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 agglutination reaction 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 |
|
|
belonging |
||
|
researched |
||
|
red blood cells with |
serum (plasma) |
|
|
standard |
with standard |
|
|
serums | ||
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 others 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
Immune reactions. Application of immune reactions in diagnostics 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 methods research (from the Latin Serum - serum and logos - teaching) 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.
None visible changes not 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 environmental factor in which the reaction occurs, and this is visible to the eye – gluing, dissolution, precipitation 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);
quantities (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 physiological solution.
During agglutination, AT particles stick together to form a flocculent sediment.
Reaction passive hemagglutination(RPGA) 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 adhered 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.) by 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” by 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. Use it as accelerated method detection of antibodies or identification of 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 studied bacteria 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:
Coliclone anti-A reagent (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 when the plate is gently rocked.
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.
Exist various ways staging 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. At positive result a ring of opaque precipitate forms at the boundary 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 precipitationfor pathogen detection 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 (causative agents 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 antigen 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 during mandatory participation 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.).
Experience result
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 It 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
Linked immunosorbent assay.
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) research hormonal status patient;
4) examination for tumor markers;
5) examination for the presence of autoimmune diseases.
In the figure, solid-phase ELISA shows known antigens (on the left) adsorbed on a well of a plate, (on the right) on wells of a 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. They add biological material patient, 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.
Positive result - change colors, in the picture - yellow.
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. The conjugate with a colloidal gold label 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 the 2nd week 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.
1. Study the material
Make 3 notes on the video
in microbiology
"Agglutination reaction and its types (RA)"
Plan:
1. Introduction………………………………………………………………………………..3
2. RA on glass……………………………………………………………………………….4
3. Test tube RA……………………………………………………………………………………….5
4. Literature used…………………………………………………………………..7
1. Introduction.
The interaction of microbial antigen and antibodies is strictly specific in nature and is aimed in the animal body at neutralizing the pathogen and its toxins. The interaction of antigen and antibodies in vitro, under certain conditions, is accompanied by visible phenomena (agglutination, precipitation, immune lysis), which allows the use of AG-AT reactions, called serological (from the Latin serum), for practical purposes. Biofactories produce antigens and immune sera (antibodies) of a known specific nature (diagnostic). Using such sera in serological reactions, it is possible to identify an unknown microorganism or, using a known antigen, to detect in the body antibodies synthesized in response to the introduction of a pathogen, and thus make a diagnosis (serological diagnosis). In addition, serological reactions can be used to assess the intensity of the immune response after vaccination or an infectious disease.
Agglutination reactions, such as indirect agglutination and Coombs, are based on the in vitro interaction of corpuscular antigens with antibodies and the ability of the resulting complexes to precipitate. Bacterial cells or soluble antigens extracted from microorganisms and sorbed on carrier corpuscles: red blood cells, latex particles, etc. are used as corpuscular antigens.
Antigenic determinants of corpuscular antigens specifically interact with homologous antibodies (specific, invisible phase of the reaction), and then antigen-antibody complexes form large conglomerates visible to the naked eye, which precipitate - an agglutinate (non-specific, visible phase of the reaction). Flagellate-free forms of microbes (Brucellae) produce granular agglutinants, while flagellated forms (Escherichia, Salmonella) produce large-cotton agglutinants, which settle to the bottom of the test tube in the form of an inverted umbrella and easily break when shaken. Antigens and antibodies interact only in the presence of an electrolyte (0.8% sodium chloride solution). The course of the reaction is influenced by the salt concentration in the electrolyte, the number of microbial cells in the suspension, serum concentration, pH, temperature and other factors.
Agglutination reaction (ra).
There are specific agglutination, which is based on the interaction of the antigen With homologous antibody , contained in the animal’s body to which this antigen was introduced (immunoagglutination); nonspecific (chemical), arising from changes in the pH of the environment, the concentration of electrolytes; spontaneous, which is observed when bacteria (in R-form) are suspended in physiological solution and when heated, which is associated with a change in the colloidal state of the bacterial cell. Antigen , involved in RA is called an agglutinogen, the antibody is called an agglutinin, and the resulting precipitate is called an agglutinate. When an agglutinate is formed, the quantitative ratio of antigen and antibodies is important (the optimum phenomenon). With an excess or deficiency of antibodies, A delay occurs.
The agglutination reaction (RA) is one of the first immunological reactions used in microbiological practice. For the first time (1895), F. Vidal used RA to diagnose typhoid fever. Later (1897), A. Wright used the same reaction to diagnose brucellosis in humans. RA has also found application in the diagnosis of pullorosis in chickens, leptospirosis, infectious abortion of mares, as well as for typing unknown microbial cultures using a known agglutinating serum. RA is highly sensitive; it can be used to detect 0.01 μg of antibody protein nitrogen in 1 ml.
Several variants of the agglutination reaction have been developed, differing in methodological execution and purpose of the study.
2. Ra on glass.
In this variant of RA, the test subjects can be either serum or antigen, but most often this option is used to identify microorganisms.
1. To identify the microorganism (m/o), a drop of a known agglutinating serum, for example Salmonella serum, and a drop of physiological solution (control) are applied separately to a fat-free glass slide. Then, using a bacteriological loop, the bacterial mass of the studied culture is taken from a colony in a Petri dish or from the surface of a slanted MPA in a test tube and suspended separately in immune serum and physiological solution until a homogeneous suspension is obtained. The result is taken into account after 2...4 minutes.
Accounting for results: there should be no changes in the control sample. If the bacterial culture specifically matches the immune serum, agglutinate flakes appear (positive result); if there is no agglutination phenomenon, it is concluded that the bacterial culture under study does not correspond to the immune serum.
2. Let us consider the detection of anittels in the test blood serum using the example of the rose bengal test used in the serodiagnosis of brucellosis. 0.3 ml of the test animal blood serum and 0.03 ml of brucellosis antigen (rose-Bengal-stained Brucella cells) are applied to a glass slide. The components are thoroughly mixed by shaking the glass and the result is taken into account after 4 minutes.
Recording of results: if the reaction is positive, pink flakes of agglutinate appear. A serological reaction of this type is classified as qualitative, since it can be used to detect antibodies to the pathogen in the animal’s blood serum, but it is impossible to assess their quantitative content.
1.1. AGGLUTINATION REACTION (RA)
AGGLUTINATION REACTION (RA)
Due to its specificity, ease of performance and demonstrativeness, the agglutination reaction has become widespread in microbiological practice for the diagnosis of many infectious diseases.
The agglutination reaction is based on the specificity of the interaction of antibodies (agglutinins) with whole microbial or other cells (agglutinogens). As a result of this interaction, particles and agglomerates are formed, which precipitate (agglutinate) in the form of flakes.
The agglutination reaction can involve both live and killed bacteria, spirochetes, fungi, protozoa, rickettsia, as well as red blood cells and other cells. The reaction occurs in two phases: the first (invisible) specific, the combination of antigen and antibodies, the second (visible) nonspecific, gluing of antigens, i.e. agglutinate formation.
An agglutinate is formed when one active center of a divalent antibody combines with the determinant group of an antigen. The agglutination reaction, like any serological reaction, occurs in the presence of electrolytes.
Externally, the manifestation of a positive agglutination reaction has a twofold character. In flagellated microbes that have only somatic O2 antigen, the microbial cells themselves stick together directly. This agglutination is called fine-grained. It occurs within 18 22 hours. v
Flagellate microbes have two antigens: somatic O2 antigen and flagellar H2 antigen. If cells are glued together by flagella, large, loose flakes are formed and this agglutination reaction is called coarse-grained. It occurs within 2 4 hours.
The agglutination reaction can be performed both for the purpose of qualitative and quantitative determination of specific antibodies in the patient’s blood serum, and for the purpose of determining the species of the isolated pathogen. v
The agglutination reaction can be performed both in an expanded version, which allows you to work with serum diluted to a diagnostic titer, and in a variant indicative reaction, which allows, in principle, to detect specific antibodies or determine the species of the pathogen.
When performing a detailed agglutination reaction, in order to detect specific antibodies in the blood serum of the subject, the test serum is taken at a dilution of 1:50 or 1:100. This is due to the fact that normal antibodies may be present in very high concentrations in whole or slightly diluted serum, and then the reaction results may be inaccurate. The material being tested in this version of the reaction is the patient’s blood.
Blood is taken on an empty stomach or no earlier than 6 hours after a meal (otherwise there may be droplets of fat in the blood serum, making it cloudy and unsuitable for research). The patient's blood serum is usually obtained in the second week of the disease, collecting 3 × 4 ml of blood sterilely from the cubital vein (by this time the maximum amount of specific antibodies is concentrated). A diagnosticum prepared from killed but not destroyed microbial cells of a specific species with a specific antigenic structure is used as a known antigen.
When performing a detailed agglutination reaction in order to determine the species and type of pathogen, the antigen is a live pathogen isolated from the material being studied. Antibodies contained in immune diagnostic serum are known. v
Immune diagnostic serum obtained from the blood of a vaccinated rabbit. Having determined the titer (the maximum dilution at which antibodies are detected), the diagnostic serum is poured into ampoules with the addition of a preservative. This serum is used for identification by the antigenic structure of the isolated pathogen.
OPTIONS OF THE AGGLUTINATION REACTION
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 required: 1) antigen (agglutinogen); 2) antibody (agglutinin) and 3) electrolyte (isotonic sodium chloride solution).
ORIENTATIVE (PLATE) 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 separately apply a drop of serum at a dilution of 1:10 1:20 and a control drop of isotonic sodium chloride solution 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 a drop of serum is noted; with negative , the serum remains uniformly cloudy.
INDIRECT (PASSIVE) HEMAGLUTINATION REACTION (RNGA, RPGA)
The reaction is performed: 1) to detect polysaccharides, proteins, extracts of bacteria and other highly dispersed substances, rickettsiae and viruses, complexes of which with agglutinins cannot be seen in conventional RA, or 2) to detect antibodies in the sera of patients to these highly dispersed substances and the smallest microorganisms.
Indirect, or passive, agglutination is understood as a reaction in which antibodies interact with antigens pre-adsorbed on inert particles (latex, cellulose, polystyrene, barium oxide, etc. or sheep red blood cells, human blood group I(0)).
In the passive hemagglutination reaction (RPHA), red blood cells are used as a carrier. Antigen-loaded red blood cells stick together in the presence of specific antibodies to this antigen and precipitate. Antigen-sensitized erythrocytes are used in RPGA as an erythrocyte diagnosticum for the detection of antibodies (serodiagnosis). If red blood cells are loaded with antibodies (erythrocyte antibody diagnosticum), it can be used to detect antigens.
Staging. A series of serial dilutions of serum are prepared in the wells of polystyrene plates. Add 0.5 ml of obviously positive serum to the penultimate well and 0.5 ml of physiological solution (controls) to the last well. Then add 0.1 ml of diluted erythrocyte diagnosticum to all wells, shake and place in a thermostat for 2 hours. v
Accounting. IN positive case erythrocytes settle at the bottom of the hole in the form of an even layer of cells with a folded or jagged edge (inverted umbrella); in negative they settle in the form of a button or ring.
1.2. NEUTRALIZATION REACTION. LYSIS,
OPSONOPHAGOCYTIC REACTION, HYPERSENSITIVITY REACTION
REACTION OF NEUTRALIZATION OF EXOTOXIN WITH ANTITOXIN (RN)
The reaction is based on the ability of the antitoxic serum to neutralize the effect of the exotoxin. It is used for titration of antitoxic serums and determination of exotoxin.
When titrating the serum, a certain dose of the corresponding toxin is added to different dilutions of the antitoxic serum. When the antigen is completely neutralized and there are no unspent antibodies, initial flocculation occurs. The flocculation reaction can be used not only for the titration of serum (for example, diphtheria), but also for the titration of toxin and toxoid. The reaction of toxin neutralization with antitoxin is of great practical importance as a method for determining the activity of antitoxic therapeutic serums. The antigen in this reaction is a true exotoxin.
The strength of the antitoxic serum is determined by conventional units of AE.
1 AE of botulinum serum its amount neutralizes 1000 DLM of botulinum toxin. The neutralization reaction to determine the species or type of exotoxin (for the diagnosis of tetanus, botulism, diphtheria, etc.) can be carried out in vitro (according to Ramon), and when determining the toxigenicity of microbial cells - in a gel (according to Ouchterlony).
Lysis reaction (RL)
One of the protective properties of immune serum is its ability to dissolve microbes or cellular elements entering the body.
Specific antibodies that cause cell dissolution (lysis) are called lysines. Depending on the nature of the antigen, they can be bacteriolysins, cytolysins, spirochetolysins, hemolysins, etc.
Lysines exhibit their effect only in the presence of an additional complement factor. Complement as a nonspecific factor humoral immunity, found in almost all body fluids, except cerebrospinal fluid and anterior chamber fluid. A fairly high and constant content of complement was noted in human blood serum and there is a lot of it in the blood serum guinea pig. In other mammals, the content of complement in the blood serum is different.
Complement is a complex system whey proteins. It is unstable and collapses at 55 degrees for 30 minutes. At room temperature, complement is destroyed within two hours. Very sensitive to prolonged shaking, acids and ultraviolet rays. However, complement is stored for a long time (up to six months) in a dried state at low temperatures. Complement promotes the lysis of microbial cells and red blood cells.
A distinction is made between the reactions of bacteriolysis and hemolysis.
The essence of the bacteriolysis reaction is that when a specific immune serum combines with its corresponding homologous living microbial cells in the presence of complement, microbial lysis occurs.
The hemolysis reaction is that when erythrocytes are exposed to a specific serum that is immune to them (hemolytic) in the presence of complement, the dissolution of erythrocytes is observed, i.e. hemolysis.
The hemolysis reaction in laboratory practice is used to determine the complement range, as well as to record the results diagnostic reactions complement fixation. Complement titer is its smallest amount, which causes the lysis of red blood cells within 30 minutes in a hemolytic system in a volume of 2.5 ml. The lysis reaction, like all serological reactions, occurs in the presence of an electrolyte.
HYPERSENSITIVITY REACTIONS (ALLERGIC)
Certain forms of antigen, upon repeated contact with the body, can cause a reaction that is specific in its basis, but includes nonspecific cellular and molecular factors of an acute inflammatory response. There are two known forms of hypersensitivity: immediate-type hypersensitivity (IHT) and delayed-type hypersensitivity (DTH). The first type of reaction occurs with the participation of antibodies, and the reaction develops no later than 2 hours after repeated contact with the allergen. The second type is realized with the help of inflammatory T cells (Tgc) as the main effectors of the reaction, ensuring the accumulation of macrophages in the area of inflammation; the reaction manifests itself after 6-8 hours and later.
The development of a hypersensitivity reaction is preceded by an encounter with an antigen and the occurrence of sensitization, i.e. the appearance of antibodies, actively sensitized lymphocytes and passively sensitized cytophilic antibodies of other leukocytes (macrophages, granulocytes).
Hypersensitivity reactions have three phases of development: immunological; pathochemical; pathophysiological.
In the first, specific phase, the allergen interacts with antibodies and (or) sensitized cells. In the second phase, biological release occurs active substances from activated cells. The released mediators (histamine, serotonin, leukotrienes, bradykinin, etc.) cause various peripheral effects characteristic of the corresponding type of reaction third phase.
Reactions hypersensitivity fourth type
Reactions of this type are caused by pathogenic intercellular interactions of sensitized T-helper cells, cytotoxic T-lymphocytes (T-killer cells) and activated cells of the mononuclear phagocyte system, caused by prolonged stimulation of the immune system by bacterial antigens, which causes a relative insufficiency of the body's immune system to eliminate from internal environment bacterial pathogens of infectious diseases. These hypersensitivity reactions cause tuberculous lung cavities, their caseous necrosis and general intoxication in patients with tuberculosis. Cutaneous granulomatosis in tuberculosis and leprosy in morphopathogenetic terms is largely composed of hypersensitivity reactions of the fourth type.
Most famous example hypersensitivity reactions of the fourth type this is a Mantoux reaction that develops at the site of intradermal injection of tuberculin to a patient whose body and system are sensitized to mycobacterial antigens. As a result of the reaction, a dense hyperemic papule with necrosis in the center is formed, which appears only a few hours (slowly) after the intradermal injection of tuberculin. The formation of a papule begins with the release of mononuclear phagocytes of circulating blood from the vascular bed into the intercellular spaces. At the same time, emigration of polymorphonuclear cells from the vascular bed begins. Then the infiltration by neutrophils subsides, and the infiltrate begins to consist predominantly of lymphocytes and mononuclear phagocytes. This differs from the Mantoux reaction from the Arthus reaction, in which predominantly polymorphonuclear leukocytes accumulate at the site of the lesion.
In type 4 hypersensitivity reactions, long-term stimulation of sensitized lymphocytes with antigens leads to pathological changes tissues to pathologically intense and prolonged release of cytokines by T helper cells. The intense release of cytokines at the loci of tissue damage causes hyperactivation of the cells of the mononuclear phagocyte system located there, many of which, in a hyperactivated state, form strands of epithelioid cells, and some merge with each other to form giant cells. Macrophages, on the surface of which bacterial and viral antigens are exposed, can be destroyed through the functioning of Tkillers (natural killers).
The fourth type of hypersensitivity reaction is induced by recognition of a foreign bacterial antigen by T helper cells sensitized to it. A necessary condition for recognition is the interaction of inducers with antigens exposed on the surface of antigen-presenting cells after endocytosis and processing of foreign immunogens by mononuclear phagocytes. Another necessary condition exposure of antigens in combination with class I molecules from the major histocompatibility complex. After antigen recognition, sensitized helper cells release cytokines and, in particular, interleukin2, which activates natural killer cells and mononuclear phagocytes. Activated mononuclear phagocytes release proteolytic enzymes and free oxygen radicals, which damage tissue.
Skin allergy tests tests to establish the body’s sensitization to allergens, determine its infection level, for example, tuberculosis, brucellosis, herd immunity, for example, to tularemia. Based on the site of allergen administration, there are: 1) skin tests; 2) scarification; 3) intradermal; 4) subcutaneous. The clinical reaction to an allergen during a skin allergy test is divided into local, general and focal, as well as immediate and delayed.
Local reactions of the mediator type GNT occur after 520 minutes, are expressed in the form of erythema and a blister, disappear after a few hours, and are assessed by the plus method by the size of the erythema, measured in mm. Local HRT reactions occur within 24-48 hours, last a long time, appear in the form of an infiltrate, sometimes with necrosis in the center, and are assessed by the size of the infiltrate in mm, also using the plus system. With cytotoxic and immunocomplex types of HNT, hyperemia and infiltration are observed after 3-4 hours, reach a maximum at 6-8 hours and subside after about a day. Sometimes combined reactions are observed.
1.3. COMPLEMENT FIXATION REACTION (FFR)
This reaction is used in laboratory tests to detect antibodies in blood serum for various infections, as well as to identify the pathogen by its antigenic structure.
The complement fixation reaction is a complex serological reaction and is highly sensitive and specific.
A feature of this reaction is that the change in the antigen during its interaction with specific antibodies occurs only in the presence of complement. Complement is adsorbed only on the “antibody antigen” complex. The “antibody antigen” complex is formed only if there is an affinity between the antigen and the antibody in the serum.
Adsorption of complement on the “antigen antibody” complex can have different effects on the fate of the antigen depending on its characteristics.
Some of the antigens undergo sharp morphological changes under these conditions, including dissolution (hemolysis, Isaev-Pfeiffer phenomenon, cytolytic action). Others change the speed of movement (treponema immobilization). Still others die without sudden destructive changes (bactericidal or cytotoxic effect). Finally, complement adsorption may not be accompanied by antigen changes that are easily observable.
According to the RSC mechanism, it occurs in two phases:
- The first phase is the formation of the “antigen antibody” complex and adsorption on this complement complex. The result of the phase is not visually visible (interaction of antigen and antibodies with the obligatory participation of complement).
- The second phase is a change in the antigen under the influence of specific antibodies in the presence of complement. The result of the phase can be visible visually or not visible (detection 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.
The result of the experiment is assessed by noting the presence or absence of hemolysis in all test tubes. The reaction is considered positive when hemolysis is completely delayed, when the liquid in the test tube is colorless and the red blood cells settle to the bottom, negative when the 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 (++++, +++, ++, +).
In the case when changes in the antigen remain inaccessible for visual observation, it is necessary to use a second system, which acts as an indicator, allowing one to assess the state of complement and draw a conclusion about the result of the reaction.
This indicator system is represented by components of the hemolysis reaction, which includes sheep erythrocytes and hemolytic serum, which contains specific antibodies to erythrocytes (hemolysins), but does not contain complement. This indicator system is added to the test tubes an hour after the main RSC is placed. If the complement fixation reaction is positive, then an antibody antigen complex is formed, which adsorbs complement on itself. Since complement is used in the amount necessary for only one reaction, and lysis of erythrocytes can only occur in the presence of complement, then when it is adsorbed on the “antigen antibody” complex, lysis of erythrocytes in the hemolytic (indicator) system will not occur. If the complement fixation reaction is negative, the “antigen antibody” complex is not formed, the complement remains free, and when the hemolytic system is added, erythrocyte lysis occurs.
1.4. DNA PROBES. POLYMERASE CHAIN REACTION (PCR),
IMMUNO ENZYME METHOD (ELISA), FLUORESCING ANTIBODY METHOD (FFA)
METHODS OF GENE PROBING
The intensive development of molecular biology and the creation of a perfect methodological base for genetic research formed the basis of genetic engineering. In the field of diagnostics, a direction has emerged and is rapidly developing to determine specific nucleotide sequences of DNA and RNA, the so-called gene probing. Such techniques are based on the ability of nucleic acids to hybridize and form double-stranded structures due to the interaction of complementary nucleotides (AT, GC).
To determine the desired DNA (or RNA) sequence, a so-called polynucleotide probe with a specific base sequence is specially created. A special label is introduced into its composition, which makes it possible to identify the formation of the complex.
Although gene probing cannot be classified as a method of immunochemical analysis, its basic principle (the interaction of complementary structures) is methodically implemented in the same ways as indicator methods of immunodiagnostics. In addition, gene probing methods make it possible to replenish information about an infectious agent in the absence of its phenotypic expression (viruses embedded in the genome, “silent” genes).
To carry out DNA analysis, the sample is denatured in order to obtain single-stranded structures with which the DNA or RNA probe molecules react. To prepare probes, either various sections of DNA (or RNA) isolated from a natural source (for example, a particular microorganism), usually presented in the form of genetic sequences in vector plasmids, or chemically synthesized oligonucleotides are used. In some cases, genomic DNA preparations hydrolyzed into fragments are used as a probe, sometimes RNA preparations, and especially often ribosomal RNA. The same indicators are used as a label as in various types of immunochemical analysis: radioactive isotopes, fluoresceins, biotope (with further development by the avidin-enzyme complex), etc.
The order of analysis is determined by the properties of the available probe
Currently, commercial kits containing all the necessary ingredients are increasingly being used.
In most cases, the analysis procedure can be divided into the following stages: sample preparation (including DNA extraction and denaturation), sample fixation on a carrier (most often a polymer membrane filter), prehybridization, hybridization itself, washing of unbound products, detection. In the absence of a standard DNA or RNAprobe preparation, it is first obtained and labeled.
To prepare a sample, it may be necessary to preliminary “grow” the test material to identify individual colonies of bacteria or increase the concentration of viruses in a cell culture. Is also carried out direct analysis samples of blood serum, urine, shaped elements blood or whole blood for the presence of an infectious agent. To release nucleic acids from cellular structures, cell lysis is carried out, and in some cases the DNA preparation is purified using phenol.
Denaturation of DNA, i.e. its transition to a single-stranded form, occurs when treated with alkali. The nucleic acid sample is then fixed to a support, a nitrocellulose or nylon membrane, usually by incubation for 10 minutes to 4 hours at 80°C in a vacuum. Further, in the process of prehybridization, inactivation of free binding sites is achieved to reduce nonspecific interaction of the probe with the membrane. The hybridization process takes from 2 to 20 hours, depending on the concentration of DNA in the sample, the concentration of the probe used and its size.
After hybridization is completed and unbound products are washed away, the formed complex is detected. If the probe contains a radioactive label, then to demonstrate the reaction, the membrane is exposed to photographic film (autoradiography). For other labels, the corresponding procedures are used.
The most promising is to obtain non-radioactive (so-called cold) probes. On the same basis, a hybridization technique is being developed, which makes it possible to establish the presence of a pathogen in section preparations and tissue punctures, which is especially important in pathomorphological analysis (in situ hybridization).
A significant step in the development of gene probing methods was the use of polymerase amplification reaction (PCR). This approach makes it possible to increase the concentration of a specific (pre-known) DNA sequence in a sample by synthesizing multiple copies in vitro. To carry out the reaction, a DNA polymerase enzyme preparation, an excess of deoxynucleotides for synthesis and the so-called primers are added to the DNA sample under study - two types of oligonucleotides with a size of 2025 bases corresponding to the terminal sections of the DNA sequence of interest. One of the primers should be a copy of the beginning of the reading region of the coding DNA strand in the reading direction 53, and the second should be a copy of the opposite end of the non-coding strand. Then, with each cycle of the polymerase reaction, the number of DNA copies doubles.
To achieve primer binding, DNA denaturation (melting) at 94°C is necessary, followed by bringing the mixture to 40-55°C.
To carry out the reaction, programmable microsample incubators have been designed to easily alternate changes in the optimal temperature for each stage of the reaction.
The amplification reaction can significantly increase the sensitivity of the analysis during gene probing, which is especially important at low concentrations of the infectious agent.
One of the significant advantages of gene probing with amplification is the ability to study submicroscopic amounts of pathological material.
Another feature of the method, more important for the analysis of infectious material, is the ability to identify hidden (silent) genes. Methods associated with the use of gene probing will certainly be more widely introduced into the practice of diagnosing infectious diseases as they become simpler and cheaper.
The ELISA and RIF methods are largely qualitative or semi-quantitative in nature. At very low concentrations of components, the formation of the antigen antibody complex cannot be detected either visually or with simple instrumental means. Indication of the antigen antibody complex in such cases can be carried out if a label is introduced into one of the initial components antigen or antibody , which can be easily detected in concentrations comparable to the determined concentration of the analyte.
Radioactive isotopes (for example, 125I), fluorescent substances, and enzymes can be used as labels.
Depending on the label used, there are radioimmune (RIA), fluorescent immune (FIA), enzyme-linked immunosorbent assay (ELISA) methods of analysis, etc. last years ELISA has received widespread practical use, which is due to the possibility quantitative determinations, high sensitivity, specificity and automation of accounting.
Enzyme immunoassay methods are a group of methods that allow the detection of the antigen-antibody complex using a substrate that is cleaved by an enzyme and produces a color.
The essence of the method is to combine the components of the antigen antibody reaction with a measured enzyme label. The antigen or antibody that reacts is labeled with an enzyme. Based on the transformation of the substrate under the action of the enzyme, one can judge the amount of the interacting component of the antigen antibody reaction. Enzyme in in this case serves as a marker immune reaction and allows you to observe it visually or instrumentally.
Enzymes are very convenient tags because their catalytic properties allow them to act as amplifiers, since one enzyme molecule can promote the formation of more than 1 × 105 molecules of catalytic reaction product per minute. It is necessary to select an enzyme that retains its catalytic activity for a long time, does not lose it when binding to an antigen or antibody, and has high specificity with respect to the substrate.
The main methods for producing enzyme-labeled antibodies or antigens and conjugates are: chemical, immunological and genetic engineering. The enzymes most often used for ELISA are horseradish peroxidase, alkaline phosphatase, galactosidase, etc.
To detect enzyme activity in the antigen-antibody complex for the purpose of visual and instrumental recording of the reaction, chromogenic substrates are used, solutions of which, initially colorless, during the enzymatic reaction acquire color, the intensity of which is proportional to the amount of enzyme. Thus, to detect the activity of horseradish peroxidase in solid-phase ELISA, 5-aminosalicylic acid, which produces an intense brown color, and ortho-phenylenediamine, which produces an orange-yellow color, are used as a substrate. To detect the activity of alkaline phosphatase and β-galatosidase, nitrophenylphosphates and nitrophenylgalactosides are used, respectively.
The result of the reaction in the formation of a colored product is determined visually or using a spectrophotometer that measures the absorption of light with a certain wavelength.
There are many options for performing ELISA. There are homogeneous and heterogeneous options.
According to the method of production, competitive and non-competitive ELISA methods are distinguished. If at the first stage only the analyzed compound and its corresponding binding centers (antigen and specific antibodies) are present in the system, then the method is non-competitive. If at the first stage the analyzed compound (antigen) and its analogue (enzyme-labeled antigen) are present, competing with each other for binding to the specific binding centers (antibodies) that are in short supply, then the method is competitive. In this case, the more test antigen the solution contains, the less the number of bound labeled antigens.
METHOD OF FLUORESCING ANTIBODIES (MFA) or IMMUNOFLUORESCENCE REACTION (RIF)
The immunofluorescence method is the method of choice for the rapid detection and identification of an unknown microorganism in the test material.
Ag + AT + electrolyte = complex glowing in UV rays
Microbe serum labeled with fluorochrome
The dye often used is fluorescein isothiocyanate FITC
When studying this method, a fluorescent microscope is used.
Staging RIF
30 μl of FITC-labeled antibody solution is applied to the smear.
Place the glass in a humid chamber and keep it at room temperature for 20-25 minutes, or in a thermostat at 37°C for 15 minutes.
Wash the glass in a running machine tap water 2 minutes, rinse with distilled water and air dry.
A drop of mounting liquid is applied to the dried smear, the smear is covered with a coverslip and microscoped using a fluorescent microscope or a fluorescent attachment to a conventional optical microscope.
