Immuno Assay PDF

Kampala International University Immuno Assay By Dr.Ghada Tajelsir Abdoun Department of Biochemistry  Radial Immundiffusion- Principle, Procedure, Results, Uses Immuno-diffusion is a technique for the detection or measurement of antibodies and antigens by their precipi...

Kampala International University Immuno Assay By Dr.Ghada Tajelsir Abdoun Department of Biochemistry  Radial Immundiffusion- Principle, Procedure, Results, Uses Immuno-diffusion is a technique for the detection or measurement of antibodies and antigens by their precipitation which involves diffusion through a substance such as agar or gel agarose.. Based on the method employed, immuno-diffusion may be: Radial immunodiffusion  Radial immunodiffusion (RID) or Manc ini method also known as Mancini immunodiffusion or single radial immunodiffusion assay. It is a single diffusion technique whereby a solution containing the antigen is plac ed into in a gel or agar surface impregnated with antibody.  Objectives of Radial Immunodiffusion To detect antigen-antibody complexes. Describe the circumstances under which antigen-antibody complexes precipitate out. Determine relative concentration of antigens.  Principle of Radial Immunodiffusion Radial immuno-diffusion is a type of precipitation reaction. It is thus based on the principles of the precipitin curve which states that antigen-antibody interact forming visible cross-linked precipitate when the proper ratio of antigen to antibody is present.  Procedure of Radial Immunodiffusion An agar containing an appropriate antiserum (antibody) is poured in plates. Carefully circular wells are cut and removed from the plates. A single or series of standards containing known concentration of antigen are placed in separate wells, while control and “unknown” samples are placed in other remaining wells. As the antigen diffuses radially, a ring of precipitate will form in the area of optimal antigen – antibody concentration. The ring diameters are measured and noted. A standard curve is prepared using the ring diameters of the standards versus their concentrations. This curve is then used to determine the concentration of the control and unknown samples.  The presence of a precipitin ring around the antigen wells indicate specific antigen-antibody interaction. Absence of precipitin ring suggest absence of reaction. The greater the amount of antigen in the well, the farther the ring will form from the well.  Applications of Radial Immunodiffusion determine the quantity or concentration of an antigen in a sample. Estimation of the immunoglobulin classes in sera. Estimation of IgG, IgM antibodies in sera to influenza viruses.  Other applications include: To determine relative concentrations of antibodies in serum. Estimate serum transferrin and alpha- feroprotein. To compare properties of two different antigens. To determine the relative purity of an antigen preparation For disease diagnosis Serological surveys  Advantages of Radial Immunodiffusion Precipitation in gels is provide more specific and sensitive results. The reaction is in the form of bands of precipitation and can be stained for better viewing. If a large number of antigens are present, each antigen-antibody reaction will give rise to a separate line of precipitation. This technique also indicates identity, cross reaction and non identity between different antigens.  Limitations of Radial Immunodiffusion Long reaction time (18-48 hours) results is influenced by the presence bound metal cations in the test samples (protein). Single diffusion method of precipitation is considered relatively wasteful than other methods. The test has been recently replaced by more sensitive and automated methods, such as enzyme-linked immunosorbent assays.  Immunoelectroph oresis Principle,Procedure, Results and Uses  Immunoelectrophoresis Immunoelectrophoresis refers to precipitation in agar under an electric field. It is a process of a combination of immuno- diffusion and electrophoresis. An antigen mixture is first separated into its component parts by electrophoresis and then tested by double immuno-diffusion. Antigens are placed into wells cut in a gel (without antibody) and electrophoresed. then cut in the gel into which antibodies are placed. The antibodies diffuse laterally to meet diffusing antigens, precipitation determination of the nature of the antigens.  Principle of Immunoelectrophoresis When an electric current is applied to a slide layered with gel, the antigen mixture placed in wells is separated into individual antigen components according to their charge and size. Following electrophoresis, the separated antigens are reacted with specific antisera placed in troughs parallel to the Antiserum present in the trough moves toward the antigen components resulting in the formation of separate precipitin lines in 18-24 hrs., each indicating reaction between individual proteins with its antibody.  Procedure of Immunoelectrophoresis Agarose gel is prepared on a glass slide put in a horizontal position. The sample is diluted 2:3 with protein diluent solution 5 μl of control and sample is applied across each corresponding slit (Control slit and Sample slit). The gel is placed into the electrophoresis chamber with the samples on the cathodic side, and electrophoresis runs for 20 mins/ 100 volts. After electrophoresis completes, 20 μl of the corresponding antiserum is added to troughs in a moist chamber and incubated for 18- 20 hours at room temperature in a horizontal position.  The agarose gel is placed on a horizontal position and dried with blotter sheets. The gel in saline solution is soaked for 10 minutes and the drying and washing repeated twice again. The gel is dried at a temperature less than 70°C and may be stained with protein staining solution for about 3 minutes followed by decolorizing the gel for 5 minutes in distaining solution baths. The gel is dried and results evaluated.  Results of Immunoelectrophoresis The presence of elliptical precipitin represents antigen-antibody interaction. The absence of the formation of precipitate suggests no reaction. Different antigens (proteins) can be identified based on the intensity, shape, and position of the precipitation lines.  Applications of Immunoelectrophoresis helps in the identification and approximate quantization of various proteins present in the serum. used in patients with suspected monoclonal and polyclonal gammopathies. used to detect normal as well as abnormal proteins, such as myeloma proteins in human serum. Used to analyze complex protein mixtures containing different antigens.  Applications of Immunoelectrophoresis The medical diagnostic of proteins hypogammaglobulinemia or overproduced (e.g., multiple myeloma). useful to monitor antigen and antigen- antibody purity and to identify a single antigen in a mixture of antigens. Used for qualitative analysis of M-proteins in serum and urine. used for diagnosis and evaluation of the therapeutic response in many disease states affecting the immune system. a  Advantages of Immunoelectrophoresis is a powerful analytical technique with high resolving power as it combines the separation of antigens by electrophoresis with immunodiffusion against an antiserum. The main advantage immunoelectrophoresis is that a number of antigens can be identified in serum.  Limitations of Immunoelectrophoresis Immunoelectrophoresis is slower, less sensitive, and more difficult to interpret than Immunofixation electrophoresis. fails to detect some small monoclonal M- proteins because the most rapidly migrat immunoglobulins present in the highest concentrations may obscure the presence of small M-proteins. The use of immunoelectrophoresis in food analysis is limited by the availability of specific antibodies.  Counter Current Immunoelectrophoresis  Counter Current Immunoelectrophoresis refers to the process in which an antigen mixture is first separated into its component parts by electrophoresis and then tested by immunodiffusion.  Counter Current Immunoelectrophoresis is a modification of immunoelectrophoresis in which antigen and antibody move in opposite directions and form precipitates in the area where they meet in concentrations of optimal proportions. It is also referred to as countercurrent or crossed- over immunoelectrophoresis. also called ‘voltage facilitated double immunodiffusion’.  Objectives To rapidly check the presence of antibodies for a particular antigen. To detect antigens and/or antibodies in serum for diagnosis of a particular disease  Principle Counter-current immunoelectrophoresis depends on the movement of antigen towards the anode and of antibody towards the cathode through the agar under the electric field. The test is performed on a glass slide in agarose gel of high electro-flow. A pair of wells is punched out where one well is filled with antigen and the other with the antibody. Electric current is then passed through the gel. The migration of antigen and antibody is greatly facilitated under the electric field, and the line of precipitation visible in 30–60 minutes, which indicates a positive reaction.  Results Precipitin line between the antigen and antisera wells indicate positive reaction or specific antigen-antibody reaction due to the presence of antibody specific to the antigen. The absence of the precipitin line indicates no reaction or the absence of any corresponding antibody-antigen. The presence of more than one precipitin line indicates the heterogeneity of the antibody for the antigen.  Applications The counter-current immuno-electrophoresis has many uses: It is a rapid and a highly specific method for detection of both antigen and antibodies in the serum, cerebrospinal fluid, and other body fluids in the diagnosis of many infectious diseases including bacterial, viral, fungal, and parasitic. detecting various antigens such as alpha-fetoprotein in serum and capsular antigens of Cryptococcus and Meningococcus in cerebrospinal fluid. , it is commonly used for Hepatitis B surface antigen (HBsAg), fetoprotein, hydatid and amoebic antigens in the serum, and cryptococcal antigen in the CSF. It is a rapid sensitive method for detecting pneumococcal capsular antigens in sputum.  Advantages A fast method of antigen-antibody detection (takes 30 minutes). More sensitive than electro- immunodiffusion (EID) because it involves simultaneous electrophoresis of the antigen and the antibody in gel in opposite directions resulting in band formation. Much faster and more sensitive than the double diffusion technique.  Limitations It is more expensive than agglutination based tests. It is believed to have decreased sensitivity, speed, and simplicity  Radioimmunoassay (RIA): Principle, Procedure, Results, Uses  Radioimmunoassay (RIA): Radioimmunoassay is one of the sensitive immunoassay techniques which helps in the determination of antigens or antibodies in a sample with the use of radioisotopes. It is an in vitro type of antigen-antibody interaction with very high sensitivity..  Radioimmunoassay (RIA) Requirements Radiolabeled antigens: The antigens are generally labeled with gamma-ray emitting isotopes such as I-125 and beta-ray emitting isotopes such as Tritium. They are also called hot antigens. Specific Antibodies: They are required in smaller amounts than antigens. Unlabeled antigens (sample antigens): They are also called cold antigens. Microtitre plates: 96 wells microtitre plate Washing Buffer solutions: Wash buffer such as 1% Trifluoroacetic acid is used.  Radioimmunoassay (RIA) Principle Antigens and antibodies bind specifically to form the Ag-Ab complex. The antigen can be labeled or conjugated with radioisotopes. The unlabeled antigens from the sample compete with radiolabeled antigens to bind on paratopes of specific antibodies. The unlabeled antigens replace labeled antigens that are already linked with the antibodies. The unlabeled antigens when bind with antibodies, increases the amount of free radiolabeled antigens in the solution. the concentration of free labeled antigens is directly proportional to the bound unlabeled antigens.  It involves a combination of three principles. An immune reaction i.e. antigen, antibody binding. A competitive binding or competitive displacement reaction. (It gives specificity) Measurement of radio emission. (It gives sensitivity  Radioimmunoassay (RIA) Procedure Specific antibodies of known concentration are fixed in the microtitre well. A known amount of hot antigens is then added to the well Washed carefully to remove any unbound antigens At this point, the radioactivity of the well will be maximum. Unlabeled antigens are then added to the well The unlabeled antigens will bind to the antibodies and there will be free labeled antigens in the well. Again washed carefully to remove the free labeled antigens. Radioactivity of wells is then measured by gamma- counter. Figure: Radioimmunoassay (RIA) Procedure. Image  Radioimmunoassay (RIA) Result Interpretation At first, the labeled antigens will bind to the antibodies hence radioactivity will be maximum. If the sample contains specific antigens of interest, it will bind to the antibodies releasing labeled antigens and hence the radioactivity of the solution will decrease. decreasing radioactivity, confirmed that the antigen of interest is present in the sample. And if the radioactivity remains the same, it can be called a negative test. With the increasing concentration of unlabeled antigens, the radioactivity decreases.  By plotting a graph of radioactivity(in percentage) vs concentration of unlabeled antigens, a standard curve is obtained. The sample to be assayed is run parallel following a similar procedure and the radioactivity measured is calibrated with the standard curve to determine the concentration of the antigen.  Radioimmunoassay (RIA) Applications used for detection of peptide hormones. Detection of different viral antigens Detection of many hormones and drugs Detection of Hepatitis B surface antigens Detection of mycotoxins Detection of the early stage of cancer  Radioimmunoassay (RIA) Advantages High specificity High sensitivity Can detect a very small amount (nanograms) of antigen or antibodies.  Radioimmunoassay (RIA) Limitations Working with radioactive substances makes it a bit risky. Disposal of radioactive substances can be problematic. Equipment and reagents are expensive. Radiolabeled substances used have a short shelf-life.  Agglutination  Agglutination: Agglutination is an antigen-antibody reaction in which a particulate antigen combines with its antibody in the presence of electrolytes at a specified temperature and pH resulting in the formation of visible clumping of particles..  Types of Agglutination Reactions Agglutination reactions can be broadly divided into three groups: Active/Direct agglutination Passive agglutination Hem agglutination  1. Active Agglutination In active agglutination, direct agglutination of particulate antigen with specific antibody occurs. Direct bacterial agglutination uses whole pathogens as a source of antigen. It measures the antibody level produced by a host infected with that pathogen. The binding of antibodies to surface antigens on the bacteria results in visible clumps Active agglutination can be of following types: A-Slide agglutination: Basic type of agglutination reaction that is performed on a slide. In this method suspension of unknown antigen is kept on slide and a drop of standardized antiserum is added or vice versa. A positive reaction is indicated by formation of visible clumps. E.g. Widal test  B-Tube agglutination: In this method serum is diluted in a series of tubes and standard antigen suspensions (specific for the suspected disease) are added to it. After incubation, antigen-antibody reaction is indicated visible clumps of agglutination.  C-Antiglobulin (Coombs) test:. When serum containing incomplete anti-Rh antibodies is mixed with Rh+ erythrocytes in saline, but does not cause any agglutination. When such erythrocytes are treated with antiglobulin or Coombs serum (rabbit antiserum against human gamma globulin), then the cells are agglutinated.  Coombs test can be direct as well as indirect. In direct method, the sensitization of red blood cells (RBCs) with incomplete antibodies takes place in vivo. Cell- bound antibodies can be detected by this test in which antiserum against human immunoglobulin is used to agglutinate patient’s RBC. in indirect method, the sensitization of RBCs with incomplete antibodies takes place in vitro. Patient’s serum is mixed with normal red cells and antiserum to human immunoglobulin. Agglutination occurs if antibodies are present in serum. Coombs test is used for detection of anti-Rh antibodies and incomplete antibodies in brucellosis and other diseases.  2. Passive Agglutination antibody or antigen is attached to certain inert carrier thereby, particles or cells gets agglutinated when corresponding antigen or antibody reacts. E.g. Antigens coated in latex particles used in ASO test. When the antibody instead of antigens is adsorbed on the carrier particle for detection of antigens, it is called reverse passive agglutination.  3. Hemagglutination Test RBCs are used as carrier particles in hemagglutination tests. RBCs of sheep, human, chick, etc. are commonly used in the test. RBCs are coated with antigen to detect antibodies in the serum Viral hemagglutination: viruses including influenza, mumps, and measles have the ability to agglutinate RBCs without antigen–antibody reactions inhibited by antibody specifically directed against the virus, and this phenomenon is called hemagglutination inhibition  4-Coagglutination test: Coagglutination is a type of agglutination reaction in which Cowan I strain of S. aureus is used as carrier particle to coat antibodies. In a positive test, protein A bearing S aureus coated with antibodies will be. agglutinated if mixed with specific antigen. The advantage of the test is that these particles show greater stability than latex particles.  Uses of Coagglutination test Detection of cryptococcal antigen in the CSF for diagnosis of cryptococcal meningitis; Detection of amoebic and hydatid antigens in the serum for diagnosis of amoebiasis and cystic echinococcosis, Grouping of streptococci and mycobacteria and for typing of Neisseria gonorrhoeae.  Applications of Agglutination Reactions Cross-matching and grouping of blood. Identification of Bacteria. E.g. Serotyping of Salmonella Typhi and Paratyphi. Serological diagnosis of various diseases. E.g Rapid plasma regains (RPR) test for Syphilis, (ASO) test for rheumatic fever. Detection of unknown antigen in various clinical specimens. E.g. detection of Vi antigen of Salmonella Typhi in the urine.  ELISA- Definition, Principle, Procedure, Types, Steps, Uses  ELISA- Enzyme-Linked Immunosorbent Assay (ELISA) is a modern molecular technique for the detection of antigen-antibody interaction with the help of an enzyme. It is one of the sensitive enzyme immunoassay techniques for the detection of the presence of antigen or antibody and quantification as well in the case of clinical diagnosis of many diseases.  Enzyme-linked immunosorbent assay (ELISA) utilizes an enzyme system to show a specific combination of an antigen with its antibody It is a method of quantifying an antigen immobilized on a solid surface. ELISA uses a specific antibody with a covalently coupled enzyme. The amount of antibody that binds the antigen is proportional to the amount of antigen present, which is determined by spectrophotometrically.  The enzyme system of ELISA consists enzyme which is labeled to a specific antibody or antige and a chromogenic substrate that is added after the antigen-antibody reaction The substrate is hydrolyzed by the enzyme attached to antigen-antibody complexes. An ELISA test uses components of the immune system (such as IgG or IgM antibodies) and chemicals for the detection of immune responses in the body. The ELISA test involves an enzyme It also involves an antibody or antigen Examples of the uses of an ELISA test include diagnosing infections such as HIV and some allergic diseases like food allergies. ELISA tests are also known as immunosorbent assays.  ELISA Requirements Coated plates (Microtitre plates): Commonly polystyrene plates. Sample diluents: Wash Buffers:. Eg. Triphosphate buffer (ph 7.40) and detergents such as Tween-20. Enzyme-linked Antibodies: AP(Alkaline Phosphatase) HRP(Horseradish Peroxidase)  Substrates: o-phenylene diamine for HRP enzyme and p-nitrophenyl phosphate for AP enzyme. Stop solution: sulphuric acid.  ELISA Principle Antigens and antibodies react specifically to form the Ag-Ab complex. Antibodies can be linked or attached to enzymes. The enzyme- linked antibodies can modify the specific substrates used to produce a color change within the preparation. The enzyme activity is measured with a colorimeter in a specific wavelength of light to determine the magnitude of the infection in the patient.  Types of ELISA They are of mainly four types: Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA  1. Direct ELISA It is the simplest and quickest of all other types of ELISA. A single enzyme-linked antibody is used which directly interacts with the antigen present in the sample.  Direct ELISA Procedure Addition of sample(containing antigen) to the well of the microtitre plate Antigen gets adsorbed to the surface of the well. Washing to remove unbound antigens The addition of an enzyme-linked antibody that with the antigens if present. Washing to remove unbound antibodies Addition of chromogenic substrate Visualization of color change and result interpretation  Direct ELISA Applications Used in the identification of biomolecules. Also for the diagnosis of infections of Mycoplasma bovis  2. Indirect ELISA It is the most popular type of ELISA in use. Antibody detection is carried out in indirect ELISA. A secondary antibody linked with the enzyme is used.  Indirect ELISA Procedure Addition of known antigens specific to the antibody of interest Careful washing to remove unadsorbed antigens or some adsorbed contaminants. Addition of serum samples to their respective wells If there is the presence of specific antibodies, they will bind to the antigens. Wash again. Addition of enzyme-linked secondary antibodies to the well Wash again Addition of substrate Visualization of color change and result interpretation  Indirect ELISA Applications Detection of HIV, Rubella, Dengue viruses, etc. To detect certain drugs in serum.  3. Sandwich ELISA Detection of antigens. Two different antibodies i.e. capture antibodies(for attachment sample antigens) and enzyme-linked secondary antibodies are required. The antigen of interest first binds with the capture antibodies and then the secondary antibodies bind with the epitope antigen forming a sandwich-like structure with antigen in between antibodies.  Sandwich ELISA Procedure Addition of capture antibodies in the microtitre well Washing Addition of serum sample(containing antigen) in the well Washing Addition of enzyme-linked antibodies in the well Washing Addition of substrate Visualization of color development and result interpretation  Sandwich ELISA Applications Mainly used for detection of Rotavirus and enterotoxins of fecal E. coli. Pregnancy tests kids are based on this technique.  Competitive ELISA The basic concept of this type is that there occurs competition of binding between the sample antibodies(if present) and enzyme-linked secondary antibodies to the antigens. If the sample contains specific antibodies of interest, they will bind to the antigens and won’t allow enzyme-linked antibodies to bind with antigens which are then washed away. Hence after the addition of substrate, there won’t be a color change indicating a positive test and vice-versa  Competitive ELISA Procedure Addition of HIV antigens in the microtitre well Washing Addition of serum sample Washing Addition of enzyme-linked HIV specific antibodies Washing Addition of substrate Visualization of color change and result interpretation  Competitive ELISA Application Mainly used for the detection of HIV  ELISA Applications sensitive and effective methods for the detection of different viral, bacterial, and fungal infections. Screening test for HIV infection. Different EISA test kits are available for the detection of dengue fever, TB, and Hepatitis B infections. Pregnancy test kits based on ELISA are also available. Qualitative and quantitative estimation of various proteins, hormones, toxins, etc. Also used in the detection of different food allergen  Advantages of ELISA Protocols are simple. Highly specific and sensitive Highly efficient Low-cost reagents No requirement of any unsafe materials such as radioactive substances as in Radio Immunoassay Limitations of ELISA Work-Intensive Preparation of enzyme-linked antibodies can be difficult and expensive. Since the antibodies are unstable so for transport and storage proper refrigeration is required.  Immunofluorescence- Definition, Principle, Types, Uses, Limitations  Immunofluorescence- Immunofluorescence is a type of assay performed on biological samples to detect specific antigens in any biological specimen or sample using fluorescence microscope. It is an effective method for visualizing intracellular processes, structures, and conditions as well. In Vitro type of Ag-Ab Interaction. Detects surface antigens or antibodies. Fluorescent dyes are used for the visualization of Ag-Ab reactions.  In the immunofluorescence test, a fluorescent dye that illuminates in UV light is used to detect/show the specific combination of an antigen and antibody. The dye usually used is fluorescein isothiocyanate, which gives yellow- green fluorescence. Immunofluorescence tests are also termed fluorescent antibody tests (FAT).  Requirements of Immunofluorescence antibodies that can bind to the antigen of interest to form the Ag-Ab complex. They can be : a. Primary Antibody: b. Secondary Antibody:.  Commonly used Fluorochromes are: Immunofluorescence microscope for visualization – Wash buffers such as PBS ( Phosphate Buffered Saline ): Helps to was away unbound antibodies.  Principle of Immunofluorescence Specific antibodies bind to the protein or antigen of interest. Antibodies could be labeled with molecules that have the property of fluorescence (fluorochromes) When light of one wavelength falls on fluorochrome, it absorbs that light to emit light of another wavelength. The emitted light can be viewed with a fluorescence microscope.  Types of Immunofluorescence Direct Immunofluorescence Test Indirect Immunofluorescence Test.  Direct Immunofluorescence Test Single antibody i.e. primary antibody is used that is chemically linked to a fluorochrome. If the antigen is present, the primary antibody directly reacts with it and fluorescence can be observed under the fluorescent microscope  Procedure of Direct Immunofluorescence Test Fixing of Specimen (Antigen) into the slide. Fluorochrome labeled antibodies are then added to the slide. Incubation and careful washing with wash buffers like PBS (Phosphate Buffered Saline) to remove other components except for the complex of antigen and fluorochrome-labeled antibody. Observed under a fluorescence microscope. Uses of Direct Immunofluorescence Test For the detection of rabies virus antigen in the skin For the detection of N. gonorrhoeae, C. diphtheriae, T. pallidum, etc. directly in appropriate clinical specimens. Advantages of Direct Immunofluorescence Test Protocols for direct IF are usually shorter as they only require one labeling step. Species cross-reactivity is minimized indirect methods as the fluorophore is already conjugated to the primary antibody  Disadvantages of Direct Immunofluorescence Test Separately labeled antibodies need to prepared for each pathogen. Requires the use of a much more primary antibody, which is extremely expensive. Less sensitive than indirect immunofluorescence.  Figure- Direct and Indirect Immunofluorescence. Image Source: Leica Microsystems.  Indirect Immunofluorescence Test Double antibodies are used i.e. primary and secondary antibodies. The primary antibody is not labeled and a fluorochrome-labeled secondary antibody is used for detection. The antigen used is known and it binds to the specific primary antibodies of interest in the sample. The secondary antibody then binds to the Fc region of the primary antibody.  Procedure of Indirect Immunofluorescence Test Fixing of a known antigen on a slide. The specimen to be tested is applied to the slide. Incubation and careful washing with PBS. A secondary antibody (e.g., fluorescently labeled anti-IgG) is added. Incubation and careful washing again with PBS. Observed under the fluorescence microscope.  Uses of Indirect Immunofluorescence Test In detection of specific antibodies for diagnosis of syphilis, amoebiasis, leptospirosis, toxoplasmosis, and other diseases. Also used in the detection of autoantibodies that cause auto immune disorders.  Advantages of Indirect Immunofluorescence Test. More sensitive than direct immunofluorescence test. Multiple secondary antibodies can bind to the Fc region of primary antibody which amplifies the fluorescence signal. Disadvantages of Indirect Immunofluorescence Test It is more complex and time-consuming than the direct IF. Cross-reactivity of secondary antibody to other agents can be problematic.  Applications of Immunofluorescence Immunofluorescence can be used on tissues or cell sections to determine presence of different biological molecules which also includes proteins, carbohydrates, etc. Also used in molecular biology for visualization of cytoskeletons such as intermediate filaments. It also plays a key role in the detection of autoimmune disorders.  Limitations of Immunofluorescence degradation of fluorochromes, It can be prevented by using higher concentration of flurochromes and decreasing exposure time to the light. Autofluorescence can occur due to some agents. It is mostly used for only fixed cells or dead cells. Expensive and require higher expertise. What is Flow Cytometry? Flow cytometry is the advancement of the immunofluorescence technique It is a computer-based advanced technology  Monoclonal Antibodies Definition, Types, Production,Applications  What are Monoclonal Antibodies? monoclonal antibodies are proteins prepared in the laboratory to target specific antigens on body cells.  what are monoclonal antibodies? Monoclonal antibodies are artificial antibodies produced from a single clone of cells by fusing B-lymphocytes to myeloma cells by somatic cell hybridization secretes -desired antibody-producing elements -known as a hybridoma. These hybridomas produce homogenous monoclonal antibodies..  Monoclonal antibodies (mAbs) have the ability to recognize unique binding sites (epitopes) found on the specific antigens. Monoclonal antibodies (mAbs) have been produced to target receptors or other foreign proteins that are present on the surface of normal cells and cancer cells. Monoclonal antibodies are used in the treatment of many diseases including some cancers. The specificity of monoclonal antibodies (mAbs) allows them to bind to cancerous cells coupled with a cytotoxic agent such as a strong radioactive agent. The radioactive agent seeks to destroy the cancer cells without harming the healthy ones  Types of monoclonal antibodies A. Murine monoclonal antibodies They were named murine because of their origin from rodent hosts (mice and rats) belonging to the Muridae family. use in the treatment of Cryptococcus neoformans.  B. Chimeric monoclonal antibodies These are structural chimeras that are made of a combination of mouse parts and human parts, by fusion. Reduce immunogenicity and increase the serum half-life when preparing them for therapeutic reasons.  C. Humanized monoclonal antibodies all regions of the mouse antibody in chimeric are replaced with human ones except for the complementarity- determining regions  D. Human monoclonal antibodies These are fully human proteins that have been manipulated by molecular biological techniques so as to modify the amino acid sequences.  Types of monoclonal antibodies based on functions 1. Naked monoclonal antibodies These are mAbs that do not have a drug or radioactive agent attached to them. they are the most common mAbs used in the treatment of cancer.  2. Conjugated Conjugated mAbs are conjugated or combined with chemotherapy drugs or a radioactive agent.. Conjugated mAbs circulate freely throughout the body until it finds and attaches (hooks) to the target antigen and delivers the antigen to immune elimination processes.  a. Radiolabeled monoclonal antibodies a small radioactive particle attached to them delivered directly to the target cells. b. Chemolabeled antibodies These mAbs have powerful chemotherapy or drugs attached to them  3. Bispecific monoclonal antibodies These are monoclonal antibody drugs that are made up of two different monoclonal antibodies attached to each other. For example, a leukemic drug  Production of monoclonal antibodies Production of monoclonal antibodies The production of monoclonal antibodies is an in vitro process by the use of tissue-culture techniques. Producing monoclonal antibodies (mAbs) is initially done by identifying a specific antigen, and immunizing an animal with the antigen multiple times. The most commonly used animal models are laboratory mice. The B-cells of the immunized animals are removed from the spleen and then fused with cancer B-cells known as the myeloma cells. The fusion of adjacent plasma membranes of the myeloma cells is done using polyethylene glycol,. Production of monoclonal antibodies  Functions and Applications of Monoclonal Antibodies Monoclonal antibodies are used in the treatment of several diseases and disorders and their application is known as immunotherapy. include: Cancers Rheumatoid arthritis Multiple sclerosis, Systemic Lupus erythematous Cardiovascular diseases, Crohn’s disease, Ulcerative colitis, Psoriasis, and Rejections associated with transplantation Monoclonal antibodies are widely used in therapies, laboratory technique studies and research  used in chemotherapy-resistant breast cancer. used in the diagnosis of several diseases by detecting specific antigens circulating in the body tissues by the use of immunoassay techniques. treatment of COVID-19. The diagnosis of many infectious and systemic diseases relies on the detection of particular antigens or antibodies in the blood, urine, or tissues by the use of monoclonal antibodies in immunoassays. treatment of B cell leukemia and in certain autoimmune disorders  Limitations of Monoclonal Antibodies Monoclonal antibodies are most easily produced by immunizing mice, but patients treated with mouse antibodies may make antibodies against the mouse,also cause a disorder called serum sickness.  Side effects of Monoclonal Antibodies Reactions depend on several factors including: Needle site reaction associated with Pain, swelling, soreness, redness, itchiness, rash Flu-like symptoms associated with chills, fatigue, nausea, vomit, fever, diarrhea, muscle ache, pain Effects of monoclonal antibodies associated: – Mouth and skin sores that can lead to serious infections, – High blood pressure, – Congestive heart failure, – Heart attacks, Inflammatory lung disease, – Mild to a severe allergic reaction.