ELISA BIO 110 Lab 1. Immunity and Disease

Transcription

1 ELISA BIO 110 Lab 1 Immunity and Disease Introduction The principal role of the mammalian immune response is to contain infectious disease agents. This response is mediated by several cellular and molecular components synthesized in advance of and during infections. Broadly speaking, there are two forms of immunity; innate and adaptive. The innate immune system is composed of soluble molecules and cellular receptors that are ever-present and poised to engage a pathogenic microorganism immediately upon infection. Many of these molecules and receptors recognize molecules commonly found in bacteria and viruses, but not produced by vertebrates, such as lipopolysaccharide (a potent toxin) and double stranded RNA (some viruses). Although the innate response is capable of engaging pathogens, it is not sufficient to contain or eliminate the pathogens. The adaptive response is responsible for specifically targeting and eliminating invading microbes. However, unlike innate immunity, the adaptive response usually takes several days to initiate and does so only in response to a given infectious agent. This response is controlled by two groups of lymphocytes; T cells that develop in the thymus and B cells that develop in the bone marrow. The innate and adaptive responses must occur together to contain infectious threats. Without the adaptive response, animals would be unable to control most infections. Without the innate response, animals would likely die from infection before the adaptive response could occur. Many human immunodeficiency diseases are a result of mutations in genes of the innate or adaptive immune systems. The Adaptive Response and Antibody Synthesis During an infection, microbes synthesize many molecules, usually proteins, that can be recognized by the adaptive immune response. These microbial molecules, termed antigens, elicit strong T cell and B cell responses. B cells participate in the immune response by secreting antibodies, which are immunoglobulins that are specific for a particular antigen. Each animal possesses about a billion B cells that specifically recognize different microbial antigens. This specificity is such that B cells that recognize West Nile virus (WNV) antigens will not recognize other microbes. Unfortunately, of these billion B cells, perhaps only a few dozen are capable of recognizing WNV. Thus, the ability of these few B cells to find WNV takes a few days. However, once the B cells interact with the virus, the cells undergo multiple rounds of mitosis, replicating as many as three times a day. This results in more than a million WNV-specific B cells in less than one week. (This rapid expansion also explains swollen glands, which are really enlarged lymph nodes because of the B cell expansion.) As the B cells start to divide they secrete antibodies into the blood that bind to WNV, which then facilitate the elimination of virus from the body because phagocytic cells, such as macrophages, internalize the antibody and virus and destroy both. Fortunately, many of the B cells remain as memory cells, poised to respond in hours after a second infection with WNV, thus can provide life-long immunity. Laboratory Detection of Specific Antibodies Since antibodies are synthesized only in response to infection, their presence or absence can be used as a diagnostic indicator of infection. In other words, people who have not been infected with WNV will not have antibodies to WNV in their blood,

2 ELISA 2 BIO 110 Lab while those who have been infected will have antibodies in their blood. This is exploited by diagnostic tests to determine a person s infection status. Many laboratory tests can be performed to gather such information, but one test in particular, the enzyme-linked immunosorbent assay (ELISA), is routinely used in infectious disease diagnoses. In this test (Figure 1), a specific antigen is coated onto special polyvinyl chloride (PVC) microtiter plates that have a high non-covalent affinity for proteins. Next, the serum sample is incubated on the plate for 30 minutes, during which time the antigen-specific antibodies will bind to the antigen. A detection reagent, which must possess enzymatic activity, is then added for another 30 minutes. This reagent binds specifically to the antibody if it is present (i.e., the person was infected). Finally a chromogenic substrate is added and incubated for 15 minutes in order to determine if the detection reagent is bound to an antibody. If it is present, a color change will result (Figure 2). Quantitative assessment of antibody levels in blood samples. ELISA can also be used as a quantitative test. In this use, log2 dilutions (e.g., 1:100, 1:200, 1:400, etc.) are made to determine the endpoint titration of an antibody sample. The endpoint is the greatest dilution that still provides a positive signal. This value is reported as the antibody titer, which is the reciprocal of the dilution (e.g., endpoint of 1:800 is a titer of Figure 2. Example ELISA plate results 800). Exercise: Detection of antibodies to Sin Nombre hantavirus. In today s exercise you will use ELISA to detect the presence of antibodies in serum samples of mice. Viral antigen will be coated onto PVC plates overnight, then stored in blocking buffer until the laboratory period. In class, two experiments will be conducted simultaneously on the same plate (exercises A and B). The first

3 ELISA BIO 110 Lab 3 exercise is to determine positive/negative status of unknown samples. The second exercise is to determine the titer of unknown samples. Materials and Methods In advance, the TA will coat ELISA plates with recombinant Sin Nombre virus nucleocapsid antigen diluted in phosphate-buffered saline (PBS). The plates will be incubated overnight in the refrigerator to permit antigen-binding to the plate wells. The plates will then be blocked in a buffer containing 0.2% porcine gelatin (digested collagen) until use in the laboratory. Blocking is necessary to saturate the wells with protein, otherwise the antibodies from the serum samples would non-specifically bind to the PVC, thus result in false-positive samples. Materials 1. Antigen coated PVC plate 2. Positive and negative controls 3. Six (6) samples diluted 1:100 in PBS for Exercise A (positive/negative testing) 4. Two (2) samples diluted 1:100 for Exercise B (titration determination) 5. PBS diluent 6. Squirt bottles with PBS-TWEEN wash buffer 7. Protein A/G-horseradish peroxidase enzyme conjugate diluted 1:1000 in PBS (detection reagent) 8. ABTS substrate and 30% H 2 O 2. TA will prepare substrate at time of use by adding 4 µl of the H 2 O 2 to 4 ml of ABTS 9. P200 micropipettes and tips Methods. Run Exercises A and B concurrently on your plate. Exercise A: Positive/Negative Testing 1. Flick and blot the ELISA plate to remove the blocking buffer. Wash the plate 3x with PBS-TWEEN (a detergent). Blot the plate on paper towels 2. In well A1, add 100 µl of the positive control (+K) serum sample. In well B1 add 100 µl of the negative control (-K) sample. In wells C1-H1 add 100 µl of the six unknown samples (1-6). Cover and incubate at room temperature for 30 minutes. 3. Wash the plate 3x with PBS-TWEEN as above to remove any unbound antibodies. 4. Add 100 µl of detection reagent/peroxidase to wells A1-H1. Cover and incubate for 30 min at room temperature. 5. Wash the plate as in step 3 to remove any unbound detection reagent. 6. Add 100 µl of activated ABTS substrate to the wells and cover and incubate 15 min. 7. Identify the positive and negative samples by color change to green. As a rule, any well that appears more green than the negative control is called positive. Exercise B: Determining the titer of unknown samples 2. Add 100 µl of PBS diluent to wells B2-H3 (total of 14 wells). Add 200 µl of sample A into well A2 and 200 µl of sample B into well A3. Serially transfer 100 µl of the serum samples down the plate to make the log 2 dilutions. Discard 100 µl from the H2 and H3 wells. At this point, all wells should have 100 µl. Incubate the plate for 30 min at room temperature. 7. Identify the greatest dilution with a positive result and determine the titer of the sample. If the last well is positive, then you must report the sample s titer as >128,000.

4 ELISA BIO 110 Lab 4 Figure 3. Plate schemat ic.

5 ELISA BIO 110 Lab 5 Results Exercise A. Record your sample as positive or negative: Sample +K -K Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Positive/Negative Exercise B. Determine the endpoint and titer of your two samples: Sample Endpoint well (A-H) Dilution of Endpoint Titer A B Questions 1. Interpret the results of your data from Exercises A and B. 2. Eighty percent (80%) of people infected with West Nile virus produce antibodies but do not show any signs of illness. What might account for this fact? 3. The detection reagent in this lab is composed of protein A, naturally made by Staphylococcus aureus, and protein G, made by some Streptococci bacteria. Why would bacteria produce these compounds? 4. HIV kills T cells that recognize specific microbes. In the advanced stages of AIDS, the titer of blood antibodies to microorganisms begins to drop dramatically, leaving the patient susceptible to the microbes. Since HIV does not affect the B cells that make antibodies, how might killing T cells cause a drop in antibody titers?