COMPLEMENT FIXATION TEST • Detects the antibodies in patient’s serum that are capable of fixing with complements. It was once very popular, now is almost obsolete. • Applications: o Wasserman test o Was also widely used for detection of complement fixing antibodies in Rickettsia, Chlamydia, Brucella, Mycoplasma infections and some viral infections, such as arboviruses, rabies, etc. o Complements are also used for various serological tests other than CFT, such as:  Treponema pallidum immobilization test  Sabin-feldman dye test for Toxoplasma  Vibriocidal antibody test. NEUTRALIZATION TEST • Neutralization tests are also less commonly used in modern days. Various examples are as follows: o Viral neutralization test o Plaque inhibition test NEUTRALIZATION TEST o Toxin–antitoxin neutralization test: Examples include Schick test Nagler’s reaction ASO test NEUTRALIZATION TEST o Hemagglutination inhibition (HAI) test: Antibodies in patient’s sera can agglutinate with the hemagglutinin antigens present on the surfaces of some viruses. Was used in past for the diagnosis of various viral diseases, e.g. influenza. NEWER TECHNIQUES • Uses a detector molecule to label antibody or antigen which in turn detects the corresponding antigen or the antibody in the sample by producing a visible effect. • Most of the newer techniques use the same principle, but they differ from each other by the type of labelled molecule used and the type of visible effect produced. Immunoassays and the types of molecule used for labeling Abbreviation Immunoassay method Molecules used for labeling Type of visible effect ELISA Enzyme linked immunosorbent assay Enzyme- substratechromogen complex Color change is detected by spectrophotometer ELFA Enzyme linked fluorescent assay Enzyme- substrate Fluorometric detection IFA Immunofluorescence Assay Fluorescent dye Emits light, detected by fluorescence microscope RIA Radioimmunoassay Radioactive isotope Emits β and γ radiations, detected by β and γ counters CLIA Chemiluminescence-linked immunoassay Chemiluminescent compounds Emits light, detected by luminometer Immunoassays and the types of molecule used for labeling Abbreviation Immunoassay method Molecules used for labeling Type of visible effect IHC Immunohistochemistry Enzyme or Fluorescent dye Color change (naked eye) or Fluorescence microscope WB Western blot Enzyme Color band (naked eye) Rapid test Immunochromatographic test Colloidal gold or silver Color band, (naked eye) Flow through assay Protein A conjugate Color band, (naked eye) Immunoferritin electron microscopy Electron dense molecules (e.g ferritin) Appears as black dot under electron microscope IEM ENzYME-LINKED IMMUNOSORBENT ASSAY (ELISA) • Enzyme immunoassay (EIA) is a term used to describe all the tests that detect either antigen or antibodies or haptens in the specimen, by using enzyme–substrate system for detection. • They can be classified as below: o Homogenous EIA o Heterogeneous EIA ENzYME-LINKED IMMUNOSORBENT ASSAY (ELISA) • Homogenous EIA o All reagents are added at one step. o Used for detection of haptens such as drugs (e.g. opiates, cocaine), but not for detection of microbial antigens and antibodies • Heterogeneous EIA o Involves multiple steps with different reagents being added at every step. o Used for detection of antigens and antibodies. o ELISA is a classical example. ELISA (ENZYME LINKED IMMUNOSORBENT ASSAY) • So named because of two of its componentso Immunosorbent- Absorbing material used (e.g. polystyrene, polyvinyl) that specifically absorbs the antigen or antibody present in serum. o Enzyme is used to label one of the components of immunoassay (i.e. antigen or antibody). Substrate-chromogen system • Substrate- chromogen system is added at the final step of ELISA. • Enzyme-substrate reaction in turn activates the chromogen to produce a color. • Sometime, the substrate is chromogenic in nature (e.g. pNPP), on reaction with the enzyme, it changes its color. Substrate-chromogen system • Color change - detected by spectrophotometry in an ELISA reader. • Intensity of the color is directly proportional to the amount of detection molecule (Ag or Ab) present in test serum. Enzyme Substrate Chromogen Horseradish Peroxidase Hydrogen peroxide Urease Urea Bromocresol β-Galactosidase ONPG ONPG Alkaline Phosphatase pNPP* pNPP* Tetramethyl benzidine (TMB) ELISA • (Ag-Ab complex)-enzyme + substrate → activates the chromogen → color change → detected by spectrophotometry ELISA reader (Biorad) ELISA washer Automated ELISA (EVOLIS system, Biorad) hers ELISA • Performed on a microtiter plate containing 96 wells (microELISA) or less commonly performed in tubes (macro-ELISA). • The microtiter plate or the tubes are made up of polystyrene, polyvinyl or polycarbonate material. ELISA • ELISA kits are commercially available; contain all the necessary reagents (such as enzyme conjugate, dilution buffer, substrate/chromogen etc). • Procedure involves a series of steps done sequentially; at each step, a reagent is being added, and then incubated followed by washing of the wells (manually or by automated ELISA washer). Types of ELISA ELISA type Used for detection of Enzyme is labeled with Direct ELISA Indirect ELISA Antigen Antibody or antigen Primary antibody Secondary antibody Sandwich ELISA Antigen Primary antibody in sandwich direct ELISA Secondary antibody in sandwich indirect ELISA Competitive ELISA Antigen or antibody Secondary antibody ELISPOT Cells producing antibody or Primary antibody cytokine Direct ELISA • Used for detection of antigen in test serum. • Primary antibody (targeted against the serum antigen) is labeled with the enzyme. Steps Explanation Step 1 Wells of microtiter plate are empty, not precoated with Ag or Ab. Step-2 Test serum (containing antigen) is added into the wells. Antigen becomes attached to the solid phase by passive adsorption. Step-3 After washing, the enzyme-labeled primary antibodies (raised in rabbits) are added. Step-4 After washing, a substrate- chromogen system is added and color is measured. Direct ELISA • Well + Ag (test serum) + primary Ab-Enzyme + substratechromogen → Color change. Indirect ELISA – Antibody detection • It differs from the direct ELISA in that the secondary antibody (Ab targeted to Fc region of any human Ig) is labeled with enzyme instead of primary antibody. Steps Explanation Step 1 Solid phase of the wells of microtiter plates are precoated with the Ag. Step-2 Test serum (containing primary Ab specific to the Ag) is added to the wells. Ab gets attached to the Ag coated on the well. Step-3 After washing, enzyme-labeled secondary Ab (anti-human immunoglobulin) is added. Step-4 After washing, a substrate-chromogen system is added and color is developed. Direct ELISA • Wells are coated with Ag + primary Ab (test serum) + secondary Ab-Enzyme + substrate- chromogen → development of color. Indirect ELISA – Antigen detection • Wells are empty, not precoated with Ag or Ab. Steps Explanation Step 1 Test antigen (serum) is added to the well. The Ag gets absorbed onto the well. Step-2 Primary antibody raised in rabbits (reagent) is added. The Ag binds to the primary antibody. Step-3 After washing, enzyme-labeled secondary Ab (anti-rabbit Ab) is added. Step-4 After washing, a substrate- chromogen system is added and color is developed. Direct ELISA • Ag (test serum) added, gets adsorbed to well + primary Ab + secondary Ab-Enzyme + substrate- chromogen → development of color. Sandwich ELISA • Detects the antigen in test serum. • So named because the antigen gets sandwiched between a capture antibody and a detector antibody. • There are two types of sandwich ELISA: o Direct - detector antibody is a primary antibody. o Indirect - detector antibody is a secondary antibody. Direct sandwich ELISA Steps Explanation Step 1 Microtiter well is precoated with the capture antibody (monoclonal Ab raised in rabbit) targeted against the test antigen. Step-2 Test serum (containing antigen) is added to the wells. Ag gets attached to the capture antibody coated on the well. Step-3 After washing, an enzyme labeled primary ‘detector antibody’ specific for the antigen is added. The detector antibody can be same as the capture antibody. Step-4 After washing, a substrate-chromogen system is added and color is developed. Direct sandwich ELISA • Wells coated with capture Ab + Ag (test serum) + primary Abenzyme + substrate- chromogen → color Indirect sandwich ELISA • Primary antibody and the capture antibody belong to different species. More so, the primary antibody is not labelled with enzyme. • Another enzyme-labelled secondary antibody targeted against the primary antibody is added. • More specific than direct sandwich ELISA. Direct sandwich ELISA • Wells coated with capture Ab + Ag (test serum) + primary Ab + secondary Ab- enzyme + substrate- chromogen → color. Competitive ELISA • Example: Indirect competitive ELISA format used for antigen detection. Steps Explanation Step 1 Primary antibody is first incubated in a solution with a serum sample containing the test antigen. Step-2 Antigen-antibody mixture is then added to the microtiter well precoated with the same type of antigen. Step-3 Free antibodies bind to the antigen coated on the well. More the test antigens present in the sample, lesser free antibodies will be available to bind to the antigens coated onto well. Competitive ELISA Steps Explanation Step-4 After washing (to remove free antibodies and antigens), enzyme-conjugated secondary antibody is added. Step-5- After washing, a substrate- chromogen system is added and color is developed. Intensity of the color is inversely proportional to the amount of antigen present in the test serum. Competitive ELISA • Can also be used for the detection of antibody in serum. • Different formats are available such as direct, indirect and sandwich formats. ELISPOT test • Modification of ELISA that allows the quantitative detection of cells producing antibodies (plasma cells) or cytokines (e.g. macrophage). • Procedure: o Microtiter well is coated with the capture antibody specific for the cytokine. o A suspension of the cell population under investigation is then added to the coated wells and incubated. o Sensitized cells capable of producing the cytokines settle onto the surface of the well. ELISPOT test o Released cytokines are bound by the capture antibodies in the vicinity of the secreting cells, producing a ring of antigenantibody complexes around each cell that is producing the cytokine of interest. o After the incubation period, the well is washed and an enzymelabeled anti-cytokine antibody is added. o After washing away unbound antibody, a chromogenic substrate that forms an insoluble colored product is added. o Colored product (purple) precipitates and forms a spot only on the areas of the well where cytokine-secreting cells have been deposited. o Quantitation-The number of colored spots corresponds to the number of cytokine producing cells present in the added cell suspension. o For quantitative detection of antibody producing cells, the procedure is similar except that the wells are coated with capture antigen which would bind to the antibodies produced by the cells. • ELISPOT test is currently used in IGRA (interferon gamma assay), for diagnosis of latent tuberculosis. ELISPOT test IgG Avidity ELISA • Principle: Avidity of IgG indicates how firmly the IgG antibody is bound with its antigen. Avidity reflect the maturity of the antibodies; which usually increases with time. o Low avidity antibodies are synthetized during the primary infection. it also occurs in immunosuppression. o High avidity antibodies are produced by memory B-cells during the secondary infection, or reactivation, recovery or in vaccinated people. IgG Avidity ELISA • Procedure: o ELISA is performed in two parallel wells of microtiter plate; one well with untreated patient serum and a second well with serum sample after treating with dissociating agents such as urea.  Low avidity antibodies dissociate from complexes and are washed away during the washing step of ELISA;  High avidity antibodies withstand urea treatment and remain bound to the antigens. IgG Avidity ELISA • Results: Avidity (%) is expressed as absorbance of the well with urea to absorbance of the well without urea. o Avidity <40%- interpreted as low avidity o Avidity >60%- interpreted as high avidity o Avidity 40-60% - interpreted as grey zone (inconclusive result); test has to be repeated. IgG Avidity ELISA • Uses: IgG avidity ELISA is useful in: o Differentiating recent and past infection- in situations where IgM disappears very soon in primary infection; or IgM persists long time even after resolution of primary infection. o Diagnosing atypical or sub clinical course of the disease. o For diagnosing congenital infection. • Applications: IgG avidity test is available for : rubella, CMV, VZV, toxoplasmosis, EBV, HIV, viral hepatitis and West Nile virus infection. Advantage of ELISA • Method of choice for detection of antigens/antibodies in serum in big laboratories as large number of samples can be tested together using the 96 well microtiter plate. • Economical, takes 2-3 hours for performing the assay. • Most sensitive immunoassay - commonly used for performing screening test at blood banks and tertiary care sites. • Specificity used to be low - But now, with use of more purified recombinant and synthetic antigens, and monoclonal antibodies, ELISA has become more specific. Disadvantage of ELISA • Small laboratories with less sample load - ELISA is less preferred than rapid tests as the later can be done on individual samples. • More time (2-3 hours) compared to rapid tests which take 10-20 min. • Needs expensive equipments such as ELISA washer and reader. Applications of ELISA • ELISA used for antigen detection -hepatitis B [hepatitis B surface antigen (HBsAg) and precore antigen (HBeAg)], NS1 antigen for dengue etc. • ELISA can also be used for antibody detection against hepatitis B, hepatitis C, HIV, dengue, EBV, HSV, toxoplasmosis, leishmaniasis, etc. ENZYME-LINKED FLUORESCENT ASSAY (ELFA) • Modification of ELISA, differs from ELISA in two ways: o Automated system, all steps are performed by the instrument. o Ag-Ab-enzyme complex is detected by fluorometric method. • VIDAS and mini VIDAS (bioMérieux) are commercially available systems based on ELFA technology. ELFA – Advantages over ELISA • • • • • Automated system Easy to perform and user friendly Less contamination Gives quantitative results More sensitive and specific ELFA – Disadvantages over ELISA • Expensive • Can run only 12–24 number of tests at a time • Can run 2–4 types of tests at a time ELFA – Uses • Can be used to detect more than 100 parameters. o Infectious diseases: Markers of hepatitis viruses and HIV (Ag and Ab), Ab to TORCH infection, measles, mumps, varicella, H. pylori and antigen to C. difficile, Rotavirus, etc. o Other use: Biomarkers (e.g. procalcitonin), hormones (e.g. thyroid), tumor markers, cardiac markers and screening for allergy. IMMUNOFLUORESCENCE ASSAY • Technique similar to ELISA, but differs by some important features: o Fluorescent dye is used instead of enzyme for labelling of antibody. o Detects cell surface antigens or antibodies bound to cell surface antigens, unlike ELISA which detects free antigen or antibody. IMMUNOFLUORESCENCE ASSAY - Principle • Fluorescence refers to absorbing high energy-shorter wavelength ultraviolet light rays by the fluorescent compounds and in turn emitting visible light rays with a low energy-longer wavelength. • Fluorescent dye (FITC) is used to conjugate the antibody and such labeled antibody can be used to detect the antigens or antigenantibody complex on the cell surface. • Types of Immunofluorescence Assays : Direct and Indirect. Direct Immunofluorescence Assay Steps Explanation Step-1 Sample containing cells carrying surface antigens is smeared on a slide. Step-2 Primary antibody specific to the antigen, tagged with fluorescent dye is added. Step-3 Slide is washed to remove the unbound antibodies and then viewed under a fluorescence microscope. Indirect Immunofluorescence Assay • Detects antibodies in sample. Slides smeared with cells carrying known antigens are commercially available. Steps Explanation Step-1 Test serum containing primary antibody is added to the slide. Step-2 Slide is washed to remove the unbound antibodies. A secondary antibody (anti human antibody conjugated with fluorescent dye) is added. Step-3 Slide is washed and then viewed under a fluorescence microscope Applications • Detection of autoantibodies (e.g. antinuclear antibody) in autoimmune diseases. • Detecting microbial antigens, e.g. Rabies antigen in corneal smear • Detection of viral antigens in cell lines inoculated with the specimens Flow Cytometry • Laser-based technology that quantitatively analyses and separates the cells as they pass through the laser beam. • Advanced and upgraded form is called as fluorescence-activated cell sorting (FACS). Properties can be determined during the flow of cells • Cell Counting: • Differentiating between two cells Properties can be determined during the flow of cells • Scattering • Sorting out of cells Flow Cytometry- Applications • Flow cytometry can be used to analyse multiple parameters of cells (e.g. leukocytes) such as cell counting, cell sorting, analysis of size, shape, granularity, DNA or RNA content of a cell, etc. Important applications includeo CD4 T cell count in HIV infected patients o Detection of leukocyte with specific markers for the diagnosis of various lymphomas. IMMUNOHISTOCHEMISTRY • Process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of using labelled antibodies binding specifically to the antigens in biological tissues. • Immunohistochemical staining can be ELISA or IFA based • Widely used in the diagnosis of abnormal cells (e.g. tumor cells). Radioimmunoassay (RIA) • Very sensitive and specific technique that is used for quantitative detection of antigens such as hormones, proteins, drugs, vitamins and microbial antigens (e.g. HBsAg) at a concentration of <0.01 µg/mL. CHEMILUMINESCENCE-LINKED IMMUNOASSAY • Emission of light (luminescence), as a result of a chemical reaction. • Principle: similar to that of ELISA; however the chromogenic substance is replaced by chemiluminescent compounds (e.g. luminol and acridinium ester) that generate light during a chemical reaction (luxogenic). • Light (photons) can be detected by a photomultiplier, also called as luminometer. CLIA • Ag-Ab-[Luminol] + [hydrogen peroxide] → Ag-Ab-[Products] + light(photons) → detected by luminometer. Advantage of CLIA • 10 times more sensitive than ELISA. • Can be further modified by using an enhancer that potentiates the chemical reaction. This gives CLIA an overall improvement of 200 folds over ELISA. • Most samples have no 'background' signal, i.e. luminol compounds do not themselves emit light. • Measurement of chemiluminescence is not a ratio unlike the measurement of fluorescence (IFA) and or color (ELISA). • Applications of CLIA are similar to those of ELISA. WESTERN BLOT • Western blot detects specific proteins (antibodies) in a sample containing mixture of antibodies each targeted against different antigens of same microbe. • Southern blot - detects DNA fragments. • Northern blot - detects mRNAs. • Eastern blot - detects the carbohydrate epitopes present on proteins or lipids. Western blot - Procedure Western blot - Applications • Excellent specificity. • Used as a supplementary test to confirm the result of ELISA or other immunoassays having higher sensitivity. • Western blot formats are available to detect antibody in various diseases such as HIV, Lyme’s disease, Herpes simplex virus infection, cysticercosis, hydatid disease and toxoplasmosis. RAPID TEST • Point of care (POC) tests, because unlike ELISA and other immunoassays, the Point of Care tests can be performed independent of laboratory equipment and deliver instant results. • Two principles of rapid tests are available: o Lateral flow assay o Flow through assay. • Both the formats are available for the diagnosis of various diseases such as malaria, hepatitis B, hepatitis C, HIV, leptospirosis, Helicobacter pylori, syphilis, etc. Immunochromatographic Test (Lateral Flow Assay) • Principle (antigen detection): o Test system consists of a nitrocellulose membrane (NCM) and an absorbent pad. o Two formats are available- cassette or strip. Immunochromatographic Test (Lateral Flow Assay) • Test band-At the test line, the Ag-labeled Ab complex is immobilized by binding to the monoclonal Ab in the test line to form a colored band. • Control band- The free colloidal gold labeled Ab can move further and binds to the anti-human Ig to form a color control band. o If the control band is not formed, then the test is considered invalid irrespective of whether the test band is formed or not. Flow Through Assay • Flow-through tests are another type of rapid diagnostic assays which differ from ICT in two aspects: o Protein A is used for labeling antibody instead of gold conjugate. o Sample flows vertically through the nitrocellulose membrane (NCM) as compared to lateral flow in ICT. • Flow-through tests can be used for both antigen and antibody detection. HIV TRIDOT test is a classical example. • Detects antibodies to HIV-I and II separately in patient’s serum. • Test system is in a cassette format, consisting of a NCM and an absorbent pad. Flow Through Assay • The NCM is coated at three regions- two test regions coated with HIV-1 & 2 antigens and a third control region coated with antihuman Ig. • Sample and buffer reagents are added sequentially from the top following which they pass through the membrane and excess fluid is absorbed into the underlying absorbent pad. • As the patient’s sample passes through the membrane, HIV antibodies, if present bind to the immobilized antigens. Flow Through Assay • Test dots- Protein-A conjugate (present in buffer) binds to the Fc portion of the HIV antibodies to give distinct pinkish purple DOT(s), separately for HIV-1 and 2 antibodies. • Control dot- Irrespective of whether the HIV antibodies are present or not, Protein-A can bind to any IgG present in serum and the IgG-protein A complex can further bind to the antihuman Ig at the control line to give a pinkish purple DOT. TECHNIQUES USING ELECTRON MICROSCOPE • Immunoferritin test: o In electron microscopy, the electron dense areas appear darker than other areas. o Electron dense molecules such as ferritin particle can be used to label an antibody which can bind to surface antigens of cells and tissues. o When visualized under electron microscope, the electron dense label absorbs more electrons and appears as small black dot, thus confirming the presence of antigen. TECHNIQUES USING ELECTRON MICROSCOPE • Immunoelectron microscopy o Viral particles appear to be clumped when mixed with specific antisera and observed under the electron microscope. o Used for finding hepatitis A virus and rotavirus particles from stool specimen.