Agglutination test using antibodies

Agglutination test using antibodies

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Agglutination test

Latex agglutination using bound antigens : by coating soluble (non - particulate ) antigens on to microscopic latex spheres, their reaction with a particular antibody can be visualized.

Haemagglutination : red blood cells can be used as agglutinating particles.

What are non-particulate antigens ? Why are they used instead of particulate ones ?

How are red blood cells used as agglutinating particles ?

If you have an antibody that is directed against, for example, a bacterial surface protein, then by mixing the bacterial cells with the antibody at a suitable stoichiometry you could observe clumping of the cells as the antibody molecules essentially cross link the cells together. This would be an example of using an agglutination (clumping) assay with a particulate antigen. You can also probably see why it is important that each cell (particle) carries many copies of the antigen for cross linking to take place effectively.

If, however, the antibody of interest is directed against a soluble protein, then mixtures of antigen and antibody are less likely to form cross-linked complexes, although it can happen and can be used for example in the Ouchterlony technique. If you want to use an agglutination test (i.e clumping of particles) with a soluble antigen then you can achieve this by attaching multiple copies of the antigen to macroscopic particles. This is what is being described in the definition of Latex agglutination in your question.

I imagine that there are ways of attaching a soluble antigen to red blood cells (e.g. by chemical cross-linking to the red blood cell surface) in which case you could observe clumping of the red blood cells when you added your antibody.

Agglutination test using antibodies - Biology

Lab 6. Agglutination

When antibodies are mixed with their corresponding antigens on the surface of large, easily sedimented particles such as animal cells, erythrocytes, or bacteria, the antibodies cross-link the particles, forming visible clumps. This reaction is termed agglutination. Agglutination is a serological reaction and is very similar to the precipitation reaction we learnt last week. Both reactions are highly specific because they depend on the specific antibody and antigen pair. The main difference between these two reactions is the size of antigens. For precipitation, antigens are soluble molecules, and for agglutination, antigens are large, easily sedimented particles. As you will see from this lab exercise, agglutination is more sensitive than precipitation reaction because it takes a lot of more soluble antigens and antibody molecules to form a visible precipitation. To make the detection of soluble antigen and antibody reaction more sensitive, a precipitation reaction can be transformed into an agglutination reaction by attaching soluble antigens to large, inert carriers, such as erythrocytes or latex beads.

Agglutination reactions have many applications in clinical medicine. Agglutination reactions can be used to type blood cells for transfusion, to identify bacterial cultures, and to detect the presence and relative amount of specific antibody in a patient’s serum. Agglutination has been commonly used to determine whether a patient had or has a bacterial infection. For example, if a patient is suspected of having typhoid fever, the patient’s serum is mixed with a culture of Salmonella typhi. If an agglutination reaction occurs, shown as clumping of the bacteria, the patient either had or has an S. typhi infection. Since certain antibodies can persist in a patient’s blood for years after the patent has recovered from the infection, a positive reaction does not mean that the patient currently has the infection. To determine whether a patient is currently suffering from typhoid fever, the amount or titer of the antibody will be determined at the onset of illness and two weeks later. If the titer of antibody in the patient’s serum has increased at least four-fold between the two tests, the patient is currently fighting off the infection, and the pathogen causing the illness is confirmed.

In this lab exercise, you will learn two different methods of employing agglutination reactions, rapid slide agglutination and microtiter test. These two tests are valuable methods commonly used in clinical laboratories. Applications of agglutination include A-B-O blood typing tests and rapid bacterial identification. The microtiter test is used to quantify the amount of antibody in patient’s blood.

Part 1. Rapid Slide Agglutination

SAFETY NOTE: This lab uses your own blood for ABO blood typing. After obtaining your drops of blood, put on gloves. Dispose of waste in the biohazard container.


1. Label three slides A, B and A+B.

2. Spray your left “ring “ finger with 70% ethanol or wipe it with an alcohol wiper and let it air dry.

3. Take a sterile lancet and puncture your fingertip. If you have calluses, aim a little to the side. DO NOT LANCET ANYONE OTHER THAN YOURSELF. When finished with your lances, place them in the BIOHAZARD CONTAINER .

4. Place a small drop of blood on each of three microscope slides. At this point, put on GLOVES. DO NOT TOUCH ANYONE ELSE’S BLOOD.

5. On the A slide, place a 20 m l of anti-A antiserum. Place anti-B on the B slide and anti-A + anti-B on the A+B slide. Mix the antisera in with the blood using a separate toothpick for each slide. Place toothpicks in the biohazard waste.

6. After several minutes, observe agglutination and determine your blood type.

Part 2. Microtiter Test

In this lab exercise, you will learn how to use the microtiter test to determine the amount of anti-sheep red blood cell (RBC) antibody.


1. In a round-bottom microtiter plate, add 50 m l of PBS to columns 2-9 in rows A-C. Leave column 1 empty.

2. Add 100 m l of rabbit anti-sheep RBC to column 1 in rows A-C.

3. In each row, transfer 50 m l from column 1 to column 2 and mix well. Now take 50 m l from column 2 and transfer to column 3. Repeat this process across the columns. Discard the final 50 ml taken from row 9.

4. Add 50 m l of a 2% suspension of sheep RBC to all wells (columns 1-9, rows A-C). Make sure the RBCs stay adequately resuspended as you are using them. Periodically invert the capped tube to keep them resuspended evenly.

5. Incubate the plate for 24 hours at room temperature.

6. Come back the next day and observe agglutination. Positive wells will exhibit a diffuse and confluent settling of the RBC and Ab, while in negative wells, all cells will roll down to the bottom and it will look more like a dot.

Immunobiology A-7

Study Questions

1. What is the difference between precipitation and agglutination? Which one of these two methods is more sensitive and why?

2. What type of structure on your red blood cells decides your blood type?

3. You used the slide agglutination test to determine your blood type. You got the following result:
Anti-serum A B AB
Agglutination - + +

What is your blood type?

If you receive type AB blood for blood transfusion, what is going to happen and why?

4. Describe how to determine whether a patient has ever had a Salmonella typhi infection, the name of method, reagents used, possible results, conclusions deduced from results and why.

5. In preparing her immunology lab, an instructor purified IgG antibodies that are specific to sheep red blood cells (SRBCs) and digested some of the antibodies into Fab, Fc, and F(ab’)2 fragments. She placed each preparation in a separate tube, labeled the tubes with a water-soluble marker, and left them in an ice bucket. When the instructor returned, she discovered that the labels had smeared and were unreadable. Determined to salvage the antibodies, she relabeled the tubes 1, 2, 3, and 4 and proceeded. Based on the test results described below, indicate which preparation was contained in each tube and explain why you so identified the contents.

a. The preparation in tube 1 agglutinated SRBCs but did not lyse them in the presence of complement.

b. The preparation in tube 2 did not agglutinate SRBCs or lyse them in the presence of complement. However, when this preparation was added to SRBCs before the addition of whole anti-SRBC, it prevented agglutination of the cells by the whole anti-SRBC antiserum.

c. The preparation in tube 3 agglutinated SRBCs and also lysed the cells in the presence of complement.

d. The preparation in tube 4 did not agglutinate or lyse SRBCs and did not inhibit agglutination of SRBCs by whole anti-SRBC antiserum.


In the Monospot test a known Epstein-Barr viral antigen is used in the test, for the identification of patient antibody to this virus (which causes infectious mononucleosis, Burkitt's lymphoma, and nasopharyngeal cancer). The interesting thing about this test is that you are identifying NONSPECIFIC antibody rather than antibody against the Epstein-Barr virus, the cause of infectious mononucleosis. This odd antibody, called &lsquoheterophile' antibody reacts against sheep and bovine RBCs. The term heterophile antibody refers to antibodies having the capacity to react with certain antigens, which are very different from the ones inducing the antibody formation. The latex particles in the MONO-LATEX REAGENT LATEX are coated with a suspension of mononucleosis antigen obtained from red blood cells from cattle. If antibodies are present in the serum or plasma and mixed with the latex, a specific reaction will result in a visible agglutination of the latex particles. The patient serum, a NEGATIVE control, and a POSITIVE control are each placed on the glass slide, then adding the reagent LATEX (plastic beads covered with antigen).


  1. Treated animal RBC is used as a carrier of antigen
  2. Passive hemagglutination: Antigens that are being bound by antibodies are not the antigen of RBC but are passively bound antigens.
  3. Examples
    1. Microhemagglutination test for Syphilis (MHA-TP)
    2. Hemagglutination treponemal test for Syphilis (HATTS)
    3. Passive hemagglutination tests for antibody to extracellular antigen of Streptococci
    4. Rubella indirect hemagglutination test for Avian Influenza
    5. Quantitative Micro Hemagglutination Test (HA)

    CLOSTRIDIUM | Detection of Enterotoxin of Clostridium perfringens

    Latex Agglutination Tests

    Two latex agglutination tests have been described: A reverse passive latex agglutination (RPLA), which is achieved after overnight incubation and a slide latex agglutination (SLAT), which requires only a few minutes.

    Reverse Passive Latex Agglutination

    RPLA is commercially available (PET-RPLA, TD930, Oxoid, Basingstoke, UK). The sensitivity is about 3 ng ml − 1 ( Table 1 ). The procedure is as follows:

    For each sample, two rows of a 96-well V type microtiter plate are used.

    Place 25 μl of PBS containing 9.5% bovine serum albumin (BSA) in each well, except in the first well of each row. The last wells only contain PBS-BSA.

    Add a 25 μl sample to the first and second well of each row.

    Serial twofold dilutions are done in each row from the second to the seventh well.

    Add 25 μl of beads sensitized with immunopurified anti-CPE antibodies to each well of the first row.

    Add 25 μl of control beads sensitized with nonimmune rabbit immunoglobulins to each well of the second row.

    Mix well by hand rotation of the plate or by using a plate shaker.

    Cover the microplate with a lid or put the microplate in a humidified chamber.

    Incubate the plate at room temperature for 20–24 h.

    The results are interpreted as follows:

    Agglutination is determined by visual inspection. This is easier with a black sheet under the microplate or with a test reading mirror.

    The results are scored as +++ (complete agglutination), ++, +, +/− or – (absence of agglutination) ( Figure 1 ).

    Figure 1 . Interpretation of the agglutination results in RPLA. + corresponds to the agglutination of latex, − corresponds to the sedimentation of particles.

    The row containing control latex must be negative. A nonspecific agglutination can be observed in some samples. A sample is considered to contain CPE when the positive agglutination in the sensitized row exceeds that in the control by two wells or more.

    Slide Latex Agglutination

    The SLAT technique consists of latex bead agglutination in the presence of CPE on a glass slide. Reagent preparation is as follows:

    Dilute latex beads (0.8 μm) 1:3 in glycine buffer (0.1 M glycine, 0.15 M NaCl, pH 8.2).

    Add anti-CPE immunoglobulins that have been purified by immunoaffinity on a Sepharose column containing immobilized CPE (13 μg ml − 1 , final concentration).

    Agitate the mixture for 1 min at room temperature, then add an equal volume of PBS-0.1% BSA, and vortex vigorously to mix the suspension.

    Use nonimmune rabbit immunoglobulins G (Sigma) for the control latex.

    Store the latex suspensions at 4 °C.

    The test procedure is as follows:

    Mix 25 μl of samples and serial twofold dilutions in PBS containing 0.1% BSA with 25 μl sensitized or control latex beads on a glass slide. Gentle rotate each mixture and record the results after 1–5 min by visual inspection.

    Score the results in a similar way to those for RPLA: ++ (complete agglutination), +, +/− or – (absence of agglutination).

    Samples containing CPE do not agglutinate control latex beads. Note that samples containing a high concentration of CPE give negative or weakly positive results, and complete agglutination is observed with diluted samples.

    The sensitivity depends on the purification of the immunoglobulins used for the latex bead preparation. When the immunoglobulin G fraction purified from rabbit anti-CPE serum is used for the sensitization of latex beads, the SLAT sensitivity with purified CPE is 100 ng ml − 1 . By using specific anti-CPE immunoglobulins purified by immunoaffinity, however, a lower limit of detection of 0.1 ng ml − 1 is attained.

    Serological Reactions and Human Body | Human Immunology

    Neutralization is a type of serological reaction which occurs between a toxin and its antibody (antitoxin) or between a virus and its antiserum. The reaction is so named, because it leads to neutralization or removal of harmful effects of the antigens.

    Among the practical applications of neutralization reaction is detection of botulin toxin in contaminated food, and the Schick test for diagnosis of diphtheria. The spore-forming obligate anaerobe, Clostridium botulinum, produces a deadly poisonous proteinaceous toxin, known as botulin toxin, in canned food when the food has not been properly sterilized. For detection of the presence of botulin toxin in food suspected to be contaminated with C. botulinum, the test material is mixed with botulin-antitoxin (immune serum containing anti-botulin antibodies) and injected into some experimental animals.

    In a control set of animals, the test material alone is injected. If the food contains botulin toxin, the animals injected with test material alone succumb, while the animals receiving test material + anti-botulin antiserum survive. If, the animals of the latter group also die, it would indicate that the food is contaminated with some other poison, but not botulin-toxin.

    A. Test material prepared from contaminated food

    B. Test material + botulin-antitoxin

    I. Injection of A alone into experimental animal —> Death A contains some poisonous material

    II. Injection of A + B into experimental animals —> No death —> Food contains botulin toxin

    III. Injection of A + B Death —> Food contains some poison, but not botulin toxin.

    The Schick test is for detection of diphtherial antitoxin in the serum of a patient suspected to have contracted diphtheria caused by Corynebacterium diphtheriae. The test is conducted by intradermal injection of a minute quantity of diphtherial exotoxin to the suspected patient. A positive Schick test is indicated by a local inflammatory response characterized by erythema, swelling and tenderness at the injection spot.

    Such a response happens because the person has no antitoxin and is free of diphtheria, but, at the same time is, susceptible to an attack of diphtheria because of absence of antitoxin in serum. On the other hand, a negative Schick test indicates the person has diphtherial antitoxin in serum which neutralizes the injected toxin. This may be due to previous immunization or an active infection.

    Serological Reaction: Type # 2. Precipitation:

    This type of serological reaction occurs between a soluble antigen and antiserum. The resultant antigen-antibody complex forms a lattice which forms a precipitate when the lattice becomes so large that it can no longer remain in a soluble state.

    Precipitation reaction can be used for detection of specific antibodies in the serum of a person suspected to be infected by a pathogen. Obviously, the antigenic substance produced by the pathogen must be present in soluble form, e.g. a toxin or a capsular polysaccharide etc.

    Precipitation reaction can be carried out either in a liquid system or an agar gel. In a liquid system, precipitation reaction leads to formation of a ring of flocculation at the interface of antigen and antibody solutions. In agar-gel, precipitation zones are produced where the antigen and antibody meet with each other as they diffuse through the gel.

    Liquid precipitation reaction is carried out in narrow tubes (4 to 5 mm in diameter). Equal quantities of the antiserum to be tested are distributed in a series of tubes to form the bottom layer. Then equal quantities of serial dilutions of a known antigen solution in normal saline are added on the top without disturbing the bottom layer.

    A zone of flocculation detected by its turbidity appears at the interface within a few hours. With a potent antiserum, a dilution of 10 -6 to 10 -7 can produce a visible ring. The ring results from combination of a large number of antigen molecules with antibodies belonging to IgG and IgM classes present in the antiserum of the patient.

    In the Ouchterlony double-diffusion technique, the precipitation reaction is carried out in petridishes containing agar or agarose gel. Wells of 1 cm diameter and 5mm depth are scooped out from a solidified gel. Antisera and solutions of antigens are placed in different wells. On incubation, both antiserum and antigen diffuse into the agar. They meet each other and form visible zones of precipitation (Fig. 10.45).

    Serological Reaction: Type # 3. Agglutination:

    Agglutination is another serological reaction occurring when the antigen present on particulate bodies reacts with antibodies to form clumps or agglutinates. This happens because particulate bodies, like bacterial cells, erythrocytes etc. having surfaced antigens are linked to each other by binding with antibodies to form a mat of antigen-antibody complex.

    Previously agglutination tests were carried out in small tubes. The antiserum to be tested was diluted in steps of 2 and distributed in a series of tubes. The particulate antigen was added and incubated for 2 to 24 hr. Agglutinates of the antigen, if formed, settled at the bottom. A practical application of agglutination test was the Widal test for diagnosing typhoid. It was performed in hanging-drops. The test is no longer used.

    The procedure of Widal test was to mix a drop of the diluted serum of the patient with a standard suspension of Salmonella typhi on a coverslip which was then inverted over a grooved slide. After varying periods of incubation, presence of clumps of bacteria was examined. Formation of clumps indicated that the serum contained specific antibody for typhoid bacilli. The procedure has now been replaced by a more sophisticated method.

    At present, microtitre plates made of plastic are used for agglutination or other tests. These plates have many shallow wells of equal capacity. Agglutination tests are conducted by two methods — one is direct and the other is indirect passive.

    In the direct agglutination test, equal quantities of two-fold dilutions of the test serum are taken in the wells of a microtitre plate, so that each well contains half the amount of antibody (presumably present in the serum) of the preceding well and double the amount of the succeeding well. Next, the particulate antigen is added to each well in equal volumes having the same concentration. The highest dilution of the serum which gives visually detectable agglutination is considered as the antibody titre of the test serum (Fig. 10.46).

    The passive indirect agglutination test is a further refinement of the technique. In this method, extremely fine polystyrene particles having an average diameter of one-tenth of that of bacterial cells are used for adsorbing either soluble antigens or antibodies present in serum. The particle-bound soluble antigen reacts with antibody forming agglutinates. Alternatively, polysterene-bound antibody reacts with particulate antigen and agglutination occurs (Figs. 10.47A and 47B).

    Agglutination of erythrocytes is known as haemagglutination. Erythrocytes have surface-bound antigens characteristic for different blood groups. When incompatible blood samples are mixed, these surface antigens bind to the antibodies present in serum and results in haemagglutination. Such antigen- antibody reaction is made use of in blood typing.

    Haemagglutination due to mixing of blood samples of groups A and B of ABO system is schematically shown in Fig. 10.48:

    Another practical application of haemagglutination is the Coombs test for detection of Rh- antibodies. These antibodies are formed in the body of an Rh-negative person when Rh-positive erythrocytes enter. Rh-antibodies can interact with Rh-antigens present on the erythrocytes of Rh- positive blood.

    To test the presence of Rh-antibodies in a person’s serum, it is mixed with Rh-positive erythrocytes resulting in coating of the erythrocytes by Rh-antibody (if present). But this does not lead to haemagglutination. Next, anti-human globulin is added to the mixture resulting in haemagglutination indicating the presence of Rh-antibody in the test serum.

    The principle involved in coombs test is shown in Fig. 10.49:

    Some viruses, like those causing mumps, measles and influenza are capable of haemagglutination without involving antibodies. The phenomenon is known as viral haemagglutination. These enveloped viruses can directly bind to the erythrocytes and cause their clumping. Specific antiviral antibodies can inhibit the haem-agglutinating activity of the viruses by combining with their attachment sites. This can be used for diagnosing the disease caused by a virus of mumps, measles and influenza group.

    A rapid assay for detecting antibody against leucocytozoonosis in chickens with a latex agglutination test using recombinant R7 antigen

    A method for detecting antibody against leucocytozoonosis in chickens by latex agglutination (LA) was developed using latex beads coated with recombinant R7 (rR7), an outer membrane antigen of second-generation schizonts of Leucocytozoon caulleryi. Compared with the agar gel precipitation test, which is widely used in Japan, LA could detect antibody induced by L. caulleryi infection with greater sensitivity. No agglutination was detected with sera from specific pathogen free or layer chickens that had not been vaccinated with oil-adjuvanted rR7 antigen, or infected with L. caulleryi, nor with sera from chickens inoculated with other pathogens, establishing the specificity of the assay. The LA test was also able to be used to quantify serum antibody induced by vaccination, which is not possible with the agar gel precipitation test. This study has shown that LA using the rR7 antigen is a simple, quick, and useful method for detecting antibody against L. caulleryi.

    Validation of in-house liquid direct agglutination test antigen: the potential diagnostic test in visceral Leishimaniasis endemic areas of Northwest Ethiopia

    Background: Visceral leishmaniasis in Ethiopia is a re-emerging threat to public health, with increased geographical distribution and number of cases. It is a fatal disease without early diagnosis and treatment thus, the availability of affordable diagnostic tools is crucial. However, due to delays caused by import regulations, procurement and late delivery of imported test kits, accessibility remains a problem in the control program. Therefore, we aimed to produce and evaluate the performance of an in-house liquid (AQ) direct agglutination test (DAT) antigen.

    Result: The AQ-DAT was produced at the Armauer Hansen Research Institute, using Leishmania donovani strain (MHOM/ET/67/L82). Sera from 272 participants 110 microscopically confirmed cases of VL, 76 apparently healthy and 86 patients who had infectious disease other than VL were tested with AQ-DAT, and standard kits: Freeze-dried DAT (FD-DAT) and rK39. Taking microscopy as a gold standard the sensitivity and specificity of the AQ-DAT were 97.3 and 98.8%, respectively. It had high degrees of agreement (k > 0.8), with a significant (P < 0.05) correlation compared to microscopy, FD-DAT, and rK39.

    Conclusion: Although further standardization is required, the in-house AQ-DAT could improve diagnostic accessibility, minimize intermittent stock outs and strengthen the national VL control program.

    Keywords: Liquid direct agglutination test North West Ethiopia Visceral leishmaniasis.

    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.

    For example, to determine patient&rsquos blood type the red blood cells of the person can be mixed with antibody to a blood group antigen. Another example is that to assay the presence of antibodies in a patient sample, the serum of the patient is mixed with the red blood cell (RBC) of a known blood type.

    Inheritance of Blood Groups:

    Blood groups for each individual are determined by genes or alleles (small packets of information in cells contained in the DNA) which are inherited from both parents. Genes for the Rh-ve and O groups from one parent are masked (i.e., they are recessive) by the presence of Rh+ve and A or B genes from the other parent. That is, O and Rh negative genes only produce an effect when there is a "double dose" of such genes, i.e., one from each parent (homozygous condition). Thus, people who are apparently A or B Rh+ve may also carry genes for the O and Rh-ve blood groups which can be inherited by their children.


    ag·glu·ti·nate / əˈgloōtnˌāt / • v. firmly stick or be stuck together to form a mass. ∎  Biol. (with reference to bacteria or red blood cells) clump together: [ tr. ] these strains agglutinate human red cells| [ intr. ] cell fragments agglutinate and form intricate meshes. ∎  [ tr. ] Linguistics combine (simple words or parts of words) without change of form to express compound ideas. DERIVATIVES: ag·glu·ti·na·tion / əˌgloōtnˈā sh ən / n.

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    Watch the video: Agglutination and Precipitation Animation (June 2022).