells
8. After sampling, place the sample in the same / different sample rack.
9. Add 25μl positive and negative controls to their respective positions.
10. Mix the control and test cell well to make the suspension homogeneous and add 1 drop( 75μl) of test cells and control cells to their respective wells, including positive and negative control cells.
11. Mix the contents of the well by slowly rotating the plate. Maintain the plate on a smooth, stable surface. Incubate for 1 hour at room temperature and read the results.

Interpretation of Hemagglutination test result

1. All control wells need to form compact buttons.If any control does not show button formation, repeat the test to rule out any technical errors.( The agglutination of the control cells and the test cells indicates the presence of anti- cell antibodies and in this case the test is not valid & should be repeated after the initial absorption of the test serum.To do this, dilute the test serum 1/4 with control cells and allow it to stand at room temperature.After the sample is centrifuged( 1000 rpm/5 min).Dilute 1/5 of the supernatant diluent.Test this dilution directly with test & control cell suspensions without further dilution).
2. If a compact button is present in a test well, the result is considered non- reactive
3. If there is a ring pattern or net characteristics of the cells in the control well and there is a compact button formation, the specimen is considered reactive for T. Pallidum Pallidum.
4. All positive tests should be repeated by TPHA.
5. If any sample is positive by repeat test also, identify the sample and separate out the sample then retest with RPR.