Chapter 6 Detection of Diseases PDF

Chapter 6 Detection of Diseases  Specificity between the individual antibody will react to only one antigen  on the surface of red blood cell, there are A, B, AB antigens (proteins) USED in blood group testing A...

Chapter 6 Detection of Diseases  Specificity between the individual antibody will react to only one antigen  on the surface of red blood cell, there are A, B, AB antigens (proteins) USED in blood group testing Antibody and Antigen DO NOT match Antibody and Antigen DO match Agglutination, which refers to the clumping of particles together, is an antigen-antibody reaction that occurs when an antigen (i.e., a molecule capable of triggering the adaptive immune response) is mixed with its corresponding antibody at a suitable pH and temperature  COVID progression IgM and IgG are so many Ig... some of them can be found in lymphatic system IgM - location - Lymphatic system IgG - location - different parts of the body https://www.aurorabiomed.com.cn/liquid-handling- apps/covid-19-igm-igg-antibody-rapid-test-kit/ COVID testing Molecular test: quantify amount of RNA from virus Antibody test : Monitor our immune response to the virus  Serological tests or antibody test determine the presence of CoV-2 specific antibodies (mainly IgM and IgG classes) in a patient’s serum. Since IgM and IgG antibodies are the first-line responses in fighting viral infection in situ, they are the most practical biomarkers of SARS-CoV-2 serological diagnostics. Rapid tests, predominantly lateral flow assay (LFA), have the capacity to identify antigen-specific total antibodies in serum samples within just 30 minutes. These tests are extremely beneficial due to low sample volume requirements, fast turn-around time, simplicity of use, as well as a low economic factor.  M line G line Control line CovID19 IgM Gold CovID19 Gold Rabbit Anti-Human IgM Antibody Anti-Rabbit IgG Antibody Antigen IgG Conjugate CovID19 IgG Anti-Human IgG Antibody Conjugate Has two mouse anti-human monoclonal antibodies (anti-IgG and anti-IgM) stripped on two test lines (M, G lines). A surface antigen from SARS-CoV-2 which can specifically bind to SARS- CoV-2 antibodies (both IgM and IgG) is conjugated to colloidal gold nanoparticles sprayed on conjugation pads. The Gold-rabbit IgG conjugates were also sprayed on conjugation pads for binding to anti- rabbit IgG antibody which is immobilized on the control line (acting as a control that the test kit is not expired)  If patient samples on sample pad has the virus, the Covid-19 IgM, IgG (from patient immune system) will bind to GoldCov-19 antigen conjugate and move to bind with corresponding mouse Anti-Human IgM or IgG Antibody lines (the M or the G line will turn red) indicating infection. During that time, the Gold Rabbit IgG Conjugate deposited on the conjugate pad will move along the liquid in the sample to bind to Anti-Rabit IgG Antibody at the Control line (C line) indicating that the test kit is still valid. Test results No COVID Early infection Later infection In between  Currently, there has been development of test kits that help reduce the steps involved, making it easier for the general public to use at home. doesn't need blood, just sample swab from nose These are called *Antigen Rapid Tests* (or *ATK* test kits). The kits are simple and require only a few items, such as a swab for sample collection, a test strip, and a solution contained in a test tube. This solution is a buffer solution, mostly made of saline (salt water), with other components that help maintain the solution's stability and break down the virus. Different brands may have slightly different equipment and usage instructions, so it’s essential to read the label every time before use.  Relies on interaction between They have complementary shape that fit together like The principle behind the ATK test for detecting the COVID-19 virus is based on an immunological testing method, specifically *antigen-antibody interaction*. The antigen we are detecting is the SARS-CoV-2 virus (the COVID-19 virus), and the antibody is a protein that specifically targets this virus. Antibodies are naturally produced by the immune system of living organisms to help remove foreign substances, like viruses, from the body.  When drop liquid containing swapped sample. If the virus is present, the antibody will bind to the virus and move towards the T (test line) and the C (control line) Sample containing virus Antibody + gold The test strip contains a membrane (similar to paper) coated with antibodies that are specific to the COVID-19 virus. The antibodies at the conjugation pad are attached to gold nanoparticles, which are very small and appear red. When the test is used, the red color of the gold nanoparticles allows us to see the result. The test begins with a swab from the nose, which is then dipped into the solution inside the test tube. A few drops of this solution are added to the test strip. Positive result: virus from sample binds to gold-labelled antibody, and move. At T: attached antibody bind to the mobile virus conjugated gold labelled antibody causing red stripe and at C, the left over virus conjugated gold labelled antibody will bind to the attached antibody causing the red stripe. Virus bind to gold conjugated antibody Positive result If the sample contains the virus, the virus particles will first bind to the gold labeled antibodies at a section called the *Conjugation Pad* and move along the test strip by. Then, the molecules reach the Test Line (T), where more antibodies specific to the virus are present. The virus (along with gold conjugated antibody) will bind to these antibodies, causing a red line to appear at the Test Line. If no virus is present, the gold labeled antibodies can move along the liquid and pass the T line without binding (no red line will appear at T) and reach the C line which will appear red.  Negative result: No virus, sample moves past T line (no binding because no virus), and at C the gold-labelled antibody will bind to the attached antibody causing red color. No virus Negative result https://sciplanet.org/content/10291 The presence of the red line at the Control Line is important because it confirms that the test worked properly. If the red line at the Control Line doesn’t appear, the test is invalid, even if there is a red line at the Test Line. The entire process is quick, and results can be seen with the naked eye within 15 minutes, which is why it’s called a *Rapid Test*. ATK (Antigen Test Kits or Antigen Rapid Test)  Converting RNA to DNA https://www.socmucimm.org/resources/news- media/pcr-the-polymerase-chain-reaction/ https://www.researchgate.net/figure/Reverse- transcription-polymerase-chain-reaction-RT-PCR- The-RT-PCR-creates-a-cDNA- copy_fig1_341062500  Schematic representation of nucleic acid detection of SARS-CoV-2 by RT-PCR assay. (a) RNA extraction, (b) reverse transcription, (c–e) PCR amplification by (c) c-DNA denaturation, (d) primer annealing, (e) primer elongation by DNA polymerase enzyme, (f) detection steps with TaqMan probe, (g) RT- qPCR instrument, (h) signal results, and (i) https://www.sciencedirect.com/science/a primers and probes for screening. rticle/pii/S0165993622002333  TaqMan PCR is a type of real-time PCR and uses a nucleic acid probe complementary to an internal segment of the target DNA. The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps the excitation spectrum of the other, resulting in “quenching” of the first fluorophore by the second. The probe is present during the PCR and if product is made, the probe is degraded via the 5′-nuclease activity of Taq polymerase that is specific for DNA hybridized to template (= TaqMan activity). The degradation of the probe allows the two fluorophores to separate, reducing quenching and increasing intensity of the emitted light.  ELISA The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique which is used to measure the concentration of an analyte (usually antibodies or antigens) in solution. Antigen/antibody of interest is absorbed onto plastic surface Antigen is recognized by specific antibody This antibody is recognized by second antibody which has enzyme attached Substrate reacts with enzyme to produce product, usually color product  Types of ELISA Enzyme-Linked Immunosorbent Assay versus Chemiluminescent Immunoassay: A General Overview  Direct ELISA : the primary recognition Ab binds directly to the interest or the analyte. Indirect ELISA: except for an extra wash step and the kinds of antibodies introduced after the buffer is removed, the indirect ELISA processes are identical to the stages of the direct ELISA. Two antibodies are required in indirect ELISA: One is a primary recognition Ab (attaches to the target protein) and the second is a subsequent enzyme-linked Ab (acts in conjunction with the main Ab). After applying the primary Ab, a step of washing is accomplished, and then, the enzyme-bound with secondary Ab is introduced and incubated. Indirect ELISA has a higer sensitivity than direct ELISA Sandwich ELISA : In a sandwich ELISA, instead of binding the enzyme labeled antibody to the antigen directly as in direct or indirect Elisa, we first attach an unlabeled capture antibody onto a plate, then add the antigen followed by detecting it with another specific labelled detection-antibody. Competitive ELISA : The competitive ELISA tests for the presence of an antibody specific for antigens in the test serum. This type of ELISA utilizes two specific antibodies: an enzyme-conjugated antibody and another antibody present in the test serum (if the serum is positive). Combining the two antibodies into the wells will allow for competition for binding to antigens. The In the negative test, the color changes are noted which is the indication of the conjugation of enzyme-bound antibodies and Ags, whereas if the color does not change, it is the indication of the Abs presence, meaning that the test is positive.  Sandwich ELISA for the detection of SARS-CoV- 2 antigens. (a) Microwell Plate coated with the capture antibody, (b) Addition of the patient sample containing the viral antigens, (c) Washing to remove bound antigens, then add primary antibodies, (d) Washing to remove the unbound primary antibodies, then the addition of the enzyme-bound secondary antibodies, (e) After washing to remove the unbound secondary antibodies, the substrate is added and converted by the enzyme into a detectable form by assuming a color that depends on the presence and concentration of the viral antigen; and (f) The ELISA reader is used to detect the presence and concentration of the viral antigen in the sample.  Western blot Western blotting is a laboratory technique used to detect a specific protein in a blood or tissue sample. The method involves using gel electrophoresis (movement of protein on gel using electricity) to separate the sample's proteins. The separated proteins are transferred out of the gel to the surface of a membrane. The membrane is exposed to an antibody specific to the target protein. Binding of the antibody is detected using a radioactive or chemical tag. A western blot is sometimes used to diagnose disease. https://www.biomol.com/resources/applications/western-blot/ Timeline for SARS-CoV-2 infection and COVID-19 positivity tests versus the molecular diagnostic assays (PCR). https://www.sciencedirect.com/science/article/pii/S0165993622002333