Agglutination reaction (from lat. agglutinatio- gluing) - gluing of corpuscles (bacteria, red blood cells, etc.) by antibodies in the presence of electrolytes.
Agglutination reaction manifests itself in the form of flakes or sediment consisting of corpuscles (for example, bacteria) “glued together” by antibodies (Fig. 7.37). The agglutination reaction is used to: determine the pathogen isolated from the patient; determination of antibodies in the patient’s blood serum; determination of blood groups.
Rice. 7.37 a, b. Agglutination reaction withIgM-antibodies (a) andIgG-antibodies (b)
1. Determination of the pathogen isolated from the patient Approximate reaction agglutination on glass (Fig. 7.38). A suspension of bacteria isolated from the patient is added to a drop of agglutinating serum (1:20 dilution). A flocculent precipitate forms.
Rice. 7.38.
An extensive agglutination reaction with a pathogen isolated from a patient (Fig. 7.39). A suspension of bacteria isolated from the patient is added to the dilutions of the agglutinating serum.
Rice. 52
2. Determination of antibodies in the patient’s blood serum
Detailed agglutination reaction with the patient’s blood serum (Fig. 7.39). Diagnosticum is added to dilutions of the patient's serum.
- Agglutination with O-diagnosticum (bacteria killed by heat, retaining O-antigen) occurs in the form of fine-grained agglutination.
- Agglutination with H-diagnosticum (bacteria killed by formaldehyde, retaining the flagellar H-antigen) is large and occurs faster.
3. Agglutination reaction for determining blood groups The agglutination reaction to determine blood groups is used to establish the ABO system (Table b) using agglutination of erythrocytes with immune serum antibodies against blood group antigens A (I), B (III). The control is: serum that does not contain antibodies, i.e. serum AB (IV) blood group; antigens contained in red blood cells of groups A (II), B (III). The negative control does not contain antigens, i.e., group O (I) erythrocytes are used.
Table 7.6. Determination of ABO blood groups
| Reaction results |
Group |
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|
belonging |
||
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researched |
||
|
red blood cells with |
serum (plasma) |
|
|
standard |
with standard |
|
|
serums | ||
Detailed agglutination reaction (RA). To determine AT in the patient’s blood serum, a extensive agglutination reaction (RA). To do this, a diagnosticum is added to a series of dilutions of blood serum - a suspension of killed microorganisms or particles with sorbed Ag. The maximum dilution giving agglutination Ag is called the serum titer.
Types of agglutination reaction (RA) to detect AT - a blood-drop test for tularemia (with a diagnosticum applied to a drop of blood and the appearance of visible whitish agglutinates) and the Huddleson test for brucellosis (with a diagnosticum stained with gentian violet applied to a drop of blood serum).
Approximate agglutination reaction (RA)
To identify the isolated microorganisms, an approximate RA is placed on glass slides. To do this, a pathogen culture is added to a drop of standard diagnostic antiserum (diluted 1:10, 1:20). At positive result perform a detailed reaction with increasing dilutions of the antiserum.
Reaction considered positive if agglutination is observed in dilutions close to the titer of the diagnostic serum.
OAS. Somatic O-Ags are heat stable and can withstand boiling for 2 hours. When interacting with AT, they form fine-grained aggregates.
N-Ag. N-Ag (flagellates) are thermolabile and quickly degrade at 100 °C, as well as under the influence of ethanol. In reactions with H-antiserum, after 2 hours of incubation, loose large flakes are formed (formed by bacteria sticking together with flagella).
Vi-Ar typhoid bacteria is relatively heat-stable (withstands temperatures of 60-62 °C for 2 hours); When incubated with Vi antiserum, a fine-grained agglutinate is formed.
Direct hemagglutination reactions
The simplest of these reactions - agglutination red blood cells, or hemagglutination, used to determine blood groups in the ABO system. For determining agglutination(or lack thereof) use standard antisera with anti-A and anti-B agglutinins. The reaction is called direct, since the Ags being studied are natural components of red blood cells.
Common with direct hemagglutination viral hemagglutination has mechanisms. Many viruses are capable of spontaneously agglutinating erythrocytes of birds and mammals; their addition to a suspension of erythrocytes causes the formation of aggregates from them.
Agglutination reaction Agglutination reaction
(RA) - a method of identifying and quantification Ag and At, based on their ability to form agglomerates visible to the naked eye. In the department of infectious diseases. diseases or for other purposes is used to identify unknown microbes and cells, to determine the presence and amount of Ab in blood and other liquids. The determination principle is based on the specificity of the interaction between Ag and Ab and consists in finding the known from the unknown. There are many options for RA: quantitative and qualitative, test tube and glass, volumetric and droplet, conventional, accelerated and express methods. To stage RA you need: 1) s-ka blood. In the variant with determining the type (var) of bacteria, industrial agglutinating tests are used, produced by immunizing rabbits. In the variant with determination of the type of Ab, a blood sample is taken from the test. people or animals. The solution must be sterile and free of suspended particles. Prepare the basic dilution in saline solution. It should be 2-4 times lower than the diagnostic titer for this disease; 2) Ag. In the version of the reaction with determination of the Ab type, industrial diagnostic kits are used; in the variant with the determination of Ag, diagnosticums are prepared themselves in the form of a 1-3 billion suspension in a saline solution of 18-20-hour agar (less often broth) test. microbe inactivated by heating in a water bath at 70°C for 1 hour or by 24-hour incubation at 37°C with formaldehyde (final concentration 0.2%); 3) electrolyte in the form of saline solution. Staging technique volumetric serial tube RA to determine the Ab titer in s-ki: several rows of working dilutions are prepared from the main dilution of s-ki. The number of rows depends on the number of diagnosticums taken into the experiment; the number and dilution factors are determined by the diagnostic titer of the suspected disease. The series must at least contain a dilution corresponding to the diagnostic Ab titer, two dilutions below and two dilutions above it. for example, if the diagnostic titer is 1:100, then with the volumetric method of staging RA the following dilutions should be prepared: 1:25, 1:50, 1:100, 1:200, 1"400; with the drip method, the first dilution (1:25) is not needed, but another higher dilution is needed - 1:800. scientific research s-ku is titrated to negative reaction. It is diluted as follows: 0.25 ml of saline solution is poured into all test tubes, except the 1st one, when the reaction is carried out in a volume of 0.5 ml, and 0.5 ml when the reaction is carried out in a volume of 1 ml. Pour 0.25 (0.5) ml of the main dilution into the 1st and 2nd test tubes, from the 2nd test tube, into the cut volume and breeding s-k and increased 2 times, 0.25 (0.5) ml is transferred to the 3rd, from the 3rd to the 4th, etc. to the last, from the cut 0.25 (0.5) ml is poured into everything to balance the volumes. Each dilution is carried out using a separate pipette. If several diagnosticums are taken into experiment, then for each of them its own series of dilutions is prepared in the same way. Diagnosticum is added to each dilution of the test tube in a volume equal to the volume of the test tube, as a result of which the dilution in each test tube is doubled. The experiment corresponds to the s-ki control (0.25 - 0.5 ml of the main dilution of s-ki and the same amount of saline solution) and the Ag control (0.25 - 0.5 ml of diagnosticum and the same amount of saline solution). If several diagnosticums are used in the experiment, then each has its own antigen control. The rack with test tubes is shaken well and placed in a thermostat at 37°C for 4 hours, and then left at room temperature until the next day, after which the PA is recorded based on the amount of sediment and the degree of clearing of the liquid. Determination of these indicators, depending on the nature of the agglutinates, is carried out with the naked eye on a dark background, in an agglutinoscope or over the concave surface of a microscope mirror. Accounting begins with controls: control C should be transparent, Ag should be uniformly cloudy (after shaking the tube). If the controls are good, establish the presence and degree of agglutination in all test tubes, which are designated by pluses: large sediment and complete clearing of the liquid - 4 pluses; large sediment and incomplete clearing of the liquid - 3 pluses; noticeable sediment and noticeable clearing of the liquid are 2 pluses. After this, the titer is determined: the highest dilution with an agglutination intensity of at least 2 pluses. Titre research s-ki are compared with the diagnostic titer for this disease. If the titer is examined. s-ki is 2 times lower than the diagnostic value, the reaction is assessed as doubtful; if the titer is equal diagnostic - how weakly positive; if it is 2-4 times higher, it is considered positive; if it is 8 or more times higher, it is considered sharply positive. With the widespread distribution of Ab healthy people To assess RA, an increase in Ab titer is used. To determine the type of Ar in serial RA, the number of rows must correspond to the number taken for identification diagnostic tests. From the main dilution of the diagnostic test, a series of successive two-fold dilutions are prepared in the same way as in RA to determine the Ab titer. Dilution factors depend on the titer of the agglutinating test. In the experiment, the presence of a dilution equal to the titer of the test is necessary, as well as 2, 4, 6, 8 times lower than it. For example, if the titer of the diagnostic test is 1 3200, then you should use dilutions 1 3200, 1 1600, 1 800, 1 400, 1 200 The same volume of tested Ag is added to the dilutions of the test, as a result, the dilution of the test increases by 2 times. 2 controls of test and Ag are added to the experiment. If in the experiment several s-k are involved, then each of them needs its own control. Upon completion of the reaction, the stand is shaken vigorously and placed in a thermostat at 37 ° C. The results are taken into account as described above. Evaluation of the reaction has features. In order to draw a conclusion about the compliance of the study. Ag taken into the experiment, the titer of the reaction must correspond to at least half the titer of the standard diagnostic test. Titers of 1 4 and below are considered as a group reaction Drip md staging of RA differs from volumetric in that s-ku is diluted in a volume of 1 ml, Ag is used in a higher concentration (10 billion/ml) and it is added 1 - 2 drops into a test tube Dilution of the drug after adding Ag is considered unchanged. Otherwise, the method of setting, recording and evaluation is similar to the volumetric method
(Source: Dictionary of Microbiology Terms)
IMMUNOMICROBIOLOGICAL STUDIES
Immunological methods are used to solve many problems:
1. Condition assessment immune system person ( immune status) by definition of quantitative and functional characteristics cells of the immune system and their products.
2. Determination of the composition and characteristics of human tissues: blood groups, Rh factor, transplantation antigens.
3. Diagnosis of infectious diseases and resistance to them by detecting and establishing antibody titers (serodiagnosis), identifying pathogen antigens in the body, and determining cellular reactions to these antigens.
4. Seroid identification of cultures of bacteria and viruses isolated from the body of humans and animals.
5. Detection in the human body and in external environment any substances with antigenic or hapten properties (hormones, enzymes, poisons, medicines, drugs, etc.).
6. Identification of immunopathological conditions, allergies, transplantation and antitumor reactions.
The process of interaction between antigen and antibody serological reactions occurs in two phases:
1) specific- the interaction phase in which a complementary combination of the active centers of antibodies (paratopes) and antigen epitopes occurs. Typically this phase lasts a few seconds or minutes;
2) nonspecific- manifestation phase, characterized external signs formation of immune complexes. This phase can develop from several minutes to several hours.
The optimal specific interaction of antibodies with antigen occurs in an isotonic solution with a pH close to neutral. The antigen-antibody reaction in an in vitro system can be accompanied by the occurrence of several phenomena
· agglutination,
· precipitation,
· lysis.
External manifestations reactions depend on the physicochemical properties of the antigen (particle size, physical state), class and type of antibodies (complete and incomplete), as well as experimental conditions (medium consistency, salt concentration, pH, temperature).
The polyvalency of antigens and antibodies ensures the formation of aggregates visible to the naked eye. This occurs in accordance with the theory of network formation, according to which other antibody and antigen molecules are sequentially attached to the resulting antigen-antibody complex. As a result, network structures are formed, which turn into aggregates that precipitate. The nature and severity of the reaction depend on the quantitative ratio of antigens and antibodies. The most intense reactions occur when the reagents are in equivalent proportions.
Prerequisite lattice formation (networks) - the presence of more than three antigenic determinants for each antigen molecule and two active centers for each antibody molecule. Antigen molecules are lattice nodes, and antibody molecules are connecting links. The region of optimal ratios (equivalence zone) of antigen and antibody concentrations, when neither free antigens nor free antibodies are detected in the supernatant after sediment formation.
Aggregates that can precipitate are formed when antigens combine with full antibodies. Incomplete antibodies(monovalent) do not cause the formation of network structures and large aggregates. To detect such antibodies, use special methods based on the use of antiglobulins (Coombs reaction).
Serological reactions, due to their high specificity and sensitivity, are used for the detection and quantification of antigens and antibodies. The amount of immunoreagents in reactions is expressed by titer - the maximum dilution of serum or antigen at which a reaction is still observed.
Serological reactions in microbiological and immunological laboratories are used for two purposes:
1) for seroidentification of microorganisms, toxins, antigens in general using a known antibody (immune diagnostic serum),
2) for serodiagnosis - determining the nature of the antibody in the patient’s blood serum for bacterial, viral, and less often other infectious diseases using a known antigen (diagnosticum).
To determine the generic, species and type of antigen, known immune tests are required. diagnostic sera. They are obtained by repeated administration to animals (usually rabbits) in increasing doses of killed or live microorganisms, their decay products, neutralized or native toxins. After a certain cycle of immunization of animals, massive bloodletting or total bleeding of the animal is performed. Blood collected in a sterile container is first placed in a thermostat at a temperature of 37°C for 4 - 6 hours to accelerate clotting, then in an icebox for a day. The resulting transparent serum is sucked into a sterile container, preservatives are added, the antibody titer is determined, checked for sterility and poured into ampoules.
Are used non-adsorbed And adsorbed diagnostic sera. Unadsorbed serums have high titers antibodies, but are capable of giving group (cross) reactions.
Adsorbed sera are characterized by strict specificity of action (they react only with a homologous antigen). Sera containing antibodies to only one specific antigen are called monoreceptor.
They also produce serums labeled with fluorochromes, enzymes, and radioisotopes, which make it possible to detect even traces of the antigen with a high degree of accuracy.
Suspensions of living or killed bacteria, their breakdown products, toxins, and viruses are used as antigens (diagnosticums) in serological reactions. In some cases, extracts or chemically isolated antigens from microorganisms and animal tissues are used.
All immunomicrobiological methods can be divided into 3 groups:
1) based on direct interaction of antigen with antibody(phenomena of agglutination, precipitation, hemagglutination, immobilization, etc.);
2) based on mediated interaction of antigen with antibody(reactions indirect hemagglutination, coagglutination, latex agglutination, carbon agglomeration, bentonite agglutination, complement fixation, etc.);
3) using labeled antibodies or antigens(fluorescent antibody method, enzyme-linked immunosorbent and radioimmunoassays and other methods).
AGGLUTINATION REACTIONS
These reactions involve antigens in the form of particles (microbial cells, red blood cells and other corpuscular antigens), which are glued together by antibodies and precipitate.
To perform an agglutination reaction(RA) three components are needed: 1) antigen (agglutinogen);
2) antibody (agglutinin)
3) electrolyte (isotonic sodium chloride solution).
Approximate agglutination reaction (RA)
An indicative, or plate, RA is placed on a glass slide at room temperature. To do this, use a Pasteur pipette to apply a drop of serum at a dilution of 1:10 to 1:20 and a control drop of isotonic sodium chloride solution separately onto the glass. Colonies or a daily culture of bacteria (a drop of diagnosticum) are introduced into both bacteriological loops and mixed thoroughly. Reactions are taken into account visually after a few minutes, sometimes using a magnifying glass (x5). With positive RA, the appearance of large and small flakes in the serum drop is noted; with negative RA, the serum remains uniformly cloudy.
Detailed agglutination reaction in order to identify the titer of specific antibodies in a patient.
Full-blown RA for serodiagnosis is made in the serum of patients. It is also diluted in an isotonic sodium chloride solution from 1:50 - 1:100 to 1:800 or 1:1600. Since lower serum titers may contain normal agglutinins found in healthy people or patients with another diagnosis (diagnostic titer). As an antigen in this reaction, diagnosticums are used - known suspensions, usually of killed bacteria.
1 ml of isotonic sodium chloride solution is first poured into agglutination tubes. 1 ml of serum diluted 1:100 is added to the first of them, and after mixing it, 1 ml is transferred to the second, from the second to the third, etc. 1-2 drops of a bacterial suspension containing 3 billion microbial bodies in 1 ml are added to the resulting two-fold dilutions of sera (from 1:100 to 1:1600 or more). The tubes are shaken and placed in a thermostat at 37°C for 2 hours, then kept at room temperature for 24 hours.
The detailed agglutination reaction is taken into account by evaluating each test tube sequentially, starting with the control ones, with gentle shaking. There should be no agglutination in control tubes. The intensity of the agglutination reaction is marked with the following signs: ++++ - complete agglutination (agglutinate flakes in an absolute transparent liquid); +++ - incomplete agglutination (flakes in a slightly opalescent liquid); ++ - partial agglutination (flakes are clearly visible, the liquid is slightly cloudy); + - weak, questionable agglutination - the liquid is very cloudy, the flakes in it are difficult to distinguish; - - absence of agglutination (the liquid is uniformly cloudy).
The serum titer is taken to be its last dilution, in which the intensity of agglutination is assessed as no less than two pluses (++)
Lecture No. 16
Dictionary
DIRECT – straight next without anything, without subsequent links.
INDIRECTLY– through other cells, not directly.
DECREASE ≠ INCREASE (cell activity).
REJECT – not accept, reject. The body rejects the transplanted organ.
Immune reactions.
Immune reactions are reactions of specific interaction (binding) of an antigen and an antibody or an antigen and a sensitized T-lymphocyte.
These reactions occur in vitro (in a test tube) and in vivo (in a living organism).
These reactions involve serum (serum), which is why they are called serological.
Immune responses are used to diagnosis of infectious diseases. In this case, the unknown component is determined by the known component, which is based on the specificity of the interaction of the antigen with the antibody. If known antibody, then you can identify (discover) an unknown antigen. If known antigen, then you can use it detect unknown antibodies.
Thus, immune responses are used:
1) for serological diagnostics(serodiagnosis) of diseases - detection in the blood serum of sick people of antibodies to a specific causative agent of an infectious disease (known antigen); if antibodies to any pathogen are present in the blood serum of a sick person, then it is this pathogen that caused this infection;
2) for serological identification (seroidentification) of microbes - to determine the type of pathogen using immune diagnostic sera (known antibodies); if antibodies bind a pathogen isolated from a sick person, then this microbe is identical to the one with which the animal was immunized receiving immune diagnostic serum .
Immune reactions occur in 2 phases:
1) specific phase– connection of the active center of the antibody with the determinant group of the antigen with the formation of antigen-antibody complexes; this phase proceeds quickly, but there is no visible effect;
2) nonspecific phase– appearance visible effect interaction of antigen and antibody - loss draft(agglutination) or cloudiness(precipitation), which allows see education antigen-antibody complexes and draw a conclusion about compliance antigen and antibody to each other.
Immune reactions include agglutination reaction (RA), precipitation reaction (RP), complement fixation reaction (CFR).
Agglutination reaction- this is the gluing and precipitation of microbial or other cells (erythrocytes) under the influence of antibodies in the presence of an electrolyte. The visible effect of the reaction (agglutination phenomenon) is the formation of a precipitate called agglutinate.
This reaction is used for serodiagnosis And seroidentification. RA is used for serodiagnosis (detection of antibodies in the blood serum of patients) typhoid fever and paratyphoid(Vidal reaction), brucellosis(Wright's reaction) tularemia and leptospirosis. RA is used for seroidentification (determining the type of pathogen isolated from a patient) when intestinal infections, whooping cough, cholera and etc.
Componentsreactions:
1. A ntigen (agglutinogen) – these are whole (not destroyed) microbial or other cells ( corpuscular, insoluble antigen). Agglutinogens- this is a suspension alive or killed microbial cells or any other cells. Antigens can be either unknown or known. An unknown agglutinogen is a microbial culture isolated from the patient’s body that needs to be determined. Known antigen – diagnosticum– diagnostic drug - suspension of the dead microbes known species in saline solution. This suspension cloudy (opaque), because microbial cells do not dissolve, but remain intact. A known agglutinogen will be used to detect unknown antibodies in the blood serum of patients.
2. Antibody (agglutinin)- found in blood serum. Antibodies can also be either unknown or known. Unknown antibodies to be determined are in the blood serum sick person. Known antibodies are found in immune diagnostic sera which are called agglutinating sera. They are used for sero-identification, i.e. to determine an unknown antigen - a type of microbial culture.
3. Electrolyte– 0.9% sodium chloride solution.
Obtaining a diagnostic test.
A pure culture of a pathogen of a known species grown on slanted agar (for example, the causative agent of typhoid fever) is washed off with 3-4 ml of an isotonic solution, placed in a sterile tube, the microbes are killed in some way (for example, by boiling and the density is determined (there should be 3 billion microbial cells in 1 ml).If microbial cells are killed high temperature, then they get O-diagnosticum (O-antigen), but if it is treated with formaldehyde, then they get H-diagnosticum (H-antigen).
Examples of diagnostics: salmonella diagnosticum, brucellosis diagnosticum, tularemia diagnosticum.
Preparation of agglutinating sera.
Animals (usually rabbits) are parenterally injected with microbial diagnosticums in increasing doses 5–7 times at intervals ( carry out hyperimmunization), and then their blood serum is taken, which contains antibodies to the microbes from which the diagnosticum is prepared. If immunization is carried out with O-diagnosticum, then O-agglutinating sera (contain O-antibodies) are obtained, if with H-diagnosticum, H-agglutinating sera are obtained.
Agglutinating sera can be unadsorbed or native and adsorbed.
Unadsorbed serums contain group antibodies to several closely related species of microbes.
Adsorbed serums contain antibodies to one or more antigens of one type of microbe. If sera contain antibodies to only one antigen, they are called monoreceptor or monovalent, if to several antigens – group adsorbed serums .
Examples of agglutinating sera:Salmonella monoreceptor H-agglutinating serum, Salmonella group adsorbed O-agglutinating serum, anticholera O-agglutinating serum, etc..
Titer agglutinating serum - the highest dilution of serum at which an agglutination reaction with an antigen is still detected (the titer depends on the amount of antibodies in the blood: the more antibodies, the higher the serum titer).
Methods for staging RA.
1. Approximate (lamellar) RA– carried out on glass. Apply 2 drops of serum and 1 drop of isotonic solution to a glass slide. A microbial culture is introduced into one of the drops of serum and into a drop of isotonic solution in a loop and mixed. A drop of isotonic solution with germs – antigen control, a drop germ-free serums – antibody control, a drop serums with microbes – experience. If the serum contains antibodies corresponding to microbial antigens that mix with it, then the antibodies and antigens will specifically bind to each other and after 1–3 minutes agglutinate flakes will appear in the test drop. The antigen control should be cloudy and the antibody control should be clear. The results of the reaction are recorded based on the appearance of agglutinate flakes . If flakes fall out, the reaction is positive, i.e. An antigen corresponds to an antibody and the antigen can be used to determine the antibody or vice versa. If cloudiness remains, the reaction is negative.
2. Detailed agglutination reaction - carried out in test tubes. First, prepare 2-fold dilutions of the blood serum of a sick person from 1:50 to 1:1600. 1 ml of isotonic sodium chloride solution is poured into 6 test tubes. 1 ml of the patient’s blood serum at a dilution of 1:50 is added to the first test tube, mixed and a dilution of 1:100 is obtained, then 1 ml of a dilution of 1:100 is transferred to the second test tube and a dilution of 1:200 is obtained, etc. Two tubes are kept for antigen and serum control. To the serum control add only serum at a dilution of 1:50, to the antigen control - only the antigen. Add 0.1 ml of antigen - diagnosticum (O- or H-) to all other test tubes and place all test tubes in a thermostat at 37°C for 18-20 hours. The results of the reaction are recorded according to the nature, amount of precipitate (agglutinate) formed and the degree of turbidity. Accounting is carried out only with the following results in the controls: serum control - transparent, antigen control - cloudy. O-antibodies give a fine-grained precipitate. H-antibodies – coarse-grained. Based on the last test tube in which the agglutination reaction is still visible, it is established diagnostic titer.
During serodiagnosis diseases, it is important not only to detect specific antibodies to a particular pathogen, but also to identify their quantity, i.e. establish such an antibody titer to When can we talk about the presence of a disease caused by this pathogen? . This titer is called the diagnostic titer. For example, to diagnose typhoid fever, you need to identify an antibody titer of 1:400, but not less. Gives even more accurate results detection of an increase in antibodies in paired sera. The patient's serum is collected at the onset of the disease and after 3 to 5 or more days. If the antibody titer increases at least 4 times, therefore, we can talk about current illness.
If a detailed agglutination reaction is performed for seroidentification, then agglutinating diagnostic sera are used, diluted to the titer or to half their titer. RA is considered positive if agglutination is detected at a dilution close to the titer of the diagnostic serum.
