Elisa Test Lab (1).docx

Name: Shannon Bernard ID#: 620086773 Date: February 18, 2019 Group 2 Title: Molecular and Serological Diagnostic Methods Aim: 

To learn about serologic and molecular techniques used in the diagnosis of laboratory infections.



To perform an ELISA.

Abstract: The aim of the experiment was for the student to learn about the serologic and molecular techniques that are used in the diagnosis of laboratory infections and also to perform an ELISA test. To carry out the experiment the reagent s were removed from the refrigerator and allow to come to room temperature before used. The samples were then vortex before used, then 50mL of 20X wash buffer type 1 to 1L with distilled water was diluted and mix. The test sera, calibrator and control sera was diluted in serum diligent. Then 100 microliter of the appropriate diluted calibrator, control and patient’s serum was add to individual well and 100 micrometer of serum diluent was add to the reagent blank well. Then the well were incubated at room temperature for 25 minutes. The liquid in each well was then shake out in a wash container, then 250-300 micrometer of wash buffer was added to each well and this was done 3 times then the liquid was game in a container. After which 100 micrometer of conjugate was added to each well the incubated at 2mroom temperature for 25 minutes. Then the washing procedure was repeated

with the buffer wash. Then 100 micrometer of chromogen was added to each well then this was incubated at room temperature for 10- 15 minutes. Then 100 micrometer of stop solution was added to each well. Then the this was read on a ELISA plate reader equipped with 450 nm filter. From results obtained it can be concluded that Ms. Strawberry test results shows a positive test for herpes simplex virus and also patient’s 3 and a negative test was obtained for patients 2. Therefore it can be concluded that patient’s 1 and 3 has the HSV IgG antibody and patient’s 2 does not. Introduction: An enzyme-linked immunosorbent assay, also called ELISA or EIA, is a test that detects and measures antibodies in your blood. This test can be used to determine if you have antibodies related to certain infectious conditions. Antibodies are proteins that your body produces in response to harmful substances called antigens. From the late 1960s, ELISA tests play a role in diagnostic research over 50 years. The origin of ELISA was the idea of finding an alternative method to substitute radioimmunoassay (RIA) in immunoassay. Before the invention of ELISA, RIA is the only method to conduct immunoassay. The First paper introduced this technique was published in 1960 by Rosalyn Sussman Yalow and Solomon Berson. However, the concerns of potential safety and health problems coming with the initiation of RIA. Scientists proposed to find another labeling method to replace radioactive label. Some of them came up with an idea to use enzyme labels in immunoassay. But, many thought it is impossible to link the enzyme, such as large molecule, to an antibody or antigen without affecting their bioactivity in conjugating reaction. Although met with skepticism and criticisms, Perlmann and Schuurs independently invented the method to prove the using of enzyme-linked immunoassay is feasible in 1966. And following in 1966-1970, other scientists continually polish and improve methods, including resolving two critical issues for ELISA, which are “reaction associated color

changing” and “removing non-specific conjugation”. Finally in 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden (Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G), and Anton Schuurs and Bauke van Weemen in the Netherlands (Immunoassay using antigen—enzyme conjugates) independently published papers that systematically introducing EIA/ ELISA methods. ELISA tests developed rapidly in the 1970s and early 1980s, and revolute into commercial clinical used products what we use now. It is firstly used in detecting autoimmune related antibodies in patients with autoimmune disease. With the help of linked enzyme, the reactions between antigens and antibodies could be showed in a particular color. That is why it is named “enzyme-linked immunosorbent assay”, because ELISA test is related to “immune” and “enzyme”. ELISA test is a typical “wetlaboratory” type test, though it uses a solid phase to detect the presence of the substance, the substance is usually in a liquid or wet sample. Generally the solid phase is a solid plate with 96 wells, while some ELISA tests are performed with 192 wells’ or 384 wells’ plate. ELISA test has been wildly used in medical diagnostic. And, it has been used in detecting plant diseases’ pathogen in plant research. In addition, many industries, such as food industry, apply ELISA test in quality control and quality assurance process. Enzyme-linked immunosorbent assay (ELISA) is a method of target antigen (or antibody) capture in samples using a specific antibody (or antigen), and of target molecule detection/quantitation using an enzyme reaction with its substrate. In ELISA, various antigenantibody combinations are used, always including an enzyme-labeled antigen or antibody, and enzyme activity is measured colorimetrically.The enzyme activity is measured using a substrate that changes color when modified by the enzyme. Light absorption of the product formed after substrate addition is measured and converted to numeric values. Depending on the antigen-

antibody combination, the assay is called a direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA etc. Direct ELISA- A target protein (or a target antibody) is immobilized on the surface of microplate wells and incubated with an enzyme-labeled antibody to the target protein (or a specific antigen to the target antibody). After washing, the activity of the microplate well-bound enzyme is measured. Indirect ELISA- A target protein is immobilized on the surface of microplate wells and incubated with an antibody to the target protein (the primary antibody), followed by a secondary antibody against the primary antibody. After washing, the activity of the microplate well-bound enzyme is measured. Although indirect ELISA requires more steps than direct ELISA, labeled secondary antibodies are commercially available, eliminating the need to label the primary antibody. Sandwich ELISA- An antibody to a target protein is immobilized on the surface of microplate wells and incubated first with the target protein and then with another target protein-specific antibody, which is labeled with an enzyme. After washing, the activity of the microplate wellbound enzyme is measured. The immobilized antibody (orange) and the enzyme-labeled antibody (green) must recognize different epitopes of the target protein. Competitive ELISAAn antibody specific for a target protein is immobilized on the surface of microplate wells and incubated with samples containing the target protein and a known amount of enzyme-labeled target protein. After the reaction, the activity of the microplate well-bound enzyme is measured. When the antigen level in the sample is high, the level of antibody-bound enzyme-labeled antigen is lower and the color is lighter. Conversely, when it is low, the level of antibody-bound enzymelabeled antigen is higher and the color, darker. The graph above and to the right illustrates the correlation between absorption and antigen levels in samples. The most popular ELISA

product is the ELISA kit. Usually, users only need to prepare the analyte sample for the experiments, all the other components, such as plates, antibodies or antigens, substrate solution, TMB solution, controls, calibrators and etc., are provided by the ELISA kit. Mumps (Parotitis) is a non-segmented, negative sense, enveloped RNA virus, a common contagious disease with relatively moderate symptoms during childhood, but increasing complications, when adults are infected. The causative agent of mumps is a virus which is a member of the Rubulavirus genus of the family Paramyxoviridæ. In Mumps diagnosis test, purified Mumps antigen is coated on the surface of microwells. Diluted patient serum is added to wells, and the Mumps IgG specific antibody, if present, binds to the antigen. All unbound materials are washed away. After adding enzyme conjugate, it binds to the antibody-antigen complex. Excess enzyme conjugate is washed off, and TMB Chromogenic Substrate is added. The enzyme conjugate catalytic reaction is stopped at a specific time. The intensity of the color generated is proportional to the amount of IgG specific antibody in the sample. The results of the test for Mumps are read by a microwell reader compared in a parallel manner with calibrator and controls.

Method: Medical Microbiology Laboratory Manual by Department of Basic Medical Sciences, University of the West Indies, Mona Campus, page 23 Changes to the method:

Results:

Sample calculation: Mean Calibrator O.D. = 1C (0.544)+ 1D(0.522) =1.066/2 =0.533 Correction factor=0.50 Cutoff Calibrator Value= Correction factor × Mean Calibrator O.D 0.50 × 0.533 = 0.267 F1 Patient sera= 0.882 ISR Value = O.D obtained for patient × Cutoff Calibrator value F1= (0.882) ÷ (0.267) = 3.30 G1= (-0.040) ÷ (0.267)= -0.15 H1= (0.842) ÷ (0.267) = 3.15 Negative Control= (-0.084) ÷ (0.267) = -0.31 E1 Positive Control= ( 0.852) ÷ (0.267) = 3.20

Discussion: The herpes simplex virus, also known as HSV, is an infection that causes herpes. Herpes can appear in various parts of the body, most commonly on the genitals or mouth. There are two types of the herpes simplex virus. HSV-1: Also known as oral herpes, this type can cause cold sores and fever blisters around the mouth and on the face. HSV-2: This type is generally responsible for genital herpes outbreaks. The herpes simplex virus is a contagious virus that can be passed from person to person through direct contact. Children will often contract HSV-1 from early contact with an infected adult. They then carry the virus with them for the rest of their lives. Infection with HSV-1 can happen from general interactions such as: eating from the same

utensils, sharing lip balm, and kissing. The virus spreads more quickly when an infected person is experiencing an outbreak. Anywhere from 30 to 95 percent of adults are seropositive for HSV1, though they may never experience an outbreak. It’s also possible to get genital herpes from HSV-1 if someone who performed oral sex had cold sores during that time. HSV-2 is contracted through forms of sexual contact with a person who has HSV-2. While HSV-2 infections are spread through contact with a herpes sore, the AAD reports that most people get HSV-1 from an infected person who is asymptomatic, or does not have sores.

Anyone can be infected with HSV, regardless of age. Your risk is based almost entirely on exposure to the infection. In cases of sexually transmitted HSV, people are more at risk when they participate in risky sexual behavior without the use of protection, such as condoms. Other risk factors for HSV-2 include: having multiple sex partners, having sex at a younger age, having another sexually transmitted infection (STI) and having a weakened immune system. If a pregnant woman is having an outbreak of genital herpes at the time of childbirth, it can expose the baby to both types of HSV, and may put them at risk for serious complications. It is important to understand that someone may not have visible sores or symptoms and still be infected by the virus. And they may transmit the virus to others. Some of the symptoms associated with this virus include: blistering sores (in the mouth or on the genitals), pain during urination (genital herpes) and itching. You may also experience symptoms that are similar to the flu. These symptoms can include: fever, swollen lymph nodes, headaches, tiredness and lack of appetite. HSV can also spread to the eyes, causing a condition called herpes keratitis. This can cause symptoms such as eye pain, discharge, and a gritty feeling in the eye.

The herpes simplex virus IgG test detects IgG antibodies that are specific to HSV types 1 and or 2 virus. The assay is based on purified recombinant glycoprotein G-1 (HSV-1) or G-2 (HSV-2) antigens. The HSV IgG antibodies does not show up until slightly after initial infection. The herpes IgM test is a type of blood test that can be used to detect early herpes (HSV) infection. This test does not detect herpes directly. Instead, it looks for a type of antibody that can be produced in response to a herpes infection, IgM. Therefore, the IgM test is used for acute infection as IgM antibodies are detectable within 7-10 days after initial infection. An enzymelinked immunosorbent assay, also called ELISA or EIA, is a test that detects and measures antibodies in your blood. This test can be used to determine if you have antibodies related to certain infectious conditions. Antibodies are proteins that your body produces in response to harmful substances called antigens. An ELISA test may be used to diagnose: HIV, which causes AIDS, Lyme disease, pernicious anemia, Rocky Mountain spotted fever, rotavirus, squamous cell carcinoma, syphilis, toxoplasmosis, varicella-zoster virus, which causes chickenpox and shingles, Zika virus and others. A positive HSV IgG test if the tests are accurate means that the patient’s body as been infected with the herpes simplex virus. The limitation to the HSV IgG test is that it may not show positive until several months after the individual has been infected. As such when an individual becomes infected the immune system tries to fight off the infection and produce antibodies specific for the infection they are fighting.

Based on the results obtained patient 1 and 3 had a positive result, while patient 2 had negative result. Therefore, both patient’s 1 and 3 had been infected with the herpes simplex virus as there ISR values were greater than or equal to 1.10 and this indicate a positive test. While patient 2 had a negative result because the value for the ISR is less than or equal to 0.90 and this indicate a positive test. However the IgG antibody was detected in Both patient’s 1 and 3. This was

expected for patient one as the examinations done on her indicate that her cervix was ulcerated and this is a sign of HSV 2. The HSV culture shows a significant growth of Neisseria gonorrhoeae and the PCR shows a positive test for N.gonorrhoeae and Trichomonas app. While the serological test also indicate that the patient have the virus. The vaginal swab was done on Me. Strawberry and this indicated as Neisseria gonorrhoeae as there was significant growth on the plate as mention before. The swab was done to detect what was causing the vaginal irritation and discharge. The high vaginal swab also show result of 3 positive pus cell, 2 epithelial cells, 3 positive gram negative cocci which could possible be N.gonorrhoeae and 2 gram negative bacilli which could possible be Gardnerella vaginalis and this can cause bacterial vaginosis and this is the most common cause of abnormal vaginal odor and discharge. It is caused by a change in the type of bacteria found in the vagina. G.vaginalis is a facultatively anaerobic Gram-variable rod that is involved, together with many other bacteria, mostly anaerobic, in bacterial vaginosis in some women as a result of a disruption in the normal vaginal microflora. It has a Gram-positive cell wall,[4] but, because the cell wall is so thin, it can appear either Gram-positive or Gram-negative under the microscope. It is associated microscopically with clue cells, which are epithelial cells covered in bacteria. G. vaginalis produces a pore-forming toxin, vaginolysin, which affects only human cells. As mention before that the PCR showed N.gonorrhoeae and Trichomonas spp. The Trichomonas spp. Could be the reason why Ms. Strawberry is having abdominal frothy mucopurulent vaginal discharge along with the odour and abdominal pain that she is feeling. The serological test shows that the both IgM were positive and the IgG was negative for both gonorrhoeae and chlamydia and this indicate that she recently got the infection as this IgM indicate acute infections.

Questions:

1. What are the differences between tests for IgM vs IgG and what is the significance of these tests? IgM is the immediate antibody that is produced once a human body is exposed to a bacteria, virus or a toxin. IgG is found throughout the body, mainly in most of the bodily fluids, while IgM is found mainly in the blood and lymphatic fluids. IgM is larger in size compared to IgG. IgM is temporary and disappears after a few weeks. It is then replaced by IgG. The herpes IgM antibodies usually are detectable by herpes blood tests within 7-10 days after initial infection. IgM levels stay high for approximately two weeks. After that, they usually decline. Therefore IgM testing is primarily considered to be useful for detecting acute infection. However antibody levels also sometimes go up during an outbreak. In contrast, HSV IgG antibodies do not show up until slightly later after initial infection. A positive herpes IgG test, if the test result is accurate, means that your body has been infected with the herpes simplex virus. Furthermore, type specific HSV IgG tests can be used to distinguish between HSV-1 and HSV-2. Type-specific tests are far more accurate than non-type-specific tests, however, they can not detect whether a particular infection is oral or genital. 2. What diseases are affecting Ms. CS at this time? The disease that Ms.CS is affected with are Chlamydia, Herpes, Trichomonas and gonorrhoeae. 3. What advice would you give this patient? 4. Why is it necessary to use a negative and positive control? Positive and negative controls are necessary to validate the immunoassay. They are used to make sure the results are accurate because we are dealing with patient samples. These

controls allows the tester to see that each step was conducted correctly and that the materials like the enzymes are not denatured. 5. Why is it necessary to wash between steps of the assay? To remove any non-specific background, such as that caused by unbound proteins or antibodies. After the final wash step, the addition of an enzyme substrate produces a measurable change to indicate the quantity of antigen in the sample. Washing also removes unbound primary and secondary antibodies. If these were not washed out, the enzyme-substrate reaction would lead to a false positive. Conclusion: It can be concluded that Ms. Strawberry has herpes simplex virus along with patient’s 3. References: Lequin, R. M. (2005). "Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA)". Clinical Chemistry. 51 (12): 2415–8. Schmidt, S. D, Mazzella, M. J, Nixon, R. A, Mathews, P. M (2012). Aβ measurement by enzymelinked immunosorbent assay. Methods in Molecular Biology. 849. pp. 507–27 Van Weemen, B.K, Schuurs, A. H. W. M. (1971). "Immunoassay using antigen—enzyme conjugates". FEBS Letters. 15 (3): 232–236 Weiland, G. (1978). "[The enzyme-linked immunosorbent assay (ELISA)--a new serodiagnostic method for the detection of parasitic infections” MMW . 120 (44): 1457–1460.