Serology for Medical Laboratory Students PDF

Serology for Medical Laboratory students Ephrem Awulachew (BSc, MSc) Basic principles of Immunologic and Serologic reactions 2 Learning Objectives At the end of the lesson, the students should be able to: List different types of serological tests State the principle of serological t...

Serology for Medical Laboratory students Ephrem Awulachew (BSc, MSc) Basic principles of Immunologic and Serologic reactions 2 Learning Objectives At the end of the lesson, the students should be able to: List different types of serological tests State the principle of serological tests Describe the application of different serological techniques Identify the advantages and drawbacks of different serological techniques Identify factors, which affect antigen-antibody 3 reaction. Outline 1.1. Introduction 1.2. Immunological technique 1.2.1. primary binding sites 1.2.2. Secondary binding sites 1.2.3. Tertiary binding tests 1.3. Factors affecting antigen antibody reactions 4 Introduction to Serology  Is the scientific study of blood serum.  It is the study of immune reaction in human blood.  The principle involved with serology is the antibody- antigen reaction.  Antigen is the substance which "provokes" the body to produce antibodies.  Antibody is the substance which f ights the invading organism. 5 Introduction to Serology…  I n serologic tests, sam ples are analyzed in a laboratory to detect either antigen or antibody.  In practice, it involves: A. Diagnostic identification of antibodies in the serum. Such antibodies are typically formed in response to an infection (against a given microorganism), against other foreign proteins (in response, for example, to a mismatched blood transfusion), or to 6 one's own proteins (in instances of autoimmune Introduction to Serology … B. Diagnostic identification of antigens in the sample Such antigens are typically whole organism, structural components of the organism (e.g., cell wall Ag, capsular Ag, f lagellar Ag, spore, nucleic acids etc.), products of microorganism (e.g., toxin, enzyme), tumor m akers, and products of infection ( e.g., Ag-Ab deposition, tissue destruction products), allergens, drugs, hormones, blood cells antigen, and self antigens. 7 Introduction to Serology…  Immune system: the structures, cells, and soluble constituents that allow the host to recognize and respond to foreign stimulus.  Secondary immune response: the cellular and humoral events that occur when an antigen is encountered for a second or subsequent time.  Specif icity: the special af finity between an antigen and its corresponding antibody.  Serum and plasma are the two most common samples for serologic tests to identify antibodies.  Serum: the fluid portion of the blood after the blood clots.  Plasma: is the fluid portion of blood from anticoagulated blood. 8 Application of serologic tests …  Serological tests may be performed for diagnostic p urp o se s w he n an i nf e c ti o n i s susp e c te d , i n rheumatic illnesses, and in checking an individual's blood type.  Serology of blood tests help to diagnose patients with certain immune def iciencies associated with the lack of antibodies. I n suc h c a se s, t e st s f o r a nt i b o d i e s w i l l b e consistently negative. 9 Application of serologic tests … Serological tests can be used in two ways: 1. A known antibody can be used to detect and measure an unknown antigen 2. A known antigen can be used to detect and measure an unknown antibody. Serologic tests are performed as: a) Qualitative tests - find out presence of antibodies or antigens in the serum. b) Quantitative tests – conducted by serial dilution dilutions.  Quantitative results are normally expressed in terms of the titer of the serum. Application of serologic tests …  Antigen tests Antigen tests often enable an early diagnosis or presumptive identifications of an infectious disease through:- Identification of a pathogen that has been isolated by culture Identification of pathogens in different samples of the patients, etc 11 Application of serologic tests … Antibody tests are used mainly:- To diagnose a microbial disease when the pathogen or microbial antigen is not present in routine specimen or if present is not easily isolated and identified by other available techniques. To screen donor blood for different infectious diseases. To monitor the effectiveness of a given treatment by measuring 12 antibody liter. Application of serologic tests … Antibody molecules combine reversibly with antigens to form immune complexes.  Ag+Ab Ag.Ab complex The detection and measurements of these reactions form the basis of serology. T h e r e f o r e s e r o l o g y - i s t h e s c i e n c e o f measuring antibody or antigen in body fluids. 13 Application of serologic tests …  Detection of either antigen or antibody may not necessarily indicate presence of diseases.  For example the presence of serum antibody indicate:  Present infection  Past infection  Carrier state  Vaccination  Presence of antigen may indicate  Present infection  Carrier state 14 Immunological techniques Three groups of immunological techniques are used to detect and measure antigen-antibody combination. Primary binding tests, Secondary binding tests and Tertiary binding tests. 15 Primary binding tests  Primary binding tests are tests that directly measure the binding of antigen and antibody (i.e.; directly measure or visualize the immune complex).  They are the most sensitive techniques in terms of the amount of detectable antigen or antibody.  It is usual to use radioisotopes, fluorescent dyes, or enzymes as labels to identify one of the reactants.  For example: Enzyme linked Immunosorbent assay (ELISA) tests and Radioimmunoassay (RIA) Western blotting Northern blotting Southern blotting Fluorescence tests 16 Primary binding tests…  Primary binding tests are widely used in the serological diagnosis of:  Bacterial,  Viral,  Fungal, and  Parasitic diseases.  They are usually sensitive and give reproducible results. 17 Immunofluorescence tests  They are widely used in the serological diagnosis of bacterial, viral, fungal, and parasitic diseases.  They are usually sensitive and give reproducible results.  Principle  Fluorescent dyes (f luorochromes) illuminated by UV lights are used to show the specif ic combination of an ant igen wit h it s ant ibody. The ant igen-ant ibody c omp lexes are seen f lu oresc ing ag ainst a dark 18 background. Immunof luorescence tests are referred to Immunofluorescence tests…  When immunof luorescence is specif ic ally used to detect an antigen by conjugating the f luorescent dye w i t h t he Fc re g i o n o f t he sp e c i f ic a nt i b o d y (immunoglobulins) against that antigen, it is referred to as Fluorescent Antibody Test (FAT).  There are two type of f luorescent antibody tests (FAT ): a. Direct 19 b. Indirect a. Direct fluorescent antibody tests (Direct FAT) Is used to detect and identify unknown antigen in specimens E.g. Viral, bacterial, fungal and parasitic antigens It is called direct test because the fluorescent dye is attached, or labeled, directly to the antibody. The f lu orochrome used is usually f lu orescein isothyocynate (FITC), which gives a yellow-green fluorescence. 20 A fluorescent substance is one that, when absorbing a. Direct FAT… Procedure A tissue or smear containing the organism (antigen) is f ix ed to a glass slide and incubated with the f luorescent antibody (antibody chemically linked to FITC). It is then washed to remove unbound antibody. E x am i ne d b y d ark- f ie l d i l l um i nat i o n und e r a microscope with UV light source. 21 The antigenic particles that have bound the labeled a. Direct FAT… Direct Fluorescent Antibody (DFA) Test
22 a. Direct FAT…  Direct FAT can be used; To identify bacteria when the numbers are very low. It may also be used to detect viruses growing in tissue culture or tissues from infected animals E.g., used to diagnose respiratory syncytial virus herpes simplex 1 and 2, Pneumocystis infections, rabies virus in the brains of infected animals or antigens of HIV on the surface of infected cells. 23 a. Direct FAT…  Advantages :  Rapid single step staining.  Easy to perform under suitable conditions.  It can be used for multiple antibodies from same host.  Disadvantages Spe c ial training is ne e d e d to pe rfo rm and re ad immunofluorescence tests.  Fluorescence microscope and high quality reagents are required.  Difficulties with the labeling procedure as each primary must be labeled individually. B. Indirect Fluorescent Antibody Test (IFAT) In the IFAT, unlabelled antibody combines with antigen and the antigen antibody complex is detected by attaching a f luorescent-labeled anti -species globulin to the antibody. The antibody, therefore, is labeled indirectly. Fluorescent-labeled antihuman globulin is used if the antibody is of human origin. 25 B. Indirect Fluorescent Antibody Test (IFAT)… The indirect FAT is used in two main ways: I. To detect and identify unknown antigen in specimen II. To detect antibodies in a patient's serum using a known antigen (microorganism). 26 Indirect Fluorescent Antibody (IFA) Test

B. Indirect Fluorescent Antibody Test (IFAT)… I. Indirect FAT to detect antigen A slide preparation of the specimen is made and unlabelled specific antibody is added. After allowing time for th e antigen and antibody to combine, the preparation is was h ed l eav i n g on l y an ti body th at h as combined with the antigen on the slide. 27 B. Indirect Fluorescent Antibody Test (IFAT)… I. Indirect FAT to detect antigen… A f luorescent labeled anti-species globulin is added and allowed to combine with the antibody. Then the excess is washed from the slide. The preparation is exam ined by f lu orescence microscopy using the correct filters. T h e a nt i g e n a nt i b o d y c o m p l e x w i l l b e se e n fluorescing brightly. 28 B. Indirect Fluorescent Antibody Test (IFAT)… II. Indirect FAT to detect Antibody  In this test, a known antigen is placed on the slide and the patient's serum is added.  The preparation is then washed and f lu orescent- labeled antihuman globulin is added to demonstrate the antigen-antibody reaction.  The preparation is examined by f lu orescence microscopy using the correct filters. 29 Advantage and Disadvantage of Indirect FAT Advantages of Indirect FAT Highly sensitive and specific. Since several labeled antiglobulin molecules will bind to each antibody molecule, the fluorescence will be considerably brighter Because the antiglobulins used are specific for each immunoglobulin class, the class of specific antibody may also be determined. Commercially produced secondary antibodies are relatively inexpensive. Disadvantages of Indirect FAT 2.Enzyme Linked Immunosorbent Assay (ELISA)  ELISA was developed in 1971, by Peter Perlmann and Eva Engvall in Sweden.  It is a common laboratory technique which is usually used to measure the concentration of antibodies or antigens in blood.  Used in the diagnosis of microbial infections.  They are specif ic, sensitive, and require only a small amount of specimen. 31  ELISA is an extremely sensitive test as little as 10 – 9 g of ELISA…  Reagents used in the ELISA are stable and have a long shelf life which makes for easy distribution to district laboratories.  The results of qualitative ELISA techniques can be read visually.  Large numbers of specimens can be tested at one time and the ELISA can be easily automated for use. ELISA…  ELISA is a plate-based assay technique designed for detecting and quantifying microorganisms, microbial peptides, proteins, Drugs, tumor markers, antibodies and hormones.  Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product.  The most crucial element of the detection strategy is 33 a highly specific antibody-antigen interaction. ELISA… Enzymes and substrates Enzymes used in ELISA techniques must be stable and soluble ; they must not be present in any quantity in the specimens being tested. The most commonly used enzymes are horseradish peroxidase, alkaline phosphatase, and beta galactosidase. A substrates used in ELISA are a chromogenic substrates that undergo an enzyme-mediated color change.  For example p-nitro phenol phosphate (paranitrophenyl phosphate). 34  This is hydrolyzed by alkaline phosphatase to inorganic phosphate and p-nitro ELISA…  ELISAs are typically performed in 96-well polystyrene plates, which will passively bind antibodies and proteins.  It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform.  Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material during the assay. 35 ELISA… Principles of ELISA ELISA uses an enzyme system to show the specific combination of an antigen with its antibody. The enzyme system consists of an enzyme which is labeled or linked to a specific antibody or antigen. A substrate which is added after the antigen antibody reaction. This substrate is acted on (usually hydrolyzed) by the enzyme attached to the antigen antibody complexes, to give a color change. The intensity of the color gives an indication of the amount of 36 
ELISA… General ELISA Procedure ELISA begins with a coating step or uses precoated plates, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate. This is followed by a blocking step in which all unbound sites are coated with a blocking agent. To detect the bound antibodies or antigens, a secondary 37 antibodies that are attached to an enzyme ELISA… General ELISA Procedure… After an incubation period, the unbound secondary antibodies are washed off. When a suitable substrate is added, the enzyme reacts with it to produce a color. This color produced is measurable as a function or quantity of antigens or antibodies present in the given sample. The intensity of color/ optical density is measured at 450nm. 39 ELISA… There are two main ways of performing ELISA a.Direct ELISA b. Double antibody technique ((Sandwich ELISA) c.Indirect ELISA d. Competitive ELISA 40 a. Direct ELISA  For direct detection, an antigen coated to a multi-well plate is detected by an antibody that has been directly conjugated to an enzyme.  This detection method is a good option if there is no commercially available ELISA kits for your target protein.  Advantages Quick because only one antibody and fewer steps are used. a. Direct ELISA…  Disadvantages Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags. Labeling primary antibodies for each specific ELISA system is time-consuming and expensive. b. Double antibody ELISA (sandwich ELISA)  Specific antibody is coated on the surface of the well of a microtitration plate (or a test tube), and the specimen is added.  After a period of incubation during which the antibody takes up (captures) the antigen from the specimen, the well is washed leaving the antigen attached to the antibody. 43 b. Double antibody ELISA (sandwich ELISA)…  Enzyme labeled specif ic antibody (often the same antiserum as that coating the well except it is enzyme linked) is added to detect the presence of the antigen.  After a further period of incubation during which the enzyme labeled antibody combines with the antigen, the well is washed and a substrate is added.  The enzyme acts on the substrate to give a color 44 change in the fluid. b. Double antibody ELISA (sandwich ELISA)…  The enzyme activity is stopped by altering the pH of the reaction or denaturing the enzyme.  By measuring the color produced, the amount of attached antibody and therefore antigen in the specimen can be estimate. Application  In developing countries, an important application of the double antibody ELISA is in the diagnosis of  Rotavirus infection in young children. 45 b. Double antibody ELISA (sandwich ELISA)… Double antibody ELISA (antigen test) 46 b. Double antibody ELISA (sandwich ELISA)… Procedure  Prepare all reagents and necessary materials  Prepare serum from non-hemolyzed blood.  Take micro titration well plates, which are coated with specific antibody.  Add positive and negative control in 4 micro titration well.  Add specimens to the micro titration well of 96.  If specimen containing antigen combines with 47 antibody. b. Double antibody ELISA (sandwich ELISA)…  Wash the plate by using automatic washer more than 4 times.  Add enzyme labeled antibody, which attaches to the antigen.  Wash the procedure.  Add the substrate, which is hydrolyzed (broken down), gives color changes.  Read the result. 48 b. Double antibody ELISA (sandwich ELISA)… Advantages of sandwich ELISA High specificity, since two antibodies are used the antigen is specifically captured and detected. Suitable for complex samples, since the antigen does not require purification prior to measurement. Flexibility and sensitivity, since both direct and indirect detection methods can be used. C. Indirect ELISA  In this technique, known antigen is attached to the inside surface of the well and patient’s serum is added.  After incubation and washing, enzyme labeled antihuman globulin is reacted with the antibody that has attached to the antigen.  The presence and concentration of antibody that has reacted with the antigen is shown by a change in color when the substrate is added.  The intensity of the color is directly proportional to the 50 concentration of antibody in the serum. C. Indirect ELISA… (Specimen) 51 Source: Kuby Immunology 2007, 5th ed C. Indirect ELISA… Procedure  A micro titration well plate is coated with known antigen.  Block all unbound sites to prevent false positive results.  Add patent’s serum for the antibody to combine with antigen.  Wash carefully by using automatic washer more than 4 times.  Add enzyme labeled antihuman globulin, which attaches to the antibody.  Wash carefully.  Add the substrate, which is hydrolyzed (broken down) by the enzyme to give a color change. 52 d. Competitive ELISA  This test is used to measure the concentration of an antigen in the sample.  In this test, antibody is first incubated in solution with a sample containing antigen.  The antigen-antibody mixture is then added to the microtitre well which is coated with antigen.  The more the antigen present in the sample, the less free antibody will be available to bind to the antigen- coated well. d. Competitive ELISA…  After the well is washed, enzyme conjugated secondary antibody specific for isotype of the primary antibody is added to determine the amount of primary antibody bound to the well.  The higher the concentration of antigen in the sample , the lower the absorbance. d. Competitive ELISA… Procedure of Competitive ELISA Antibody is incubated with sample containing antigen. Antigen-antibody complex are added to the microtitre well which are precoated with the antigen. Wash the plate to remove unbound antibody. Enzyme linked secondary antibody which is specific to the primary antibody is added. Wash the plate, so that unbound enzyme-linked antibodies are removed. Add substrate which is converted by the enzyme into a Reading ELISA results  If it is qualitative ELISA  Read by naked eye. The presence or absence of antigen is seen as a simple color change.  If it is quantitative antibody techniques  Read either By measuring the intensity of color in a spectrometer (spectrophotometer) or By testing dilutions of the test serum and determining the highest dilution that shows a color change. 56 Reading ELISA results …  By using a spectrophotometer, spectrofluorometer, or electrochemical device, the results can be read and recorded.  The amount of color produced is proportional to the amount of primary antibody bound to the antigen proteins on the bottom of the wells. Radio Immunoassay (RIA)  One of the most sensitive techniques for detecting antigen or antibody is RIA  1st developed by two endocrinologist called S.A. Berson and Rosalyn Yalow, in 1960 to determine levels of insulin-anti-insulin complexes in diabetics. 58 Radio Immunoassay (RIA)… a. Conventional RIA Is a competitive immunologic procedure. It measures very low concentrations of antigens (or antibodies) by using radioactively labeled antigens as competitors. Radioactive isotopes such as 3H , 14C, 35S, 30P or 125I can be used for labeling Radioactive isotopes are molecules with unstable nuclei 59 and therefore emit radiation spontaneously. Radio Immunoassay (RIA)…  It is highly sensitive m ethod to detect low concentration of unknown (unlabeled) antigen  RIA is used to assay: Hormones, Drugs, Enzymes, Microbial antigens e.g. hepatitis B antigen, carcinoembryonic and α- feto protein antigen. 60 It also used to the detection of antibody Radio Immunoassay (RIA)…  RIA technique utilizes three components  Patient antigen o The specific compound we wish to determine.  Labeled antigen o The same compound as above to which is attached a radioactive label.  Antibody o Specific for the sample and labeled antigen. 61 Radio Immunoassay (RIA)… Practical procedure of RIA In a conventional RIA, a patient sample containing the antigen of interest is incubated simultaneously with a fixed amount of labeled antigen and a known amount of excess specific antibody. During this incubation time, labeled and unlabelled antigens compete for a limited number of antibody binding sites. After incubation, bound and free materials are separated from one another. By determining the amount of radioactive labeled antigen in 62 either the bound or free fraction, the amount of patient antigen Radio Immunoassay (RIA)…  In RIA, standards are run to establish a standard curve for final evaluation.  An antigen and labeled antigen compete for limited binding sites, the extent of binding of each depends on their relative concentrations.  RIA obeys the law of mass action, a small quantity of labeled Ag is bound as the quantity of sample antigen is increased.  Therefore, the concentration of sample antigen is inversely proportional to the amount of radioactive labeled Ag bound to the specific antibody.  The higher the count per minute (cpm), the lower is the concentration of sample Ag and vice-versa. 63 Radio Immunoassay (RIA)….  There are two assay approaches in conventional RIA i. Liquid phase Assay ii. Solid phase Assay 64 Radio Immunoassay (RIA)… i. Liquid phase assay: The sample, labeled antigen and the specific antibody are added to the mixture in a solution form. After completion of incubation with the ligand of interest (analyte), a bound-free separation step is performed using different techniques.  Precipitate the antigen-antibody complexes by adding a "second“ antibody directed against the first. 65  Radio Immunoassay (RIA)… ii. Solid phase assay In this assay, ✓The specif ic antibody is added either in a suspension or the antibody is covalently bound to the inside wall of the reaction tube.  Se paratio n o f the bo und - fre e frac tio n is re alize d by centrifugation or magnetic separation followed by decanting the supernatant or by simply pouring off the reaction mixture if coated tube is used.  The bo und frac ti o n i s the n washe d ad e q uate l y wi th appropriate buffered wash solution and made ready for 66 counting. Radio Immunoassay (RIA)… b. Immunoradiometric Assay (IRMA) (Sandwich Immunoassay)  Developed with the objective of solving the problems associated with conventional RIA  Physical separation methods (centrifugation decanting procedures) may distort the equilibrium between bound-free fractions, spillage loss of labeled antigen.  Frequent non-specified binding.  Lack of sensitivity for very low concentration levels of a particular analyte. 67 Radio Immunoassay (RIA)… Basic principles of Immunoradiometric Assay Specific antibody or antigen is bound to the inside wall of a tube. The same specific antibody is labeled with radioisotope (in antibody determinations, antigen is labeled with radioisotope). The antigen is specifically attached to the wall bound antibody without competition. 68 Radio Immunoassay (RIA)… Basic principles of Immunoradiometric Assay… As there is excess amount of bound antibody present, virtually all-available Ag molecules in the sample can be bound → increased sensitivity! As an excess labeled antibody is added, practically all bound Ag can be detected. In contrast to RIA (conventional), in IRMA there is a direct relationship between amount of bound radioisotope 69 and the concentration of sample antigen. 4. Western blot test (WB) Is an assay technique for protein. Used as a confirmatory test and very specific for protein analysis. Antibodies to proteins may yield an indeterminate Western blot. Western Blotting is an effective and widely used technique for the separation of a specific protein from a complex sample or mixture of proteins. It is also known as immunoblotting because antibody probes are utilized to detect the target protein on the 70 4. Western blot test (WB)… Western Blotting Principle Proteins are separated based on shape and size by SDS-PAGE electrophoresis. The separated proteins are then transferred to nitrocellulose or nylon membrane. On the membrane, they are probed with antibodies that are specific to the protein of interest. Then antigen and antibody complex formation occurs. The complex can be detected either by autoradiography or secondary antibody linked with an enzyme. 4. Western blot test (WB)… SDS PAGE is a prerequisite for western blotting. Proteins get spilt up by their size by a process called SDS-polyacrylamide gel electrophoresis. A primary antibody that target a protein is then used to probe and wash the membrane with transferred protein. This primary antibody treated membranes are then reacted with a secondary antibody, usually an antibody enzyme conjugate (e.g. horseradish 4. Western blot test (WB)… The target protein is visualized as band on blotting paper, X-ray film or imaging system. For example: bands corresponding to p24 and p55 are detected early in sero conversion followed by glycoprotein bands. 4. Western blot test (WB)… Procedure of Western blotting: Load sample suspected to contain protein. Proteins are separated on the basis of their size, shape and charge in an electrophoresis. Blotting (transferring) protein to filter paper from gel electrophoresis. Then the blot is stained as proteins are not directly visible in the gel. Dyes like coomassie blue, silver stain or deep purples are used. Then the blot is imaged with suitable instrument and a permanent record can be made after staining. Incubate the blot with one or more antibodies. Primary antibodies are specific depending on the antigens to be detected. The secondary antibody (monoclonal or polyclonal) is linked to an enzyme that is used to indicate the location of the protein. 4. Western blot test (WB)… Western Blotting Applications It is used as a confirmatory test for HIV. Determination of size and quantity of protein in a sample Serodiagnosis of tubercular meningitis and neurocystocircosis Separation of protein from a complex mixture Detection of autoimmune diseases Southern blotting  Discovered by a scientist named in E.M. Southern, who 1st described it in 1975.  Used to detect specific DNA sequences.  Used in clinical lab to detect gene mutations responsible for various diseases and etiology of a disease.  Used to detect carriers of the fragile X 77 syndrome and sickle cell anemia for genetic Southern blotting  A cloned gene or a PCR fragment can be used as a probe for finding segments of DNA that have the same or a very similar sequence.  The genomic DNA is extracted and digested with a restriction enzyme.  These fragments can be separated into groups of fragments of the same length by using (agarose) gel electrophoresis.  DNA is negatively charged, so migrate toward the positive pole. 78  The electric field causes the molecules to move through Southern blotting…  After fractionation, the gel is soaked in alkali to denature the double-stranded DNA and then blotted onto a piece of porous positively charged membrane , where they stay in the same relative positions.  After having been soaked in alkali to separate the DNA strands and link the DNA to the membrane, the membrane is placed in a hybridization buffer containing the probe.  The single-stranded probe will find and bind to its 79 Southern blotting…  Unbound probe is removed by washing steps. The probe must be labeled such that it can be readily located on the membrane once it has bound to its complementary target sequence.  Probes can be labeled with radioactive atoms (for example by adding a 32P-atom).  Radioactive probes can be detected by autoradiography (X-ray sensitive film).  Fluorescent probes can be detected by irradiation with appropriate wavelength UV light and monitoring the 80 longer wavelength that is emitted in response. Northern blotting  Northern blot hybridization is a technique used to detect and quantify a given mRNA species within a mixture of RNAs in vitro. Steps in Northern blotting:  The RNAs in the mixture are separated according to size by electrophoresis in an agarose gel.  The RNAs are transferred from the gel to a membrane filter. 81 This transfer step is also called blotting.  Northern Blotting…  The target mRNA is detected by incubating the membrane with a specific, radiolabeled nucleic acid probe.  The probe anneals by base pairing with the specific mRNA species of interest.  The membrane serves as the solid support that allows separation of specifically bound probe from excess probe, which is simply washed away.  82 Probe that is bound to RNA on the membrane is then Northern Blotting… 83 Northern Blotting…  The probe has three essential features: It consists of a sequence complementary to the mRNA of interest.  It is single-stranded. It contains a radioactive label so that it is readily detected. To accurately quantitative the mRNA species of interest, the mRNA, of probe must exceed the 84 amount of mRNA on the filter. Northern Blotting…  Applications of Northern blotting :  Detect multiple forms of an mRNA derived from a single gene.  Monitor changes in the expression of a gene during different stages of growth or development. Detect differences in gene expression in different cell types.  Compare the amounts and sizes of mRNAs of genes involved in various cellular processes (e.g., cell cycle regulation). 85 Used in the diagnosis of viral diseases. Secondary binding tests  Secondary binding tests are tests that detect and measure the consequences (secondary effect) of antigen-antibody interaction.  These consequences include:  Precipitation  Agglutination  Neutralization  Activation of the complement system.  They are usually less sensitive than primary binding 86 tests, but may be easier to perform. Agglutination tests  It is a fundamental and most commonly used reaction in medical serology laboratory.  Agglutination is simply the clumping of cells into aggregates.  Agglutination is a result of the combination of an antibody's binding sites with antigen binding sites of the cells.  When an antibody combines with a corpuscular 87 antigen (e.g. bacteria, virus, blood cell or inert part Agglutination tests… Principle of agglutination tests Agglutination is the visible clumping together of bacteria, cells, or particles, when combining with its specific antibody. The resulting clumps are referred to as agglutinates. In general, to detect antibody in patients serum a known antigen suspension is added or to detect antigen known specific antibody is added. 88 Agglutination tests… Advantages of agglutination tests serological tests. They are:  Simpler to perform.  Require no special equipment.  Usually less expensive. 89 Agglutination tests… Agglutination tests can be performed: i. On slides or tiles ii. In tubes iii. In micro titration plates 90 Agglutination tests… i. Slide or tiles agglutination tests Rapid and easily performable techniques Gives a reaction in minutes or even seconds. Not usually as sensitive as tube or micro titration techniques. Specificity depends on the reagent used. There two methods of slide agglutinations: A. Active 91 B. Passive Agglutination tests… a. Active agglutination slide tests  Direct agglutination of microbial antigen with its corresponding antibody. E.g. the slide agglutination of salmonella, shigella or vibrio cholera using specif ic antibody, or the agglutination of leptospiral antigen by leptospiral antibodies present in a patients serum in acute leptosprosis. 92 Agglutination tests… Slide agglutination tests are used: To identify bacteria from cultures are dif ficult to standardize and control. T o c h e c k a u t o - a g g l u t i n a t i o n ( f a l s e agglutination) due to the organis m not emulsifying well or the fluid evaporating. 93 Agglutination tests… b. Passive agglutination slides tests  S p e c i f ic an ti b od y or k n ow n an ti g e n i s attached to inert particles or cells.  When the known antigen or antibody combines with its corresponding antibody or antigen in the specimens the particles or cells are used only to show that an antigen antibody 94 reaction has occurred. Their role in these Agglutination tests… The substances and cells used as carriers in passive slide agglutination test include:  Latex particles  Carbon particles  Stabilized staphylococcal cells 95 Agglutination tests… Latex particles: Are polystyrene particles that can be coated with either known antigen or specific antibody. Carbon particles: These are coated with cardiolipin antigen and used in the rapid plasma reagin (RPR) 96 card test to screen for cardiolipin antibodies Agglutination tests…  Stabilized staphylococcal Cells:  Most strains of Staphylococcus aureus produce on their outside surfaces a substance called protein A on to which specific antibody can be bound.  Killed staphylococcal cells coated with antibody can be used to identify bacteria and detect soluble extracellular bacterial antigens in specimens and body fluids.  The term coagglutination (COAG) is used to describe the agglutination of antibody coated staphylococcal 97 Agglutination tests… ii. Tube agglutination tests  In tube tests, agglutination occurs in a larger volume of fluid and in fully controlled conditions.  Tube tests are usually more sensitive than slide tests.  In this tube agglutination test, serum is diluted serially and then antibody level is measured by adding standard antigenic suspension.  The temperature and time of agglutination must be correct. 98  For example used in the investigation of enteric fever Agglutination tests… iii. Micro titration agglutination tests  Are performed in micro titration plates.  Now replaced several tube agglutination tests because they are more sensitive, more economical, and easier to perform, and usually give quicker results. 99 Agglutination tests…  Other forms of agglutination is an agglutination that involved red blood cells.  Agglutination reactions involving red cells are called hemagglutinations  There are different types of hemagglutinations 1. Hemagglutination inhibition antibody test (HAI) 2. Indirect (passive) hemagglutination test (IHA) 3. Reverse passive hemagglutination test (RPHA) Hemagglutination inhibition antibody test (HAI)  This technique is used: To detect antibodies against Arboviruses, Influenza viruses, Measles, viruses, and Rubella virus.  These viruses are able to agglutinate red cells because they posses hemagglutinins on their outer 101surfaces. Hemagglutination inhibition antibody test (HAI)…  In the hemagglutination inhibition antibody test, the patient's serum is reacted with a suspension of known viral antigen.  If the corresponding antibody is present, it will coat the haemagglutinins (antigens) on the viral particles and so prevent hemagglutination when red cells are added.  Controls must be included to show the agglutinating activity of the antigen and absence of hemagglutination by the serum.  Sera should be treated to destroy non-specific inhibitors 102 Indirect (passive) hemagglutination test (IHA)  The indirect hemagglutination (IHA) test is a passive agglutination test in which known antigen is coated on treated red cells. Carrier red cells  The cells are formalin fixed and treated with tannic acid to make the antigen adhere, antigen coated red cells are referred to sensitized cells. 103 Indirect (passive) hemagglutination test (IHA)…  In the IHA test, the sensitized red cells are added to dilutions of the patient's serum.  If the serum contains the corresponding antibody in sufficient concentration, the red cells will be agglutinated and settle to form an even covering in the bottom of the well.  If the sensitized cells are not agglutinated they will settle and form a red button in the bottom of the well.  E.g., Treponema pallidum hemagglutination (TPHA) 104 Reverse passive hemagglutination test (RPHA) This technique is used to identify viruses that do not hemagglutinate. It performed by reacting viral specimens with red cells coated with specific viral antibody. If the corresponding antigen is present, the red cells will be agglutinated. 105 Measurement of antibody in serum (antibody titer)  To diagnose microbial diseases by their antibody response.  To confirm active infections it is usually necessary to show three or four-fold rise in the serum antibody level.  This is because the patient may already have agglutinating antibodies in their serum from a previous infection, or following natural or acquired immunization.  It is therefore, necessary to test two specimens (paired sera).  The first collected within 5 days of the onset of Measurement of antibody in serum (antibody titer)  The antibody level is measured by diluting the serum  This dilution is usually by using a doubling dilution technique (i.e. 1 in 2, 1 in 4, 1 in 8, 1 in 16, etc).  A standardized antigen suspension is then added.  Following incubation time, the tubes are examined for agglutination.  The last tube to show a clear supernatant with a coarse deposit is the end-point of the test.  The dilution of the serum at this end-point is known as the titer. Prozone effect  It is a condition of high antibody titer that block agglutination of Ag-Ab.  When testing a serum with a high antibody titer, it is possible for only the higher dilutions to show agglutination.  This is referred to as the prozone reaction, or phenomenon.  It is thought to be due to a high level of Ig A (blocking antibody), non-specific inhibitory factors or antibody Precipitation tests  A precipitation occur when a soluble antigen reacts with antiserum in presence of electrolytes (NaCI) at a suitable temperature and pH.  Antibodies that aggregate soluble antigens are called precipitins.  Precipitation is a process where soluble antigens bind with their specif ic antibody at an optimum temperature and pH, resulting in the formation of an insoluble precipitate (insoluble lattice). 109 Precipitation tests…  Precipitate settles to the bottom of the tube in aqueous solution or appears as an opaque white line in gel.  Precipitation is sensitive in detection of antigens  As little as 1 g of protein can be detected by precipitation tests.  Precipitation is relatively less sensitive for the detection of antibodies.  The amount of precipitate formed is greatly influenced by the:  Relative proportions of antigens and antibodies Precipitation tests… For precipitation to take place, the antibody must be bivalent, and the antigen must be either bivalent or polyvalent. Precipitation takes place in the zone of equivalence, where the concentration of antigen and antibody is equal. Unlike agglutination reactions precipitation reaction involve a small, soluble antigen. When the antibody-antigen rxn occurs, a few small, 111 Precipitation tests…  Precipitation tests are used to detect and identify antigens in  specimens  extracts and  cultures  They are also used to detect and quantify antibodies in serum.  Compared with agglutination tests, precipitation te c hni q ue s re q ui re m o re e x p e ri e nc e i n the i r 112 Precipitation tests… Precipitation tests are performed, in aqueous solution, or in semisolid media such as agar or agarose, or non gel support medium such as cellulose acetate. Agarose are largely used as an immunodiffusion medium. Agarose is a transparent, colorless, neutral gel. Precipitation tests…  Antigen and antibody must be in an appropriate concentration relative to each other. 1.Antigen access: Too much antigen prevents efficient crosslinking/lattice formation. 2.Antibody access: Too much antibody prevents efficient crosslinking/lattice formation. 3.Equivalent Antigen and Antibody: Maximum amount of lattice (Precipitate) is formed Precipitation tests…  The zone of antibody excess is known as the prozone phenomenon and the zone of antigen excess is known as the postzone phenomenon.  Thus, for precipitation reactions to be detectable, they must be run in the zone of equivalence.  When instead of sedimenting, the precipitate remains suspended as floccules, the reaction is known as flocculation. Precipitation tests… Prozone phenomenon Prozone phenomenon is a false negative response resulting from high antibody titer which interferes with formation of antigen antibody lattice. The Prozone phenomenon may be either due to presence of excess antibodies in serum (e.g., Brucellosis, secondary syphilis , Human immunodeficiency virus (HIV) co-infection, and pregnancy) or blocking antibodies or to non-specific inhibitors in serum. The monovalent antibodies (blocking antibodies) or absence of cross linking of antigen by bivalent antibodies both prevent Precipitation tests… Types of precipitation test  There are three main types of precipition techniques: a. Tube precipitation test b. Gel diffusion tests c. Counter immunoelectrophoresis tests 118 a. Tube precipitation  A clear solution containing the test antigen is carefully layered on to a clear antiserum in a pr eci pi ti n tu be o r ca pi l l a r y tu be ( mi cr o hematocrit).  Fo l l o wi ng a per i o d o f i ncuba ti o n, i f the corresponding antigen to antibody is optimal, a line of visible precipitating antibody and antigen 119 a. Tube precipitation…  Ring test and flocculation tests are examples of precipitation in solution. Ring test:  In this test, the antigen solution is layered over antiserum in a test tube.  Precipitation between antigen and antibody in antiserum solution is marked by the appearance of a ring of precipitation at the junction of two liquid layers.  E.g., C-reactive protein (CRP) and Streptococcal grouping by the Lanoefield methods are the examples of ring test. a. Tube precipitation… Flocculation test: Flocculation test may be performed in a slide or tube. In this test, a drop of antigen solution is added to a drop of patient’s serum on a cavity slide and the result is recorded after shaking the slide. In a positive test, the floccules appears which can be seen by naked eye or demonstrated well under the microscope. a. Tube precipitation… Disadvantage Needs large amount of antiserum The test is not very sensitive. Use To detect extracellular antigens in CSF, especially H-influenza type b antigen To group - hemolytic streptococci 122 b. Gel diffusion tests  When an antibody and its antigen are placed in different regions of an agar gel, they diffuse toward each other and form an opaque band of precipitate at the junction of their diffusion fronts.  P re c i p i t a t i o n c a rri e d o ut i n g e l i s k no w n a s ‘immunodiffusion’.  Gels are prepared from 1-2% agar or agarose solution in a suitable buffer, such as barbitone buffer.  Either (single) or both (double) reactants diffuse due 123 b. Gel diffusion tests…  The rate of diffusion is affected by the size of the particle, temperature, gel viscosity, amount of hydration and interaction between the matrix and reactants.  When both antigen and antibody diffuse through the agar this is referred to as double diffusion.  When only the antigen or antibody diffuse, with the corresponding antigen or antibody is being contained in the agar, this is called single diffusion. b. Gel diffusion tests… Double gel diffusion (Ouchterlony)  Antigen and antibody diffuse towards each other and where they meet in optimal proportion a visible line of precipitation forms.  The thickness of the line of precipitation is a semi quantitative measure of the amounts of antigen and antibody that combine. 125 b. Gel diffusion tests… Example: Elek gel technique Used to detect toxogenic strains of C. diphtheria Biken test Used to detect toxin- producing fecal E. coli (ETEC) 126 b. Gel diffusion tests… Single gel diffusion I n th i s te c h n i qu e , s p e c i f ic an ti b od y i s incorporated into the agar gel and wells are cut to contain the antigen, which diffuses radially. A ring of precipitation forms around a well that contains the corresponding antigen. The higher the concentration of antigen, the 127 c. Counter immunoelectrophoresis (CIE)  In this precipitation techniques speed of movement can be increased by applying electric current. Also called  Countercurrent-electrophoresis (CEP)  Immuno electroosmophoresis (IEOP)  Electro immunodiffusion. 128 Counterimmunoelectrophoresis (CIE)  It is used to increase the speed with which the antigen and antibody travel in the agar gel.  In this test, specific antibody is placed in a well at the positive electrode (anode) end of the plate and the unknown antigen in a well at the negative electrode (cathode) end.  An electric current is applied and the antibody and antigen move towards each other.  A line of precipitation forms where the two meet in optimal proportion. Complement fixation tests  In general, complement f ixation tests (CFT) are best performed in reference laboratories where facilities exist for the careful standardization and control of reagents, which these tests require.  Used to detect and quantify antibody that does not agglutinate or precipitate when reacted with its antigen, but can be demonstrated by its use, or fixation, of complement. 130 Complement fixation tests…  Antigen-antibody reactions lead to immune complex formation that produces complement fixation.  Complement fixation is when complements are bound or fixed to the antigen antibody complexes.  When these complexes are on bacteria, red cells or other cells, the complement brings about the lysis of the cells involved.  This may be exploited to determine the amount of antigen or antibody present in the patient sample. Complement fixation tests…  In the CFT, the patient's inactivated serum is serially diluted and reacted with known antigen in the presence of complement.  If the corresponding antibody is contained in the serum it will combine with the antigen and use up the complement.  This will leave no complement to hemolyze the antibody coated red cells that are added.  The highest dilution of serum that prevents hemolysis Complement fixation tests  Method Ag mixed with test serum to be assayed for Ab Standard amount of complement is added Erythrocytes coated with Abs is added Amount of erythrocyte lysis is determined Ag No Ag Ag Patient’s Y serum Y Ag Y Y Y Y Y Y Y Y 133 Tertiary binding tests  Tertiary binding tests measure the consequences of immune responses in vivo.  These tests are much more complex than primary and secondary tests but their results ref le ct the practical significance of the immune response. E.g. measurement of the protective effects of antibody. 134 1.3. Factors affecting antigen antibody reaction  Specificity  Cross reactivity  Temperature  pH  Ionic strength  Concentration  Intermolecular specificity 135  Review questions Wr it e t he d iffe re nc e b e t w e e n p re c ip it at ion and agglutination tests How does the zonal reaction affects test results? Write the advantage and disadvantage of serological test compared with other laboratory techniques for infectious disease. 136 Reference 1. Naville J. Bryant Laboratory Immunology and Serology 3 r d edit ion. Serological services Ltd.Toronto,Ontario,Canada,1992 2. Tizard. Immunology an introduction,4 th edition , Saunders publishing,1994 3. Mary Louise.Immunology and Serology in Laboratory medicine 3rd edition 137