Title 18. DIAGNOSTIC TESTS.pdf ✅

PHARMD GURU Page 1 INTRODUCTION TO DIAGNOSTIC TESTS:  Diagnostic test is a procedure of examining, and confirming the presence of any disease in a suspected patient.  There are different types of diagnostic tests among which identification of organism present in the patient through culture is a time consuming process.  Another method adopted is identification of antigen or antibody. This antigen - antibody reaction tests are called as serological tests.  The serological tests are direct and indirect tests.  In direct serological test, the organism i.e., antigen is identified when reacted with a specific antibody.  Indirect test is used to identify the kind of antibody is present by taking a known antigen.  Different types of antigen — antibody tests are performed to diagnose the organism and disease like agglutination test, precipitation test, neutralization test, complement fixation test, immunofluorescence test, and radio immuno assays etc.  Other diagnostic tests includes: Skin tests (Schick's test), Delayed type of hypersensitivity test (Tuberculin or Mantoux peripheral smear test), Allergic tests (patch test or prick test). SCHICK'S TEST:  Schick's test is a method for determining the susceptibility to diphtheria which is an acute infectious and serious contagious disease caused by the bacillus, Corynebacterlum diphtheriae.  Schick's test is an antigen - antibody reaction test.  The test is introduced by a scientist Bela Schick.  The procedure for Schick's test is: a very minute amount of the toxin of about 0.1 ml is taken and injected intradermally to the fore arm of the patient. And similarly an inactivated toxoid which is heated at 70°C for 30 minutes is injected to another forearm of the patient taken as control. DIAGNOSTIC TESTS
PHARMD GURU Page 2  If the skin around the injected area becomes red and swollen, indicates a positive result because the person lacks antibodies against the toxin and hence is susceptible to the disease and the control arm shown no reaction.  The reaction is observed on 1st, 4th and on 7th day. If the skin around the injected area shows no redness or swelling on both the arms - it indicates the negative test.  In some cases, a pseudo-positive test can be observed where a red colored inflammation is observed within 24 hours after injection on both the arms and it disappears within 4 days.  Sometimes a combined reaction is observed, in which initially it looks like pseudo reaction where a red colored inflammation is observed and disappears within 4 days but only on the control arm. This person is indicated as susceptible and hypersensitive to diphtheria.  This test is not in practice now a days, as the children have been immunized earlier with the toxoid. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA):  Enzyme-linked Immuno Sorbent Assay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.  In ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme that can convert to some detectable signal.
PHARMD GURU Page 3  The enzyme labelled immunoglobulins develops the signals due to the hydrolysing enzymes which have a chromogenic substrate which will give a colour or reaction which can be observed by using microplate reader having 96 wells.  The ELISA has been used as a diagnostic tool for detecting hepatitis and HIV and also has applications in food industry.  The ELISA can be divided as competitive ELISA and non-competitive ELISA. The non-competitive ELISA is again classified as direct, sandwich, and indirect methods. THE COMPETITIVE ELISA:  It is a competitive binding process executed by sample antigen and another added antigen.  In this method, the antibody is coated on the microplate. Now the enzyme labelled as well as unlabelled antigens are added.  Now the antigens will compete for binding to the specific antibody.
PHARMD GURU Page 4  If unlabelled antigens are present in high concentration, the less amount of labelled antigen forms complex with the antibody.  When a particular substrate is added, the labelled antigen antibody complex will show a reaction which can be determined.  For competitive ELISA, the higher the sample antigen (unlabelled) concentration, the weaker the eventual signal. DIRECT ELISA: This is the simplest type of ELISA in which, the antigen is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added to block all the other binding sites. Now the enzyme linked antibody is added which adsorbs the antigen. Then a specific substrate is added which reacts with the enzyme and can be determined with a colour change. SANDWICH ELISA:  It identifies the antigens between two layers of antibodies. Hence named as 'Sandwich'.  In this method, the plate is coated with antibodies and antigenic sample is added which forms antigen-antibody complexes.  Now enzyme linked antibody is added which will bind to the antigen i.e., the Ag is stuck between two antibodies.  Then a specific substrate is added which binds to the enzyme and gives a reaction which can be detected by a colour or fluorescent or electrochemical signal. INDIRECT ELISA:  In this method, the antigen is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added to block all the other binding sites.  Now the antibody is added which adsorbs the antigen.  Then, a secondary antibody (anti antibody) linked to enzyme is added to the plate which detects the antibody that is adsorbed to the antigen.  Then the specific substrate is added which reacts with the enzyme and thus the antigen is determined.