Approximate agglutination reaction (RA). Agglutination reaction (ra) Microbiology reactions
An agglutination reaction (RA) is the adhesion and precipitation of microbes or other cells under the influence of antibodies in the presence of an electrolyte. The resulting precipitate is called an agglutinate.
RA is used:
1. To detect antibodies in the patient’s blood serum (serodiagnosis).
2. To determine the type and serovar of a pure culture of pathogenic microorganisms isolated from a patient (serotyping).
The agglutination reaction is used to determine antibodies in the blood serum of patients, for example, with typhoid fever and paratyphoid fever (Vidal reaction), brucellosis (Wright, Heddleson reaction), tularemia, leptospirosis and other infectious diseases, as well as to determine the pathogen isolated from a patient (intestinal infections , whooping cough, etc.). RA is used to determine blood groups, Rh factor, etc.
The reaction requires the following components:
1. The antigen (agglutinogen) must be corpuscular, that is, it is a suspension of living or killed microorganisms (diagnostics m), erythrocytes or other cells. Typically, a daily culture of microorganisms grown on agar slants is used. The culture is washed off with 3 - 4 ml of isotonic solution, transferred to a sterile tube, and the density is determined. The suspension must be homogeneous and contain up to 3 billion microbial cells per 1 ml. The use of a suspension of killed microbes - diagnosticums - facilitates the work (prepared in production).
2. Antibodies (agglutinins) are found in the patient’s serum (during serodiagnosis) or in agglutinating serum (during serotyping). Agglutinating sera are obtained by immunizing rabbits with killed bacteria.
Agglutinating titer serum is called its highest dilution, in which it reacts with the corresponding antigen under certain experimental conditions.
Agglutinating sera can be native (non-adsorbed) and adsorbed. Native sera in small dilutions interact not only with the type of microorganisms with which the animal was immunized to obtain the serum, but also with related types of microorganisms, since they contain group antibodies (antibodies to microorganisms that have common antigens). Native sera are used for a detailed agglutination reaction (for serodiagnosis), which takes into account not only the presence of the reaction, but also the dynamics of the increase in antibody titer.
If group antibodies are extracted (adsorbed) from native serum by interaction with related bacteria that have group antigens, adsorbed sera are obtained. Adsorbed sera can be monoreceptor (or type-specific), containing antibodies to only one antigen receptor. Polyvalent sera consist of a mixture of several adsorbed or non-adsorbed sera. Adsorbed sera are used for the glass agglutination reaction.
When animals are immunized with motile bacteria with the H-antigen, H-agglutinating sera containing H-antibodies are obtained (for example, Salmonella monoreceptor H-agglutinating serum). By immunization with O-antigen, O-agglutinating sera containing O-antibodies are obtained (for example, Salmonella group adsorbed O-agglutinating serum, anticholera O-agglutinating serum). By immunization with H- and O-antigens, sera with H- and O-antibodies are obtained.
Moreover, O-agglutinins produce a fine-grained agglutinate, and H-agglutinins produce a coarse-grained sediment.
3. Electrolyte - isotonic NaCl solution (0.9% sodium chloride solution prepared in distilled water).
There are two main methods for performing an agglutination reaction: a reaction on glass (sometimes called an indicative or plate reaction) and a detailed reaction (in test tubes)
Setting up an agglutination reaction on glass. Two drops of serum and a drop of isotonic sodium chloride solution are applied to a fat-free glass slide. Diagnostic agglutinating serum is taken in one dilution, which, depending on its titer, is 1:10, 1:25, 1:50 or 1:100. The culture of the microorganism under study is added into one drop of serum and a drop of isotonic solution using a loop and mixed thoroughly. A drop of sodium chloride with microorganisms is antigen control, a drop of serum without microorganisms is serum control. You cannot transfer the culture from a drop with serum to a drop with NaCl. The reaction takes place at room temperature for 1-3 minutes. If the serum control remains clear, uniform turbidity is observed in the antigen control, and agglutinate flakes appear in the drop where the culture is mixed with serum, then the result is considered positive. If there is uniform turbidity in the drop with serum and antigen, then this is a negative result. The reaction is more clearly visible against a dark background.
Serum
1. antigen control
2. serum control
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 diagnosis (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 infectious disease;
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 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 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 with high temperature, then an O-diagnosticum (O-antigen) is obtained, but if it is treated with formalin, then an H-diagnosticum (H-antigen) is obtained.
Examples of diagnostics: salmonella diagnosticum, brucellosis diagnosticum, tularemia diagnosticum.
Preparation of agglutinating sera.
Animals (usually rabbits) are parenterally administered 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 added in a loop into one of the drops of serum and into a drop of isotonic solution and mixed. A drop of isotonic solution with germs – antigen control, drop germ-free serums – antibody control, 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 based on 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 determined 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.
Immune reactions. Application of immune reactions in the diagnosis of infectious diseases.
PLAN:
Types of immune reactions.
Conditions for conducting serological reactions.
Serum requirements.
The concept of positive and negative results.
MAIN CONTENTS:
Types of immune reactions.
Immunological reaction – This is the interaction of an antigen with an antibody, which is determined by the specific interaction of the active centers of the antibody (paratope) with epitopes of antigens.
General classification of immunological reactions:
serological reactions – reactions between antigens (Ag) and antibodies (Ig)
in vitro ;
cellular reactions with the participation of immunocompetent cells;
allergy tests – detection of hypersensitivity.
Serological reactions: 1) definition, 2) phases, 3) goals, 4) general classification.
1) Definition
Serological research methods (from the Latin Serum - serum and logos - study) using the antigen-antibody reaction.
2) Phases
2 phases of interaction:
I. Specific (visible) – occurs quickly, antibodies combine with their corresponding antigens. During this phase, determinant groups of antigens (AG) and active centers of antibodies (AT) interact.
The forces involved in the formation of the AG + AT complex are:
pendant;
van der Waals
Hydrogen bonds.
There are no visible changes in this phase. Electron microscopy shows the AG+AT complex in the form of a lattice.
II. Nonspecific – occurs slowly, the resulting antigen-antibody complex reacts with an additional nonspecific factor of the environment in which the reaction occurs, and this is visible to the eye – gluing, dissolution, precipitation of sediment flakes, etc. In the presence of an electrolyte, the charge decreases, solubility decreases, visible conglomerates are formed , precipitating (agglutinate).
3) Setting goals :
a) to identify the antigen (antibody known diagnostic serum):
serological identification (species identification);
serotyping (determination of serovar) – determination of the strain;
in pathological material (rapid diagnostics);
in pure culture:
b) to detect antibodies (Ig) (antigen is known-diagnosticum):
presence (qualitative reactions);
quantity (increase in titer - “paired serum” method).
4) General classification serological reactions :
a) simple (2-component: Ag+Ig):
RA agglutination reactions (with corpuscular antigen);
PR precipitation reactions (with soluble antigen);
b) complex (3-component: Ag+Ig+C);
c) using a tag.
Variants of agglutination and precipitation reactions
Agglutination reaction :
Agglutination reaction (RA) is an immune reaction of the interaction of a suspension of antigens (erythrocytes, bacteria) with antigens in a physiological solution.
During agglutination, AT particles stick together to form a flocculent sediment.
The passive hemagglutination reaction (RPHA) is a type of agglutination reaction in which an antibody or antigen erythrocyte diagnosticum (erythrocytes with AT or AG adsorbed on their surface) is used.
Red blood cells act as passive carriers in this reaction.
The evaluation of the results of the RPGA is carried out as follows:
- at positive reaction passively glued red blood cells cover the bottom of the U- or V-shaped hole in an even layer with scalloped edges (“umbrella”);
- at negative reaction (in the absence of agglutination), red blood cells accumulate in the central recess of the hole, forming a compact “button” with sharply defined edges.
The hemagglutination inhibition test (HAI) is used in the diagnosis of viral infections. Some viruses contain a protein called hemagglutinin on their surface, which glues red blood cells together. The addition of specific antiviral antibodies blocks viral hemagglutinin - there is no hemagglutination.
The indirect hemagglutination reaction (IRHA), or Coombs reaction, is used to determine incomplete antibodies. The addition of antiglobulin serum (AT against human Ig) enhances the results of the reaction. RNGA is used to determine the Rh factor.
To perform an agglutination reaction (RA), three components are required:
1) antigen (agglutinogen) AG;
2) antibody(agglutinin) AT;
3)
electrolyte
(isotonic sodium chloride solution).
Ag + AT + electrolyte = agglutinate
Agglutination (from Latin agglutinatio - gluing) - gluing of corpuscles (bacteria, red blood cells, etc.) with antibodies in the presence of electrolytes - sodium chloride.
RA manifests itself in the form of flakes or sediment consisting of corpuscles (for example, bacteria, red blood cells) “glued together” with antibodies.
RA is used for:
Direct microbial agglutination reaction (RA).
In this reaction, antibodies (agglutinins) directly agglutinate corpuscular antigens (agglutinogens).
They are usually represented by a suspension of inactivated microorganisms (microbial agglutination reaction).
To determine the type of microorganisms, usestandard diagnostic agglutinating serum ( famous AT ).
The most common are lamellar (approximate) and expanded RA.
The plate RA is placed on the glass. It is used as an accelerated method for detecting antibodies or identifying microorganisms.
Components:
1. standard diagnostic agglutinating sera (AT);
2. pure culture under study from the patient;
3. saline solution.
In the pure culture under study, antigens (AG) are in the form of particles (microbial cells, erythrocytes and other corpuscular antigens), which are glued together by antibodies and precipitate.
Example:
Staging indicative agglutination reactions (RA ) on glass for the purpose of identifying coliform bacteria.
Apply drops onto a glass slide:
1
dysentery
;
2
-th drop: - agglutinating serum against pathogenstyphoid fever
;
(1-2 diagnostic sera)
3
-th drop: - saline solution (control).
Add the tested pure bacterial culture to each drop. Stir.
Result
:
positive
- presence of agglutinate flakes,
negative
- absence of agglutinate flakes
Conclusion:The bacteria studied are causative agents of typhoid fever (antigens were determined).
To determine AT in the patient’s serum (serological diagnosis), a standard microbialdiagnosticum , containing suspensionfamous microbes or their antigensAG .
Determination of ABO blood groups (hemagglutination reaction (HRA)) – agglutinate red blood cells.
Reaction components:
1. AG (red blood cells) test blood
2. AT (erythrotests - zoliclones)
Set of zoliclons:
Reagent Tsoliklon anti-A (pink)
Coliclone anti-B reagent (blue)
Reagent Tsoliklon anti-AV (colorless)
3. electrolyte (saline solution)
Determination technique:
1
.
One drop (0.1 ml) of anti-A, anti-B and anti-AB zolicone is applied to the wells of the tablet (for control).
2.
Next to each drop of the reagent, a small (0.05-0.01 ml) drop of the blood being tested is applied.
Then a drop of zoliclone is mixed with a drop of blood using an individual clean glass rod.
3.
The agglutination reaction develops during the first 3-5 seconds with gentle rocking of the plate.
The reaction results are taken into account 2.5 - 3 minutes after mixing the drops. From left to right in the wells are anti-A, anti-B, anti-AB.
A positive result is the appearance of a granular sediment (agglutinate).
positive RA (+)
Negative - no sediment.
negative RA(-)
4.
Analysis of results.
O(I) α β – no agglutination
A(II) β – agglutination with anti-A
B(III) α – agglutination with anti-B
AB(IV)O – agglutination with anti-A, with anti-B
Schematic representation of agglutination.
Ag antigens on erythrocytes (detectable) + antibodyAT(zoliclon) diagnostic serum
Accounting for agglutination in tablets
Precipitation reaction:
Precipitation reaction is an immune reaction of the interaction of antigen in a soluble state with antigen in a physiological solution.
During precipitation, a macromolecular immune complex is formed, which is manifested by the transition of a transparent colloidal solution into an opaque suspension, or precipitate.
The amount of both reagents must be in strictly defined proportions, since an excess of one of them reduces the result.
There are various ways to perform the precipitation reaction.
1. The ring precipitation reaction is carried out in precipitation tubes with a small diameter. The immune serum is added to the test tube and the soluble antigen is carefully layered. AG and AT mix due to the thermal movement of molecules, and they interact. If the result is positive, a ring of opaque precipitate forms at the interface of the two solutions.
2. The Ouchterlony double immunodiffusion reaction is carried out in an agar gel, into the wells of which either an AG solution or an AT solution is added according to the scheme. AG and AT diffuse into the gel towards each other and, if the reaction is positive, form immune complexes visible as precipitation lines.
Precipitation reaction –this is formationand precipitation of the soluble molecular antigen-antibody complex as a cloud called precipitate. It is formed by mixing antigens and antibodies in equivalent quantities.
RA components:
precipitating serum (known AT-precipitin);
test serum (unknown precipitinogen antigen);
physical Solution.
The precipitation reaction is carried out either in special narrow test tubes (ring precipitation reaction), or in Petri dishes in gels, nutrient media, etc.
Ring-pricipitation reaction
Statement and recording of reaction resultsring precipitationto detect the causative agent of anthrax (Ascoli reaction).
Staging .
1. The material under study (leather, wool, felt, bristles, cloth, meat, soil, animal feces, etc.) is boiled in saline solution for 5-45 minutes. to obtain an isotonic extract (extract). Filtered.
2. Precipitating anti-anthrax serum is poured into a test tube.
3. Carefully layer the test material (extract) onto it.
Accounting .
Within the next 10 minutes. In positive cases, a ring of turbidity appears at the interface between serum and extract (ring precipitation). The Ascoli reaction is very sensitive and specific
With its help, it is possible to quickly identify materials infected with anthrax.
Precipitation reaction in agar
Statement and recording of resultsprecipitation reactions in agarto determine the toxigenicity of corynebacteria (pathogens of diphtheria)
Staging
Placed on phosphate peptone agar in a Petri dish.
1. Place a strip of sterile filter paper moistened along the middle of the cup.antitoxic serum.
2. After drying, at a distance of 1 cm from the edge of the strip, plaques with a diameter of 10 mm are seededselected crops.
In one cup you can sow from 3 to 10 crops, one of whichcontrol, must be knowntoxigenic.
The crops are placed in a thermostat.
Accounting
The analysis is carried out after 24-48-72 hours.
Positive result - (culturetoxigenic) - at some distance from the strip of paper appearprecipitate lines, « tendril arrows", which are clearly visible in transmitted light.
The figure shows the precipitation reaction in agar to determine the toxigenicity of diphtheria bacilli. Medium cultures did not form “arrow-tendrils”; these are not toxicogenic pathogens.
Strains of the causative agent of diphtheria can be toxigenic (producing exotoxin) and non-toxigenic. The formation of an exotoxin depends on the presence in bacteria of a prophage carrying a tox gene encoding the formation of an exotoxin.
In case of illness, all diphtheria pathogens are tested for toxigenicity - production of diphtheria exotoxin using the precipitation reaction in agar
Complex serological reactions ( 3-component: Ag+Ig+C):
Complement fixation reaction (CFR).
The reaction is carried out in two stages.
At the first stage, AT interacts with AG and complement; at the second stage, an indicator is added - the hemolytic system (a mixture of erythrocytes and anti-erythrocyte serum).
If the result is positive, at the first stage, the antibodies form an immune complex with the antigens, which binds the complement of the reaction mixture.
In this case, the red blood cells of the hemolytic system added at the second stage are not destroyed.
Otherwise, unbound complement causes lysis of indicator red blood cells.
To carry it out, five ingredients are needed: AG, AT and complement (first system), sheep erythrocytes and hemolytic serum (second system) (Fig. 1).
The reaction occurs in two phases (Fig. 3).
First phase - interaction of antigen and antibodies with the obligatory participation of complement.
Second - identification of reaction results using an indicator hemolytic system (sheep red blood cells and hemolytic serum). The destruction of red blood cells by hemolytic serum occurs only if complement is added to the hemolytic system. If complement was previously adsorbed on the antigen-antibody complex, then hemolysis of erythrocytes does not occur (Fig.).
Result of the experiment
assessed (Fig. 2), noting the presence or absence of hemolysis in all tubes. The reaction is considered positive when hemolysis is completely delayed, when the liquid in the test tube is colorless and red blood cells settle to the bottom, negative - when red blood cells are completely lysed, when the liquid is intensely colored (“varnish” blood).
The degree of hemolysis delay is assessed depending on the intensity of the color of the liquid and the size of the red blood cell sediment at the bottom (++++, +++, ++, +).
Rice. 4. Statement and result of RSC.
Conclusion:Antibodies were detected in the test serum.
RSK allows you to detect antibodies to any strain of the same serotype of the virus.
Diagnostic value has:
a fourfold increase in antibody titer in paired sera (during an influenza epidemic);
a twofold increase in the blood serum of patients with a characteristic clinical picture.
Reactions using a tag :
These methods are highly sensitive. Dyes, radioactive isotopes, enzymes, etc. are used as labels for antigens or antibodies.
RIF – immunofluorescence reaction
The immunofluorescence reaction is based on light indication of the antigen-antibody complex
Enzyme immunoassay.
A modern laboratory test that searches for specific antibodies in the blood or antigens to specific diseases in order to identify not only the etiology, but also the stage of the disease.
ELISA results can be given qualitatively and quantitatively.
Currently, ELISA is used in the following situations:
1) search for specific antibodies to any infectious disease;
2) search for antigens of any diseases (infectious, venereological);
3) study of the patient’s hormonal status;
4) examination for tumor markers;
5) examination for the presence of autoimmune diseases.
In the figure, solid-phase ELISA - known antigens (on the left) adsorbed on the well of the plate, (on the right) on the wells of the plate known antigens
Advantages of the ELISA method:
1) High specificity and sensitivity of the ELISA method (more than 90%).
2) The ability to determine the disease and track the dynamics of the process, that is, comparing the number of antibodies in different time periods.
3) Availability of ELISA diagnostics in any medical institution.
Relative disadvantage: Detection of an immune response (antibodies), but not the pathogen itself, conjugated to a tag enzyme.
ELISA test (general mechanism):
The basis of enzyme immunoassay is the immune reaction of antigen and antibody with the formation of an immune complex: antigen-antibody, which results in a change in the enzymatic activity of specific marks on the surface of the antibodies.
Reaction components:
1. AG(AT) known - on the well of the tablet.
2. AT (AG) being studied.
3. AT with enzyme, specific to the AT(AG)-AG(AT) complex
4. chromogenic substrate that interacts with the enzyme
5. stop solution
Main stages of ELISA
1. On the surface of the wells of the plate there is a purified antigen of a specific pathogen. The patient's biological material is added to them, and a specific reaction occurs between this antigen and the desired antibody (immunoglobulin). A complex is formed.
2. A conjugant is added – AT with the enzyme. The conjugant is specific to the AT-AG complex of the first stage. The enzyme is activated.
3. The substrate is added and the active enzyme reacts with it, changing the colorless color of the solution.
4. A stop solution is added to stop the enzyme-substrate interaction.
Accounting.
A positive result is a change in color, yellow in the picture.
Immunochromatographic analysis
The immunochromatographic analysis method (ICA, rapid tests) is a high-quality preliminary screening method that allows you to quickly, within a few minutes, carry out analysis under any conditions, incl. "field".
The advantages of ICA include:
Speed and ease of use;
Small sample volumes, lack of sample preparation;
Cheapness for the manufacturer and consumer;
Possibility of producing tests in large volumes;
Ease of reading and interpretation of the result;
High sensitivity and reproducibility;
Possibility of quantitative determination;
Possibility of using portable readers compatible with a computer;
Possibility of multi-analysis.
Components (applied to the test strip):
1. A conjugate labeled with colloidal gold is specific to the detected antigen.
2. AT test line – specific to the AT-AG complex
3. Abs of the control line are specific to the conjugate.
ICA setting:
1.Apply the sample to the designated starting area of the strip.
2. Obtaining the result in the form of the appearance of colored stripes in place of the test and control lines.
Accounting
Positive – when the test line is stained.
Negative - if there is no staining of the test line.
Invalid – if the control line is not stained.
General mechanism of ICA:
1. The sample is introduced onto the starting field (sample pad) and is associated with the conjugate (a specific body with a colored label), which are located on the conjugate pad. As a result, a colored complex is formed.
2. The resulting colored immune complex moves under the action of capillary forces along the nitrocellulose membrane And interactswith AT test line.The result is a single colored pink-red stripe.
3. AT (conjugate) not bound on the tested bandmoves further and reaches the control line, communicates with the AT of the control line.As a result, a second colored stripe appears.If the analysis is carried out correctly, the Control line should always appear, regardless of the presence of the test antigen (antibody) in the biological fluid sample.
2. Conditions for conducting serological reactions.
1. The presence of homologous - corresponding to each other antigen and antibody.
2. Clean, dry dishes.
3. A certain ratio of drugs (most often equal).
4. Mandatory presence of an electrolyte (isotonic NaCl solution).
5. pH neutral or close to slightly alkaline.
6. Temperature +37°C or room temperature (necessarily positive).
7. Antigen control and serum (antibody) control are carried out.
3 Serum requirements
The serum should be completely transparent without any admixture of cells.
They usually receive it at 2 weeks of illness, when antibodies are already available.
Blood is taken in an amount of 3-5 ml on an empty stomach or 6 hours after a meal.
To obtain serum, the blood is left for 1 hour at room temperature or centrifuged. The serum is sucked out very carefully so as not to capture the formed elements.
Immune serums are obtained from the blood of people or animals (usually rabbits and horses), immunized according to a certain scheme with the corresponding antigen (vaccine). Serums are usually prepared in production.
4. The concept of positive and negative results.
RA.
With a positive reaction, passively glued red blood cells cover the bottom of the hole in an even layer with scalloped edges (“umbrella”); in the absence of agglutination, red blood cells accumulate in the central recess of the hole, forming a compact “button” with sharply defined edges (see pictures above).
RP.
If the result is positive, a milky ring forms at the interface of the two solutions (see pictures above).
ELISA.
A change in the color of the solution occurs with a positive reaction.
RSK.
Delayed hemolysis - the reaction is positive; if complement is free, hemolysis is observed - the reaction is negative(see pictures above).
Results of the Wasserman reaction:
a - complete delay of hemolysis (+ + ++);
b - pronounced delay in hemolysis (+ ++);
c - partial delay of hemolysis (++);
d - slight delay in hemolysis (+);
d - complete hemolysis (-).
The reaction is positive with partial, pronounced and complete delay of hemolysis, determined by the degree of staining of the contents of the tubes from light pink to bright red; non-hemolyzed erythrocytes subsequently form a red precipitate.
Homework:
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Agglutination is the gluing of bacteria as a result of the interaction of specific ATs with them. To carry out RA, three components are required: 1) AG (agglutinogen); 2) AT (agglutinin); 3) electrolyte solution (isotonic sodium chloride solution). Only corpuscular antigens (bacteria, red blood cells, antigen-loaded latex particles) take part in the agglutination reaction.
Agglutination reaction on glass. A drop of diagnostic serum is applied to a fat-free glass slide with a pipette (serum dilution 1:10 - 1:20). Using a bacteriological loop, a pure culture of the microorganism under study is taken from the surface of the slanted agar, transferred to a drop of serum and mixed. The result of the reaction is taken into account with the naked eye after 3-5 minutes. If the reaction is positive, the appearance of flakes (large or small) is noted in a drop of serum, clearly visible against a dark background when the slide is shaken. In case of a negative reaction, the liquid remains uniformly cloudy.
Agglutination reaction in test tubes. 1 ml of physiological solution is added to a row of test tubes. An equal volume of the blood serum being tested is added to the first test tube. Serial two-fold dilutions of serum are prepared (serum titration), after which 2 drops of a suspension of inactivated bacteria (diagnosticum) are added to each test tube. The tubes are placed in a thermostat at 37 °C for 2 hours. The reaction proceeds with the formation of small flakes, invisible to the naked eye, so the results are recorded under slight magnification in a special device - an agglutinoscope. The intensity of agglutination is taken into account according to the “four plus” system: complete agglutination - 4+, partial agglutination - 3+ or 2+, questionable result - +. The last dilution in which agglutination of 2+ is observed is taken as the antibody titer in the test serum.
An agglutination reaction in test tubes (an extensive agglutination reaction) is carried out to determine the titer of antibodies to the causative agents of typhoid and paratyphoid fever (Vidal reaction), brucellosis (Wright reaction), and typhus (Weigl reaction).
Agglutination (from Latin agglutinatio - gluing) - gluing (connection) of antigen-bearing corpuscular particles (whole cells, latex particles, etc.) with molecules of specific antibodies in the presence of electrolytes, which ends in the formation of flakes or sediment (agglutinate) visible to the naked eye. The nature of the sediment depends on the nature of the antigen: flagellated bacteria produce a coarse flocculent sediment, flagellate and noncapsular bacteria produce a fine-grained sediment, and capsular bacteria produce a stringy sediment. A distinction is made between direct agglutination, in which the own antigens of a bacterial or any other cell, such as erythrocytes, directly participate in the interaction with specific antibodies; and indirect, or passive, in which bacterial cells or erythrocytes, or latex particles are carriers not of their own, but of foreign antigens (or antibodies) sorbed on them to identify antibodies (or antigens) specific to them. The agglutination reaction mainly involves antibodies belonging to the IgG and IgM classes. It occurs in two phases: first, a specific interaction between the active center of antibodies and the antigen determinant occurs; this stage can occur in the absence of electrolytes and is not accompanied by visible changes in the reacting system. For the second stage - the formation of an agglutinate - the presence of electrolytes is necessary, which reduce the electrical charge of the antigen + antibody complexes and accelerate the process of their gluing. This phase ends with the formation of an agglutinate.
Agglutination reactions are performed either on glass or smooth cardboard plates, or in sterile agglutination tubes. Agglutination reactions (direct and passive) on glass are usually used as an accelerated method for detecting specific antibodies in the patient’s serum (for example, with brucellosis) or for serological identification of the pathogen. In the latter case, well-purified (adsorbed) diagnostic sera are usually used, containing only monoreceptor antibodies or a set of them to various antigens. The undoubted advantage of the agglutination reaction on glass is the simplicity of its implementation and the fact that it takes several minutes or even seconds, since both components are used in concentrated form. However, it has only a qualitative value and is less sensitive than a test tube. An extensive agglutination reaction in test tubes gives more accurate results, because it allows you to determine the quantitative content of antibodies in the serum (establish its titer) and, if necessary, register the fact of an increase in antibody titer, which has diagnostic value. To set up the reaction, a serum diluted in a certain way with a 0.85% NaCl solution and an equal volume (usually 0.5 ml) of a suspension of a standard diagnosticum (or test culture) containing 1 billion bacteria in 1 ml are added to agglutination tubes. The results of the agglutination reaction are recorded first after 2 hours of incubation of the tubes at 37 °C and finally after 20-24 hours according to two criteria: the presence and size of the precipitate and the degree of transparency of the supernatant liquid. The assessment is carried out according to the four-cross system. The reaction is necessarily accompanied by serum and antigen control. In cases where a detailed agglutination reaction in a test tube is performed for serological identification of the pathogen, it has diagnostic value if the reaction is assessed as positive when the diagnostic serum is diluted to at least half its titer.
It must be taken into account that when mixing solutions of homologous antigens and antibodies, visible manifestations of the agglutination reaction are not always observed. A precipitate forms only at certain optimal ratios of both reaction components. Outside these limits, with a significant excess of antigen or antibodies, no reaction is observed. This phenomenon is called the “prozone phenomenon.” It is observed both in the agglutination reaction and in the precipitation reaction. The appearance of a prozone in immune reactions is explained by the fact that the antigens involved in them, as a rule, are polydeterminant, and IgG antibody molecules have two active centers. With an excess of antibodies, the surface of each antigen particle is covered with antibody molecules so that there are no free determinant groups left, so the second, unbound active center of the antibodies cannot interact with another antigen particle and bind them to each other. The formation of a visible agglutinate or precipitate is also suppressed when there is an excess of antigen, when not a single free active center of the antibodies remains, and therefore the antigen + antibody + antigen complexes can no longer enlarge.
Options for accelerated agglutination reactions. Passive hemagglutination reaction and its variants
The classic agglutination reaction involves the use of corpuscular antigens. However, soluble antigens may also be involved. To make this possible, such antigens are adsorbed on immunologically inert particles. Particles of latex or bentonite can be used as a carrier, but currently animal or human erythrocytes are most often used, improving their adsorbing properties by treating them with solutions of tannin, formalin or benzidine. Red blood cells that have adsorbed an antigen on themselves are called sensitized by this antigen, and the immune reaction in which they participate is called an indirect or passive hemagglutination reaction (IRHA, or RPHA), since red blood cells participate in it passively.
RPGA is placed in special polystyrene plates with holes having a hemispherical bottom. When using it for serological diagnostics, two-fold dilutions of the test serum are prepared in these wells in physiological solution, and then a suspension of sensitized erythrocytes is added to it as a diagnostic agent. The results are recorded after 2 hours of incubation at 37 °C using the four-cross system. With a positive reaction, agglutinated red blood cells settle to the bottom of the hole and evenly cover it in the form of an inverted umbrella. If the reaction is negative, the red blood cells also settle, the liquid becomes clear, and the sediment looks like a small “disc” in the center of the well. The serum titer in RPHA is considered to be its last dilution, which still gives pronounced hemagglutination without significant signs of the presence of a “disc”.
RPGA can also be used as an accelerated method of bacteriological diagnostics to detect directly in the test material unknown bacteria, viruses, toxins, for example, plague pathogens, staphylococcal enterotoxins, etc. With this version of RPGA, erythrocytes that have adsorbed known for their specificity are used as a known component of the reaction antibodies - antibody erythrocyte diagnosticum. If the test material contains a sufficient amount of a known antigen, RPGA will be positive.
Options for using RPHA are: antigen neutralization reaction (RNAg), antibody neutralization reaction (RNAb), passive hemagglutination inhibition reaction (PHA). For these reactions, antigen and antibody erythrocyte diagnostics are used. Two mutually controlling unidirectional reactions can be used simultaneously, for example, RPHA with an antigen diagnosticum and RNAg with an antibody erythrocyte diagnosticum.
The antibody neutralization reaction (RNAb) consists of mixing a suspension containing the desired antigen with a specific immune serum containing known antibodies in appropriate volumes and incubating at 37 °C for two hours. After this, an antigenic erythrocyte diagnosticum is added. The mixture is shaken and left at room temperature. The results are taken into account after 3-4 hours and finally after 18-24 hours. If the test material contains antigen, it will bind antibodies (neutralize them), and therefore hemagglutination will not occur.
The antigen neutralization reaction (RNAg) is performed using the same principle. Only in this case are antibodies detected in the test material. A specific antigen added to such a test material will bind to the antibodies contained in it, i.e., neutralization of the antigen by antibodies will occur, and therefore hemagglutination will not occur when adding an antibody erythrocyte diagnosticum.
Coagglutination reaction. It is one of the options for a passive, i.e., accelerated agglutination reaction on glass mediated by antibody-carrying cells. This reaction is based on the unique property of Staphylococcus aureus, which contains protein A in its cell wall, to bind to the Fc fragments of IgG and IgM. In this case, the active centers of antibodies remain free and can interact with specific determinants of antigens. A drop of a 2% suspension of staphylococci, sensitized with appropriate antibodies, is applied to a glass slide, and a drop of a suspension of the bacteria being studied is added. If the antigen matches the antibodies, a clear agglutination of the staphylococci loaded with antibodies occurs within 30-60 s.
Latex agglutination reaction (LAR). The carrier of antibodies in this diagnostic system are small standard latex particles. The reaction is performed using the micromethod in wells on glass. The main condition for the successful staging of PAH is strict adherence to the quantitative ratios of the system components: 10 μl of a latex preparation sensitized with antibodies is added to 50 μl of the test material. The specificity of PAH is controlled using three control tests contained in commercial test systems: a known positive reaction, a known negative reaction, and quality control of latex suspensions using PAH-unsensitized (not carrying antibodies) latexes with the test material. In our country, polystyrene monodisperse latexes with different particle diameters (0.3; 0.66; 0.75; 0.8 μm) are used as carriers of specific antibodies. LAG is used for rapid detection of microorganisms or their antigens in the test material.
Immunomagnetic detection of antigens. One of the options for accelerated agglutination reaction on glass is associated with the use of supermagnetic polymer particles coated with specific antibodies. One such particle binds up to 107-108 microbial cells, due to which the sensitivity of this method reaches 5 CFU/ml. Immunomagnetic detection of microorganisms can be used in combination with CPR.
Aggregate hemagglutination reaction (AHA). Allows you to quickly detect in the blood of patients both freely circulating antigens (antigenemia) and antigens associated with antibodies - circulating immune complexes (CIC). For RAHA, red blood cells sensitized with appropriate antibodies are used. The addition of the patient's blood serum, which contains antigens, to sensitized erythrocytes on which antibodies are fixed, leads to gluing (agglutination) of erythrocytes and immune complexes.
Antiglobulin Coombs test (R. Coombs reaction). Full (divalent) antibodies are determined using direct and passive agglutination reactions. Incomplete (monovalent, blocking) antibodies are not detected by these methods, since, when they combine with the antigen, they block it, but cannot cause aggregation of the antigen into large conglomerates. Incomplete (blocking) antibodies are those in which only one active center functions; the second active center does not work for an unknown reason. To detect incomplete antibodies, a special Coombs reaction is used (Fig. 72). The reaction involves: the patient’s serum, in which incomplete antibodies are determined, corpuscular antigen-diagnosticum, antiglobulin serum containing antibodies to human globulin. The reaction occurs in two stages:
Interaction of antigen with incomplete antibodies. There are no visible manifestations. The first stage is completed by washing the antigen from the remaining serum of the patient.
Interaction of antiglobulin serum obtained as a result of immunization of an animal with human globulin with incomplete antibodies adsorbed on the antigen. Due to the fact that antiglobulin antibodies are bivalent, they bind two monovalent antibodies of separate complexes of antigen + incomplete antibody, which leads to their gluing and the appearance of a visible precipitate.
Agglutination is the gluing and precipitation of microbes or other cells under the influence of antibodies in the presence of an electrolyte (isotonic sodium chloride solution). Groups of glued bacteria (cells) are called agglutinate. The following components are required for the agglutination reaction:
1. Antibodies (agglutinins) that are found in the serum of a sick or immune animal.
2. Antigen - a suspension of living or killed microbes, red blood cells or other cells.
3. Isotonic (0.9%) sodium chloride solution.
The agglutination reaction for serodiagnosis is used for typhoid fever and paratyphoid fever (Vidal reaction), brucellosis (Wright and Heddleson reaction), tularemia, etc. The antibody is the patient's serum, and the antigen is a known microbe. When identifying microbes or other cells, their suspension is used as an antigen, and a known immune serum is used as an antibody. This reaction is widely used to diagnose intestinal infections, whooping cough, etc.
Methods for staging RA
Approximate RA on glass
Deployed RA
(volume method)
Coagglutination reaction
Unfolded RA on glass (seroidentification)
Agglutination reaction on glass. Two drops of specific (adsorbed) serum and a drop of isotonic sodium chloride solution are applied to a fat-free glass slide. Non-adsorbed serums are pre-diluted in a ratio of 1:5 - 1:100. Drops must be applied to the glass so that there is a distance between them. The culture is thoroughly ground with a loop or pipette on glass, and then added to a drop of isotonic sodium chloride solution and one of the serum drops, stirring in each until a homogeneous suspension is formed. A drop of serum without culture is a serum control.
Attention! You cannot transfer the culture from the serum to a drop of isotonic sodium chloride solution, which is an antigen control. The reaction takes place at room temperature for 1-3 minutes. If the serum control remains transparent, a uniform turbidity is observed in the antigen control, and agglutinate flakes appear in the drop where the culture is mixed with serum against the background of a clear liquid, the reaction result is considered positive.
Diagnostic Physiological
serum + culture solution + culture
Detailed agglutination reaction (volume method). Serial, most often twofold, dilutions of serum are prepared. The method is called volumetric. To determine the antibody titer in blood serum, take 6 tubes. Pour 1 ml of the original serum dilution 1:50 into the first test tube and add 1 ml of saline solution into all 6 test tubes using a graduated pipette. The first test tube will yield a serum dilution of 1:100 with a volume of 2 ml. Transfer 1 ml from the first test tube to the second test tube, where the dilution becomes 1:200. So make a series of serial dilutions of the serum in the first 5 test tubes (1:100, 1:200, 1:400, 1:800, 1:1600). From the fifth test tube, pour 1 ml into the disinfectant solution. Add 2 drops of diagnosticum to all 6 test tubes. The sixth tube is a culture control, as it contains only saline solution and diagnosticum.
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serum control |
control diagnostic-kuma |
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Patient's serum | |||||||
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dilution 1:50 | |||||||
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Such control is necessary to exclude spontaneous agglutination of the culture. The tubes are shaken and placed in a thermostat at a temperature of 37°C for 2 hours, and then left for a day at room temperature, after which the results of the agglutination reaction are recorded. When performing an agglutination reaction with the sera of children in the first months of life, due to the functional inferiority of antibody formation, it is necessary to identify lower antibody titers, which is taken into account when diluting the serum. The initial serum dilution is 1:25. In the first test tube, a dilution of 1:50 is obtained, then 1:100, etc.
If the reaction result is positive, the test tubes show stuck cells in the form of grains or flakes against the background of a clear liquid. The agglutinate gradually settles to the bottom in the form of an “umbrella”, and the liquid above the sediment becomes clear. The antigen control is uniformly cloudy.
Based on the nature of the sediment, fine- and coarse-grained (flaky) agglutination is distinguished. Fine-grained agglutination is obtained when working with O-sera. Coarse-grained - when motile microbes interact with flagellar H-sera. It occurs faster than fine-grained, and the resulting sediment is very loose and easily broken.
The intensity of the reaction is expressed as follows:
All the cells have settled, the liquid in the test tube is completely transparent. The result of the reaction is sharply positive;
There is less sediment, the liquid does not clear completely. The result of the reaction is positive;
There is even less sediment, the liquid is cloudier. The result of the reaction is doubtful;
There is a slight sediment at the bottom of the test tube, the liquid is cloudy. Questionable reaction result;
There is no sediment, the liquid is uniformly cloudy, as in the antigen control. Negative reaction result
