ELISA: Enzyme-linked Immunosorbent Assay PDF

ELISA Enzyme Linked Immunosorbent Assay Introduction Immuno- assay Its a technique that use the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample( urine ,saliva, tears, or any bio-chemical materials Antigens A subst...

ELISA Enzyme Linked Immunosorbent Assay Introduction Immuno- assay Its a technique that use the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample( urine ,saliva, tears, or any bio-chemical materials Antigens A substance(strange biologic - particles, ) when enter into the body lead to stimulates production of an antibody Analyte The biochemical compound in sample that detect or analyzed in immunoassays either Antibody or Antigen Antibodies: are immunoglobulins ( gamma globulin) proteins that are found in blood serum and are manufacturing by the immune system when exposed to foreign biological materials , such as bacteria and viruses. Or other strange biochemical materials  What Are Antibodies and How Are They Produced? Antibodies are large glycoprotein molecules produced by B-lymphocytes Lymphocytes : are white blood cells originated from the hematopoietic stem cells in the bone marrow. Which differentiated into B-lymphocytes (B-cells) and T-lymphocytes (T-cells). its necessary to mention that the immune system is made of two parts – humoral immunity (antibody- mediated) and cell-mediated immunity. Humoral Immune System (Antibody-Mediated) B-lymphocytes produce large glycoproteins called antibodies as a responses to antigens and then mark those antigens-antibody complex to be destroyed by the T-lymphocytes. Each B-cell makes its own distinct antibody in response to a specific antigen which comes in contact with it. Each antibody is designed to bind to a specific surface binding site or epitope on the antigen. There are millions of different types of antibodies circulating in an individual’s bloodstream and they are based on exposure to antigens in environment. Structure of An Antibody Over 80% of human glycoprotein antibodies are in the immunoglobulin class IgG.They are shaped like a Y and are found in the blood, lymph, and intestine. IgG molecules have a molecular weight of 150,000 Daltons and are made of 1. 2 long (heavy) chains coded from chromosome 14, 2. 2 short (light) chains coded from either chromosome 2 or 22, 3. These chaine are connected by di-sulphide bonds. The antibody recognises and bind to the specific antigen and determinant specific region on the the antigen surfaces (antigen binding sight ).  Antigen or protein statuse : It might be present naturally in the body like hormones Is might be manufactured in special disease. in some normal physiological status like 1. (HCG) human chorionic gonadotrophin hormone which is produced by cells of the placenta in pregnancy 2. It might be found in the blood in some types of cancer 3. Sometimes It Is not present in the body in normal condition but represent as a strang materials like drugs or genetic modefied proteins The Methods for Antibody Production in lab. 1. to produce small quantities of antibody, Specific antibodies are produced by injecting an antigen into some small mammals , such as a mouse, rat or rabbit, twice within 3 weeks 2. to produce large quantities of antibody inject antigens in goat, sheep, horse. to obtained the anti-sera which got a polyclonal antibodies— multiple antibodies that bind in more one site to the same antigen—in the serum,  3.To obtain specific antibody for a specific antigen, (monoclonal antibodies) this done by combined the (antibody-secreting B- lymphocytes cell) with cancer cell (hybridomas), the new cell will continually grow and secrete same antibody; these antibodies are called monoclonal antibodies attach only with specific antigen binding sight.  what is ELISA? It Is a biochemical immunology technique used to detect the presence of an antibody or an antigen in a sample (hormones ,drugs ,some toxins ,(GM- CROPs)genetic modified crops ). Non copetetion ELISA Requirements.  Antigens (Ag) fixed to a solid surface ' -- immobilized 96-wells plastic plate  Antibodies (Ab) in solution to be tested ' i.e. urine or serum, saliva ,tears,  Enzyme-conjugated Anti-immunoglobulin ' Antibody against the antibodies being tested for. Enzyme linked (conjugated) to the Ab  Substrate binds to enzyme and produces color  Color intensity proportional to bound enzyme-Ab Eliza equipments. 1. Eliza reader 2.Eliza washer 3. washer solutions 4. Eliza monitor 5.Eliza kits Indirect ELISA 1. it is similar to direct ELISA in which an antigen is immobilized on a plate, 2. primary detection antibody is added and binds to the specific antigen. forming antigen-antibody complex 3. then secondary antibody conjugate with (horseradish-peroxidase) the enzyme linked was added a to this complex 4. Substrate then added to produces a signal proportional to the amount of antigen bound in the well. By reaction with conjugated enzyme to produce cooler 5. The color was estimated by spectrophotometer as a reflection for sample concentration. Sandwich ELISA: 1. Sandwich ELISA are the most common type of ELISA. When Capture antibody coated on a microplate and Two specific antibodies are used forming a sandwich shape with the antigen. 2. A conjugated enzyme-detection antibody added and binds to an additional epitope on the target protein(antigen). 3. Substrate is added and produces a signal that is proportional to the amount of analyte present in the sample.this method called direct sandwich 4. when It required to additional antibody then it called indirect sandwich 5. Sandwich ELISAs are highly specific, since two antibodies are required to bind to the protein of interest. 6. It to Use: Determining very small analyte concentration in a biological sample such as Aflatoxins. Competitive ELISA ; Competitive ELISAs are commonly used when the protein of interest is too small to efficiently sandwich with two antibodies characterized by : 1. Similar to a sandwich ELISA, a capture antibody is coated on a microplate. 2. Instead of using a conjugated detection antibody, a conjugated antigen is used to complete for binding with the antigen present in the sample. 3. Note: that more antigen present in the sample ,meaning : less conjugated antigen(standard) will bind to the capture antibody. (opposite reflection) 4. Substrate is added and the signal produced is inversely proportional to the amount of protein present in the sample.  recommendation : 1. Before starting the work read kit instruction carefully to avoid any disturbance and finally loss because its expensive : 2. There are 96 well plate is labeled carefully and the first wells are used to draw the standard curve 3.The sample is added to well of the Eliza plate in duplicate or triplicate in order to get true results when calculated 4.The quality control samples which is provided with the kit is treated as the test samples that mean in assistance with( negative and positive samples) that included with kits  Analysis of the results(clasic method): After reading the results (the absorption )by using spectrophotometer the standard curve is drawn were the concentration on the X-axis and the absorbance on the Y-axis Absorption nm Concentration ng/ml Analysis Results using soft wear : The standards concentrations is specified on the x-axis and the reading of each standard is specified on the y-axis and the standard curve is drawn Results-cont  This standard curve is used to determine the unknown concentration of each sample by finding the opposite concentration to the absorbance Absorption nm Concentration ng/ml Some Facts about the Results-:  The quality control sample concentration is determined from the standard curve and if the result is in the range given by the kit manufacturer the results could be accepted by reaction  The initial Positive results appeared directly by change the color of the solution of sample in Eliza wells, but the negative results is determined by the color of sample solutions is still colorless because there is no reaction is occur between antibody and antigen.