Precipitation Reaction is a reaction in which two soluble substances react together in a solution and form an insoluble solid substance. This insoluble solid substance is called precipitate.
In chemical reaction, it occurs when the concentration of dissolved ions becomes more than their solubility limit. This limit is controlled by solubility product constant (Ksp). When the ions exceed this limit, they join together and separate out from the solution as solid crystal.
In immunology, precipitation reaction is the reaction between soluble antigen and specific antibody. It is a non-covalent binding reaction. When antigen and antibody are present in proper amount, they form large cross linked lattice. This proper proportion is known as zone of equivalence.
The formed lattice becomes insoluble and comes out from the liquid medium. It may be seen as a visible ring, band or mass. Thus, in both chemistry and immunology, the main feature of precipitation reaction is the conversion of soluble reactants into visible insoluble solid product.
Principle of Precipitation Reaction
Principle of Precipitation Reaction is based on the formation of insoluble product from completely soluble reacting substances. In this reaction, the soluble particles combine together and form an aggregated solid substance. This solid substance is called precipitate.
In chemical precipitation reaction, it is based on solubility product (Ksp). When the concentration of dissolved ions becomes more than their solubility limit, the ions start to come together. This condition is written as Q > Ksp. Then the ions form an ordered solid crystal lattice and separate out from the solution.
In immunological precipitation reaction, it is based on lattice hypothesis. Here soluble multivalent antigen and bivalent antibody react with each other under suitable temperature, pH and electrolyte condition. The antigen and antibody must be present in proper equal proportion. This condition is known as zone of equivalence.
During this process, antigen and antibody cross-link continuously with each other. A large three dimensional macromolecular lattice is formed. When this lattice becomes large and insoluble, it comes out from the liquid as visible precipitate. Sometimes it may remain suspended as floating clumps, called floccules.
Mechanism of Antigen-Antibody Precipitation
- Introduction of reactants – Soluble multivalent antigen and specific bivalent antibody are mixed in a liquid or semi-solid medium under suitable pH, temperature and electrolyte condition.
- Initial binding – In this step, antibody binds with epitopes of antigen by rapid non-covalent interaction and forms small soluble antigen-antibody complex.
- Cross-linking at equivalence – The antigen and antibody must be present in proper equivalent proportion, which is called zone of equivalence. Here bivalent antibody bridges different antigen molecules together.
- Lattice formation – During this process, continuous cross-linking forms a large three dimensional interconnected network called lattice.
- Loss of solubility – The lattice gradually increases in size and becomes too large to remain soluble in the medium.
- Visible precipitation – Finally, the insoluble antigen-antibody lattice separates from the solution as visible solid precipitate, turbid suspension or floating floccules.
Requirements for Precipitation Reaction
- Supersaturated condition – In chemical precipitation reaction, the concentration of reacting ions must be more than the solubility limit. This condition is written as Q > Ksp. Then the insoluble compound is formed.
- Soluble reactants – In immunological precipitation reaction, both antigen and antibody must be soluble in nature. Insoluble antigen normally gives agglutination reaction, not precipitation reaction.
- Bivalent antibody – The antibody must have at least two antigen binding sites. Monovalent Fab fragment cannot form proper cross-linking, so lattice is not formed.
- Bivalent or polyvalent antigen – The antigen must have at least two same epitopes or many different epitopes. This helps in binding with more than one antibody.
- Zone of equivalence – The antigen and antibody must be present in proper equivalent proportion. In excess antibody, prozone phenomenon occurs and in excess antigen, postzone phenomenon occurs. In both condition visible precipitate is not formed.
- Suitable environment – The reaction needs suitable temperature, pH and electrolyte concentration. These conditions help in proper antigen-antibody binding and formation of visible precipitate.
Lattice Formation Process in Precipitation Reaction
A. Immunological Lattice Formation
- Presence of multivalent reactants – The antibody must be bivalent and antigen must be bivalent or polyvalent. The antigen should have two or more epitopes. Monovalent Fab fragment cannot form bridge, so lattice is not formed.
- Mixing in equivalent proportion – The antigen and antibody must be present in balanced amount. This condition is known as zone of equivalence. In excess antibody, prozone occurs and in excess antigen, post-zone occurs.
- Bridging of antigen molecules – In this step, one bivalent antibody binds with two separate antigen molecules. It forms bridge between antigen molecules by specific non-covalent binding.
- Cross-linking reaction – During this process, many antigen and antibody molecules continuously join with each other. Antibody binds antigen and antigen again binds another antibody.
- Three dimensional lattice formation – The continuous cross-linking spreads in all direction and forms a large net like structure. This structure is called lattice.
- Loss of solubility and precipitation – As the lattice becomes large, it cannot remain soluble in aqueous medium. Then it separates from the solution as visible precipitate or floating floccules.
B. Chemical Lattice Formation
- Exceeding solubility limit – In chemical precipitation, the ionic product of reacting ions becomes more than the solubility product (Ksp). This condition is written as Q > Ksp.
- Ion aggregation – After crossing solubility limit, opposite charged cations and anions start to come together. They aggregate in the solution.
- Crystal lattice formation – The aggregated ions arrange in a fixed ordered pattern. This forms a solid crystal lattice.
- Precipitation of crystal – The crystal lattice grows and becomes large and heavy. Finally it settles down at the bottom of solution as chemical precipitate.
Zone Phenomenon in Precipitation Reaction
Zone phenomenon is the condition in which the amount of visible precipitation depends on the relative proportion of antigen and antibody. The precipitation reaction occurs properly only when antigen and antibody are present in suitable amount. If any one of them is present in excess amount, the precipitate is not formed properly.
Prozone phenomenon is the zone of antibody excess. In this zone, antibody is present in very high concentration than antigen. The excess antibodies bind separately with the epitopes of antigen and saturate them. So the antibodies cannot cross-link different antigen molecules together and proper lattice is not formed.
Due to absence of proper lattice formation, visible precipitation is inhibited in prozone. The reaction may show no precipitate even if both antigen and antibody are present. This is referred to as prozone effect.
Zone of equivalence is the zone where antigen and antibody are present in balanced and equivalent proportion. This is the most suitable zone for precipitation reaction. In this zone, maximum cross-linking takes place between antigen and antibody.
During this process, a large three dimensional lattice network is formed. The lattice becomes insoluble and comes out from the solution as maximum visible precipitate. Thus, the zone of equivalence gives the best precipitation reaction.
Postzone phenomenon is the zone of antigen excess. In this zone, antigen is present in very high concentration than antibody. The excess antigens bind with all available binding sites of antibody. So antibodies cannot act as bridge between separate antigen molecules.
As a result, lattice formation is prevented in postzone also. The visible precipitation is inhibited or absent. Thus, both prozone and postzone give poor precipitation due to improper antigen-antibody proportion.
Types of Precipitation Reactions
A. Immunological Precipitation Reactions
These reactions occur between soluble antigen and antibody. They are classified according to the medium where the reaction takes place.
1. Precipitation in Solution
In this type, the reaction takes place in liquid medium. The visible precipitate is formed at the place where soluble antigen and antibody meet in suitable proportion.
- Ring test is a precipitation reaction in liquid medium. In this test, antigen solution is carefully layered over antiserum in a narrow tube. A visible ring of precipitate is formed at the junction of antigen and antibody.
- Flocculation test is another precipitation reaction in liquid medium. In this test, antigen and antibody are mixed on a slide or in a tube. The precipitate remains suspended as visible floating clumps called floccules.
2. Precipitation in Agar (Immunodiffusion)
In this type, the reaction takes place in agar gel. Antigen and antibody diffuse through the gel and form visible precipitin line or ring at the zone of equivalence.
- Single diffusion in one dimension (Oudin method) occurs when antibody is mixed in agar gel in a tube and antigen is layered above it. The antigen diffuses downward in one direction and forms precipitin line.
- Single diffusion in two dimensions (Radial immunodiffusion or Mancini method) occurs when antibody is mixed uniformly in agar plate. Antigen is placed in a well and diffuses radially in all direction. A circular precipitin ring is formed.
- Double diffusion in one dimension (Oakley-Fulthorpe method) occurs when antigen and antibody are separated by a middle layer of plain gel. Both diffuse toward each other and form a precipitate band.
- Double diffusion in two dimensions (Ouchterlony method) occurs when antigen and antibody are placed in separate wells in agar gel. They diffuse radially toward each other and form precipitin bands where they meet.
3. Precipitation in Agar with Electric Field
In this type, electric current is used with agar gel. The electric field helps in movement of antigen or antibody and precipitin line or arc is formed faster.
- Immunoelectrophoresis is a method in which mixture of antigens are first separated in agar gel by electric current. Then antibody is added in a parallel trough. The antibody diffuses and forms separate precipitin arcs with separated antigens.
- Counter-current electrophoresis is a method in which electric current drives negatively charged antigen and positively charged antibody toward each other. A precipitin line is formed rapidly in gel.
- Rocket immunoelectrophoresis is a method in which antigen moves into agar gel containing antibody. The precipitate forms a cone or rocket shaped structure. The height of rocket is proportional to antigen concentration.
B. Chemical Precipitation Reactions
These reactions occur between soluble ions. The ions combine and form insoluble ionic solid when their concentration becomes more than solubility limit.
1. Standard precipitation
In standard precipitation, soluble cations and anions combine in aqueous solution. When their concentration crosses the solubility limit, they form solid crystal lattice. This lattice comes out as precipitate.
2. Selective or fractional precipitation
In selective precipitation, different ions are separated from a mixture. Specific reagent is added step by step. Ions precipitate one by one according to their solubility limit.
3. Coprecipitation
Coprecipitation is the process where soluble impurities are carried down with the main precipitate. It may occur by surface adsorption, mixed crystal formation, occlusion or mechanical entrapment.
4. Post-precipitation
Post-precipitation is a secondary precipitation process. In this, sparingly soluble impurity slowly crystallizes on the surface of already formed main precipitate. It forms separate solid layer after primary precipitate formation.
Step by step Procedure of Precipitation Reaction
A. Procedure for Chemical Precipitation
- In this procedure, the given sample is first weighed properly. Then it is dissolved in a suitable solvent to make a clear solution. The analyte must be present in soluble form before starting the precipitation reaction.
- The interfering substances are removed from the solution by suitable separation method. This step is important because other ions may also form precipitate with the reagent and affect the actual result.
- After this, the condition of the solution is adjusted. The pH, concentration and dilution of the solution are maintained according to the requirement of the reaction. Sometimes the solution is heated to get better crystal formation.
- In this step, suitable precipitating reagent is added slowly into the solution with constant stirring. The reagent is generally added in hot and dilute condition. Slow addition helps the small nuclei to grow into proper crystal.
- The formed solid precipitate is then separated from the remaining liquid. The liquid part is called mother liquor. The separation is usually done by filtration.
- The separated precipitate is washed with suitable washing solution. The washing solution may contain electrolyte. It removes the impurities from the surface of precipitate and prevents peptization.
- Finally, the precipitate is dried or ignited. After drying, it is weighed accurately. From the weight of precipitate, the amount of analyte present in the sample is calculated.
B. Procedure for Immunological Precipitation
- In this procedure, the serum sample containing antigen or antibody is collected first. A small measuring pipette is used for taking the required amount of serum.
- The collected serum is then delivered into the pre-cut wells of the immunodiffusion plate. The sample should be placed carefully inside the well without spilling on the agar surface.
- After sample addition, the plate is kept for incubation. The plate is incubated according to the required test condition. During this period, antigen and antibody diffuse through the gel and react with each other.
- After incubation, the plate is observed for visible precipitin ring. Usually the result is read after 18 to 48 hours. The diameter of the ring is measured carefully.
- The measured ring diameter is compared with the reference curve. The standard curve is prepared from known samples. Then the amount of unknown antigen or antibody is calculated from this curve.
Interpretation of Results in Precipitation Reaction
A. Immunological Precipitation Results
1. Ring Test
In ring test, positive result is shown by the formation of visible white ring at the junction of antigen and antibody liquid layers. This ring indicates specific antigen-antibody precipitation reaction.
If no white ring is formed at the junction, the result is taken as negative. It indicates that visible precipitation reaction has not occurred.
2. Flocculation Test
In flocculation test, positive result is indicated by visible suspended clumps in the solution. These clumps are called floccules.
If the solution remains clear and no suspended clumps are seen, the result is negative. It shows absence of visible antigen-antibody reaction.
3. Radial Immunodiffusion
In radial immunodiffusion, a precipitin ring is formed around the well containing antigen. The formation of ring indicates positive reaction.
The diameter or area of the ring is directly proportional to the concentration of antigen in the sample. Larger ring means higher antigen concentration.
4. Ouchterlony Double Immunodiffusion
In Ouchterlony double immunodiffusion, results are interpreted by the pattern of precipitin lines formed between wells.
In pattern of identity, precipitin lines from adjacent wells fuse and form a smooth continuous arc. It indicates that tested antigens are identical.
In pattern of non-identity, two precipitin lines cross each other without fusion. It indicates that antigens are completely different and they have no common epitopes.
In pattern of partial identity, a continuous arc is formed with a spur. It indicates that antigens share some common epitopes, but one antigen has additional unique epitopes.
5. Rocket Immunoelectrophoresis
In rocket immunoelectrophoresis, positive result is shown by a cone shaped or rocket shaped precipitation line from the loading well.
The height and area of the rocket is directly proportional to the amount of antigen present in the sample. Higher rocket indicates more antigen concentration.
6. Immunoelectrophoresis
In immunoelectrophoresis, formation of distinct elliptical precipitin arcs confirms specific antigen-antibody interaction.
If precipitin arc is absent, it indicates no specific reaction between antigen and antibody.
B. Chemical Precipitation Results
1. Positive Identification
In chemical precipitation, positive result is indicated by spontaneous formation of insoluble solid after adding specific reagent. The colour of precipitate helps to identify the target ion.
Yellow precipitate may indicate lead or barium ion. White precipitate may indicate silver or chloride ion. Reddish brown precipitate indicates iron ion.
2. Absence of Precipitate
If the solution remains clear after adding reagent, it indicates negative result. It means the target ion is absent or present in very low concentration.
In this condition, the ionic product is less than solubility product constant (Ksp). It is written as Q < Ksp, so precipitate is not formed.
Factors Affecting Precipitation Reaction
A. Factors Affecting Chemical Precipitation
- Temperature – Temperature affects the solubility of solid substance. In most solids, increase in temperature increases solubility. So precipitation may decrease or already formed precipitate may dissolve.
- Concentration of ions – Precipitation occurs only when the ionic product of reacting ions becomes more than solubility product constant (Ksp). This condition is written as Q > Ksp. If Q < Ksp, precipitate is not formed.
- Common ion effect – When a common ion is added in the solution, the solubility of the precipitate decreases. This promotes precipitation of the compound.
- pH of solution – The pH affects precipitation of salts of weak acids or weak bases. In acidic condition, precipitates containing basic anions like carbonate and sulfide become more soluble.
- Complexing agents – Complexing agents form soluble complex with metal ions. This increases the solubility of the compound and may dissolve the precipitate.
- Ionic strength – Presence of other non-reacting electrolytes increases the ionic strength of solution. These ions form ionic atmosphere around reacting ions. So attraction between ions decreases and precipitation is reduced.
- Nature of ions – The charge and size of ions affect precipitation. Ions with high charge and small size have strong attraction. So they combine easily and form precipitate.
B. Factors Affecting Immunological Precipitation
- Proportion of reactants – The ratio of antigen and antibody is the most important factor. Precipitation occurs best in zone of equivalence. Excess antibody causes prozone and excess antigen causes postzone. In both conditions lattice is not formed properly.
- Valency of reactants – The antibody must be at least bivalent and antigen must be bivalent or polyvalent. This is needed for cross-linking and formation of insoluble lattice.
- Temperature and pH – Antigen-antibody reaction needs suitable temperature and pH. These conditions help in proper non-covalent binding and visible precipitate formation.
- Presence of electrolytes – Suitable electrolytes are required for immunological precipitation reaction. They help in formation of insoluble antigen-antibody complex.
- Gel properties – In immunodiffusion, agar gel affects the reaction. Gel viscosity, agar concentration, hydration and interaction of gel matrix with reactants affect diffusion and precipitation.
- Particle size – The size of antigen and antibody particles affects their diffusion through gel pores. Smaller particles diffuse faster, while larger particles diffuse slowly.
Applications of Precipitation Reaction in Immunology
- Precipitation reaction is used for diagnosis of syphilis. Flocculation precipitation tests are used to detect antibodies related to syphilis. The important tests are VDRL (Venereal Disease Research Laboratory) slide test and Kahn tube test.
- Ascoli’s thermoprecipitin test is used for detection of anthrax. It is a liquid based ring precipitation test where specific antigen and antibody reaction gives visible precipitate.
- Lancefield grouping technique is used for classification of different strains of β-haemolytic Streptococcus. It is based on ring precipitation reaction and detects specific bacterial antigens.
- Elek’s gel precipitation test is used for detection of Corynebacterium diphtheriae infection. This test is based on Ouchterlony double immunodiffusion method.
- Precipitation methods are used for detection of many viral, fungal and parasitic infections. These include smallpox, influenza, fungal infections, amoeba antigens, cryptococcal antigens and Hepatitis B surface antigen.
- Radial immunodiffusion, nephelometry and turbidimetry are used for measurement of serum proteins. These methods are used to estimate immunoglobulins, albumin, transferrin, acute phase proteins and complement components like C3 and C4.
- Immunoelectrophoresis is used to detect abnormal proteins in patient serum. It is useful in diagnosis of multiple myeloma, monoclonal gammopathy, polyclonal gammopathy and hypogammaglobulinemia.
- Immunonephelometry is used to measure rheumatoid factors in serum. It is also used for quantitative estimation of coagulation factors in blood.
- Precipitation reactions are used for detection of C-reactive protein (CRP) by ring test. Rocket electrophoresis is used to estimate the amount of Anti-streptolysin O (ASO) proteins.
- Tube flocculation precipitation test is used in laboratories for quantitative standardization of biological toxins. It is also used for standardization of their corresponding antitoxins and toxoids.
Advantages of Precipitation Reaction in Immunology
- Precipitation reaction is simple and easy to perform. The basic procedure does not need very complex instruments and can be done in ordinary laboratory condition.
- It gives visible result by formation of precipitate, ring, band, arc or floccules. So the reaction can be observed directly in many tests.
- It is useful for diagnosis of many diseases. It is used in detection of syphilis, anthrax, diphtheria and many microbial infections.
- It is used for grouping of microorganisms. Lancefield grouping of β-haemolytic Streptococcus is done by precipitation reaction with specific antigens.
- It is useful for standardization of toxins and antitoxins. Tube flocculation test is used to standardize biological toxins, antitoxins and toxoids.
- It is useful for measurement of serum proteins. Radial immunodiffusion, nephelometry and turbidimetry are used to estimate immunoglobulins, albumin, transferrin and complement proteins.
- It helps in detection of abnormal serum proteins. Immunoelectrophoresis is used in conditions like multiple myeloma, monoclonal gammopathy and hypogammaglobulinemia.
- In agar gel, the precipitin bands are clear and stable. These bands show identity, non-identity or partial identity between different antigens.
- The gel plates used in immunodiffusion can be preserved for long time. So the result can be studied and compared later also.
- It is useful for measuring small amount of antigen or antibody. In rocket immunoelectrophoresis, the height of rocket gives the amount of antigen present in the sample.
- It can be made faster and more accurate by using instruments like nephelometer and turbidimeter. These methods measure turbidity or scattered light formed by antigen-antibody precipitate.
- It is useful in routine clinical laboratory because many samples can be tested. Automated systems can perform precipitation based tests rapidly and continuously.
Limitations of Precipitation Reaction
Limitations of Immunological Precipitation
- Precipitation reaction is less sensitive than agglutination reaction. Very small amount of antigen or antibody may not give visible precipitate.
- It is a slow reaction. Initial antigen-antibody complex may form quickly, but visible precipitate formation takes long time. It may take several hours or one to two days.
- It needs proper proportion of antigen and antibody. Precipitation occurs only in zone of equivalence. In excess antibody prozone occurs and in excess antigen postzone occurs, so precipitate is not formed.
- It needs polyvalent reactants for lattice formation. Antigen must have multiple epitopes and antibody must be at least bivalent. Without these, lattice is not formed properly.
- Some advanced methods need trained person and special skill. Immunoelectrophoresis needs agar gel, electric field and proper interpretation of precipitin arcs.
Limitations of Chemical Precipitation
- The precipitate may be contaminated by soluble impurities. In coprecipitation, impurities are trapped inside or with the main precipitate.
- In post-precipitation, another sparingly soluble impurity slowly crystallizes on the surface of already formed precipitate. This makes the precipitate impure.
- Contamination of precipitate gives wrong result in gravimetric analysis. The final mass becomes inaccurate because impurity is also weighed with the required precipitate.
- Many organic precipitating reagents have limited solubility in water. They may need to be added in slight excess, and this may cause further contamination.
- Some precipitates do not have a stable drying or ignition form. So the final weighed form becomes uncertain and calculation may not be accurate.
- Some precipitates are difficult to handle during filtration and separation. They may not wet properly with water, float on the liquid surface or creep up the side of glass vessel.
Clinical and Diagnostic Significance of Precipitation Reaction
- Precipitation reaction is used in diagnosis of syphilis. VDRL (Venereal Disease Research Laboratory) slide flocculation test and Kahn tube flocculation test are used to detect syphilis related antibodies.
- Ascoli’s thermoprecipitin test is used for detection of anthrax antigens. It is a type of ring precipitation test and gives visible precipitate at the junction of antigen and antibody.
- Lancefield grouping technique is used to classify different strains of β-haemolytic Streptococcus. It is based on ring precipitation reaction with specific bacterial antigens.
- Elek’s gel precipitation test is used for detection of Corynebacterium diphtheriae. This test uses Ouchterlony double immunodiffusion method and shows precipitation line in agar gel.
- Various precipitation methods are used to detect viral, fungal and parasitic infections. These include Hepatitis B surface antigen, smallpox, influenza, fungal infections, amoeba antigens and cryptococcal antigens.
- Radial immunodiffusion, rocket electrophoresis, turbidimetry and nephelometry are used for estimation of serum proteins. These methods measure immunoglobulins, albumin, transferrin, acute phase proteins and complement components like C3 and C4.
- Immunoelectrophoresis is used to detect abnormal serum proteins in patient serum. It helps in diagnosis of monoclonal gammopathy, polyclonal gammopathy, multiple myeloma and hypogammaglobulinemia.
- Precipitation reactions are used for detection of specific biomarkers. C-reactive protein (CRP) is detected by ring test and Anti-streptolysin O (ASO) proteins are estimated by rocket electrophoresis.
- Immunonephelometry is used to measure rheumatoid factors in serum. It helps in clinical diagnosis and monitoring of rheumatoid related immune condition.
- Nephelometry is used in automated coagulometers for estimation of coagulation factors in blood samples. It helps in coagulation profiling of patient.
- Tube flocculation precipitation test is used for standardization of biological toxins and toxoids. It is also used against their corresponding antitoxins in laboratory work.
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