Week 13 Precipitation Reactions PDF

Al-Quds University Faculty of Health Professions Department of Medical Laboratory Sciences Immunology & Serology I 0202308 Dr....

Al-Quds University Faculty of Health Professions Department of Medical Laboratory Sciences Immunology & Serology I 0202308 Dr. Rasmi Abu-Helu (Ph.D., Immunology) Copyright ©2021 F.A. Davis Company  Precipita*on Reac*ons Copyright ©2021 F.A. Davis Company  Chapter Overview § Precipita*on curve § Immunoturbidimetry and nephelometry § Passive immunodiffusion techniques § Electrophore*c techniques Copyright ©2021 F.A. Davis Company  Learning Outcomes (Cont.) AOer finishing this chapter, student should be able to: § Define the term precipita*on § Name and describe various types of precipita*on assays. § Describe the principle, advantages, and disavantages of nephelometry § Describe turbidimetry Copyright ©2021 F.A. Davis Company  Precipita*on Reac*ons § Soluble an*gen is combined with soluble an*body to produce insoluble complexes that are visible. § Require an*gen and an*body to have: Mul*ple binding sites for one another Equivalent concentra*ons Copyright ©2021 F.A. Davis Company  Precipita*on Reac*ons (Con.) § When a soluble Ag combines with its Ab in the presence of electrolytes (NaCl) at a suitable temperature & pH, the Ag-Ab complex forms an insoluble precipitate. § When instead of sedimenVng, the precipitate remains suspended as floccules, the reacVon is called FlocculaVon. § It can take place in liquid media or in gels such as agar, agarose or polyacrylamide. Copyright ©2021 F.A. Davis Company  Mechanism of Precipita*on § LaLce forma*on - laLce hypothesis by Marrack Copyright ©2021 F.A. Davis Company  Precipita*on Curve: Zone of Equivalence § Number of mul*-valent sites of an*gen and an*body are approximately equal. § For op*mal precipita*on, reac*ons must be run in the zone of equivalence. Copyright ©2021 F.A. Davis Company  Precipita*on Curve: Prozone § Prozone phenomenon (an*body excess) An*gen combines with only one or two an*body molecules. No cross-linkages are formed. False-nega*ve reac*ons may occur as a result of high an*body concentra*on. If a false-nega*ve reac*on is suspected, dilu*ng out the an*body and performing the test again may produce a posi*ve result. Copyright ©2021 F.A. Davis Company  Precipita*on Curve: Postzone § Postzone phenomenon (an*gen excess) Small aggregates are surrounded by excess an*gen. No laLce network is formed. The presence of a small amount of an*body may be obscured, causing false-nega*ve results. ‒ Test may be repeated about a week later with another specimen to give more *me for an*body produc*on. ‒ If the test is nega*ve again, it is unlikely that the pa*ent has the an*body. Copyright ©2021 F.A. Davis Company  Precipita*on Curve: Zone of Equivalence § Op*mum precipita*on occurs if the zone of equivalence, in which the number of mul*valent sites of an*gen and an*body are approximately equal Copyright ©2021 F.A. Davis Company  Applica*ons of Precipita*on Tests § Very sensitive in Ag detection (1µg of protein) § Forensic – identification of blood & seminal stains § Food adulteration Copyright ©2021 F.A. Davis Company  Types of Precipita*on Tests § Ring test § Slide / Tube floccula*on test § Immunodiffusion (precipita*on in gel) § Electroimmunodiffusion Copyright ©2021 F.A. Davis Company  Types of Precipita*on Tests (Con.) 1- RING TEST o Simplest o Layering Ag solution over a column of an*serum in a narrow tube o Ppt forms at the junc*on. o e.g. Ascoli’s thermoprecip*n test ü Lancefield grouping of streptococci 2- SLIDE TEST – A drop each of Ag & an*serum are placed on a slide & mixed – floccules appear e.g. VDRL test for syphilis Copyright ©2021 F.A. Davis Company  Types of Precipita*on Tests (Con.) 3- TUBE TEST o Tube floccula*on o Standardiza*on of toxins& toxoids 4- IMMUNODIFFUSION (Precip. in Gel) o Dis*nct & stable band(s) of ppt. o Can be stained o Number of different ags. in the mixture can be iden*fied Copyright ©2021 F.A. Davis Company  Immunoturbidimetry and Nephelometry § Automated methods § Immunoturbidimetry: A measure of the turbidity or cloudiness of a solu*on Measures reduc*on in light intensity due to the reflec*on, absorp*on, or sca[ering of light Light transmi[ed through suspension of par*cles The greater the complexes, the decrease in light passage Use a spectrophotometer or automated clinical analyzer to measure Copyright ©2021 F.A. Davis Company  Immunoturbidimetry and Nephelometry § Nephelometry Measures light sca[er as immune complexes form by par*cles Greater size and amount of complex, more light sca[er Light measured as index of the concentra*on of the solu*on Used in quantofoca*on of: üSerum proteins, IgG, IgA, IgM and IgE üComplement proteins Nephelometry is more sensiVve than tubidimetry Copyright ©2021 F.A. Davis Company  Nephelometry is more sensiVve than tubidimetry § Nephelometry can be used to detect either an*gen or an*body, but it is usually run with an*body as the reagent and the pa*ent an*gen as the unknown. § Nephelometry provides accurate and precise quan*ta*on of serum proteins, and due to automa*on, the cost per test is typically lower than other methods. Copyright ©2018, Elsevier Inc. All rights reserved. 18 Copyright ©2021 F.A. Davis Company  Passive Immunodiffusion § The precipita*on of an*gen–an*body complexes can also be determined in a support medium such as a gel. § Reactants are added to the gel, and an*gen– an*body combina*on occurs by means of diffusion. § When no electrical current is used to speed up this process, it is known as passive immunodiffusion. Copyright ©2021 F.A. Davis Company Copyright ©2021 F.A. Davis Company  Precipita*on Reac*ons in Agar Gel § Ag. and Ab. diffuse through the agar gel and precipitate at the zone of equivalence § Speed of travel is influenced by Molecular size § Assays include: o Single immunodiffusion o Double immunodiffusion o Radial Immunodiffusion (RID) o Countercurrent immunoelectrophoresis (CIE) o Immunofixa*on electrophoresis o Rocket immunoelectrophoresis Copyright ©2021 F.A. Davis Company  Single Immunodiffusion § Single diffusion in one dimension (Oudin procedure) Antigen Precipitation band Ab in agar gel Copyright ©2021 F.A. Davis Company Double Immunodiffusion (Ouchterlony Diffusion) § A double-diffusion technique § Slide or tube § Wells are cut into a gel, and both an*gen and an*body diffuse out radially. § A line of precipitate forms where an*gen and an*body meet in equivalent amounts. § Three possible pa[erns: iden*ty, par*al iden*ty, noniden*ty. Copyright ©2021 F.A. Davis Company  Ouchterlony Double Diffusion Passive Immunodiffusion § One of the older, classic immunochemical techniques. § In this technique, both an*gen and an*body diffuse independently through a semisolid medium in two dimensions: horizontally and ver*cally. § Wells are cut in a gel, and reactants are added to the wells. § Common errors: overfilling of wells, irregular well punching, gel drying, increased room temp., and contamina*on via bacteria or fungi Copyright ©2021 F.A. Davis Company  Ouchterlony Double Diffusion (Con.) n A_er an incuba*on period of between 12 and 48 hours in a moist chamber, precipi*n lines form where the moving front of an*gen meets that of an*body. n The density of the lines reflects the amount of immune complex formed. Copyright ©2021 F.A. Davis Company  Results Copyright ©2021 F.A. Davis Company  Ouchterlony Double Diffusion (Con.) n Most Ouchterlony plates are set up with a central well surrounded by four to six equidistant outer wells. n Mul*specific an*body is placed in the central well, and different an*gens are placed in the surrounding wells to determine if the an*gens share iden*cal epitopes. Copyright ©2021 F.A. Davis Company  Ouchterlony Double Diffusion (Con.) § Several pa[erns are possible § Fusion of the lines at their junc*on to form an arc represents serological iden*ty, or the presence of a common epitope. § A pa[ern of crossed lines demonstrates two separate reac*ons and indicates that the compared an*gens share no common epitopes, or nonidenVty. Copyright ©2021 F.A. Davis Company  The Three Ouchterlony Pa[erns Ouchterlony double diffusion is s*ll used to iden*fy fungal an*gens such as: Aspergillus, Blastomyces, Coccidioides, and Candida. Copyright ©2021 F.A. Davis Company  Radial Immunodiffusion (RID) § A manual, single-diffusion technique § An*body is in the support gel, and an*gen is placed in a well cut into the gel. § An*gen diffuses out and reacts with an*body to form a ring of precipita*on around the well. § End-point (Mancini) method – reac*on goes to comple*on § Kine*c (Fahey) method–faster; measurements taken before reac*on is complete Copyright ©2021 F.A. Davis Company  RID Endpoint Method Results § The square of the diameter is propor*onal to the an*gen concentra*on. § Pa*ent results are determined from a standard curve. § Uses: es*ma*on of Ig classes in sera & screening of sera for Abs. Copyright ©2021 F.A. Davis Company  Electrophoresis § Electrophoresis is the migra*on of charged solutes or par*cles in an electrical field. o Diffusion can be combined with electrophoresis to speed up or sharpen the results. o Electrophoresis separates molecules according to differences in their electric charge when they are placed in an electric field. Copyright ©2018, Elsevier Inc. All rights reserved. 32 Copyright ©2021 F.A. Davis Company  Serum Protein Electrophoresis (SPE) § Serum protein electrophoresis results in the separa*on of proteins into five frac*ons using cellulose acetate or agarose gel as a support medium. § The immunologic applica*ons of electrophoresis include iden*fica*on of monoclonal proteins in serum or urine. Copyright ©2018, Elsevier Inc. All rights reserved. 33 Copyright ©2021 F.A. Davis Company  SPE (Cont.) (Insert Figure 11.2) Copyright ©2018, Elsevier Inc. All rights reserved. 34 Copyright ©2021 F.A. Davis Company  SPE (Cont.) (Insert Figure 11.3) Copyright ©2018, Elsevier Inc. All rights reserved. 35 Copyright ©2021 F.A. Davis Company  SPE (Cont.) Copyright ©2018, Elsevier Inc. All rights reserved. 36 Copyright ©2021 F.A. Davis Company Countercurrent Immunoelectrophoresis (CIE) § Method o Ag and Ab migrate toward each other by electrophoresis o Used only when Ag and Ab have opposite charges - + Ag Ab § Qualita*ve o Rapid o Detect Abs. to infec*ous agents and microbial Ags. o Replaced by easier agglu*na*on tests Copyright ©2021 F.A. Davis Company  Immunoelectrophoresis n Immunoelectrophoresis is a double- diffusion technique that incorporates electrophoresis current to enhance results. n Typically, the source of the an*gens is serum, which is electrophoresed to separate out the main protein frac*ons. n An*serum is placed in troughs, and the gel is incubated for 18 to 24 hours. Copyright ©2021 F.A. Davis Company  Immunoelectrophoresis n Precipi*n lines develop where specific an*gen– an*body combina*on takes place. n These lines or arcs can be compared in shape, intensity, and loca*on to that of a normal serum control to detect abnormali*es. n This procedure has been used as a screening tool for the differen*a*on of many serum proteins, including the major classes of immunoglobulins Copyright ©2021 F.A. Davis Company  Immunoelectrophoresis Copyright ©2021 F.A. Davis Company  Immunoelectrophoresis (Con.) § InterpretaVon o PrecipiVn arc represent individual anVgens + - Ag Ag Ab Ag Ab Copyright ©2021 F.A. Davis Company  Immunofixa*on Electrophoresis (IFE) § A double-diffusion technique § Proteins in pa*ent serum, urine or CSF are electrophoresed, then an*body is applied directly or or contained in a cellulose acetete strip to the gel. § Precipitates form where an*gen–an*body combina*on has taken place. § It is performed to visualize increased or decreased produc*on of anVbody classes and to differen*ate monoclonal and polyclonal immunoglobulins in serum or urine. Copyright ©2021 F.A. Davis Company  Immunofixa*on Electrophoresis (Con.) n Pa*ent serum is applied to six lanes of the gel, and a_er electrophoresis, five lanes are overlaid with one each of the following an*bodies: an*body to gamma, alpha, or mu heavy chains and to kappa or lambda light chains. n The sixth lane is overlaid with an*body to all serum proteins and serves as the reference lane. Copyright ©2021 F.A. Davis Company  Immunofixa*on Electrophoresis (Con.) n Hypogammaglobulinemias will exhibit faintly staining bands, while polyclonal hypergammaglobulinemias show darkly staining bands in the gamma region. n Monoclonal bands, such as found in Waldenström’s macroglobulinemia or multiple myeloma, have dark and narrow bands in specific lanes Copyright ©2021 F.A. Davis Company  Sample IFE Results § One of the best-known adapta*ons of this technique is the Western blot, o used as a confirmatory test to detect an*bodies to HIV-1 Copyright ©2021 F.A. Davis Company  Rocket Immunoelectrophoresis n One-dimension electroimmunodiffusion, an adapta*on of radial immunodiffusion, was developed by Laurell. n An*body is distributed in the gel, and an*gen is placed in wells cut in the gel, just as in RID. n Electrophoresis is used to facilitate migra*on of the an*gen into the agar. Copyright ©2021 F.A. Davis Company  Rocket Immunoelectrophoresis (Con.) n The end result is a precipi*n line that is conical in shape, resembling a rocket, hence the name rocket immunoelectrophoresis. n The height of the rocket, measured from the well to the apex, is directly in propor*on to the amount of an*gen in the sample. n This technique has been used to quan*tate Igs. Copyright ©2021 F.A. Davis Company  Rocket Immunoelectrophoresis (Con.) § Used to quan*fy Ags. § Ags. are electrophoresed in agar-containing Ab § A pH is selected so that Abs. are immobile § Ag.-Ab. form precipitate “Rocket” shape § Height of the rocket is propor*onal to the ag. concentra*on in the sample. Copyright ©2021 F.A. Davis Company  Western Blot n A mixture of HIV anVgens is placed on a gel and electrophoresed to separate the individual components. n The components are then transferred to nitrocellulose paper by means of bloLng or laying the nitrocellulose over the gel so that the electrophoresis pa[ern is preserved. Copyright ©2021 F.A. Davis Company  Western Blot n Pa*ent serum is applied to the nitrocellulose and allowed to react by incuba*on. n The strip is then washed and stained to detect precipi*n bands. n An*bodies to several an*gens can be detected in the pa*ent sample. Copyright ©2021 F.A. Davis Company  Errors in Electrophoresis n Applica*on of current in wrong direc*on n Amount of current applied wrong q Too much or too li[le n Incorrect pH of buffer n Incorrect amount of *me n Concentra*ons of reagents an samples Copyright ©2021 F.A. Davis Company  Comparison of Precipita*on Techniques TECHNIQUE APPLICATION SENSITIVITY PRINCIPLE (μg Ab/ml) Nephelometry Immunoglobulins, 1–10 Light that is sca[ered at complement, C- an angle is measured, reac*ve protein, indica*ng the amount of other serum an*gen or an*body proteins present. Radial Immunoglobulins, 10–50 An*gen diffuses out into immunodiffusion complement a gel that is infused with (RID) an*body. Measurement of precipi*n ring diameter indicates concentra*on of an*gen. Copyright ©2021 F.A. Davis Company  Comparison of Precipita*on Techniques (con*nued) TECHNIQUE APPLICATION SENSITIVITY PRINCIPLE (μg Ab/ml) Ouchterlony double An*bodies to 20–200 Both an*gen and diffusion complex an*gens, an*body diffuse out such as fungal from wells in a gel. The an*gens pa[erns of precipitate lines formed indicate the rela*onship of an*bodies. Immunofixa*on Over- or Variable Electrophoresis of serum electrophoresis (IFE) underproduc*on followed by direct of an*body; applica*on of reagent presence of an*sera to the gel. monoclonal an*body Copyright ©2021 F.A. Davis Company