Reference Schaechter chapters 45 and 55 Schaechter chapter 32 for Polio vaccines. There is a table that compares live and killed vaccines. Brooks chapter 30 for viral vaccines (page ) Brooks. Selected chapters with subheading “Prevention and control by vaccines”: chapters 15, 16, 19,24,27,33,34,35,36, 37,39,40 and 42.

Medical Diagnostic in Microbiology Medical Microbiology SBM 2044

Diagnosing pathogens Why is this important? To guide the selection of appropriate therapy for the cause of an infection. Valuable information can be generated in four ways: –Microscopic examination of patient samples –Cultivation and identification of microorganisms from patient samples –Measurement of a pathogen-specific immune response in the patient. –Detection of pathogen-specific macromolecules in patient samples.

Terminology: Sensitivity: the likelihood that it will be positive when the pathogen is present Specificity: the likelihood that it will be negative if the pathogen is not present

Identification by Microscopy Based on: – morphologic features – staining properties – movement Helminthic and protozoal infections are routinely diagnosed by microscopy.

FIGURE 58-2 The cross- reactivity of three types of immunoglobulin preparations. Antibodies raised to react with bacterium A may cross-react with bacterium B. Polyclonal antisera are cross-reactive because bacteria A and B have protein antigens in common. Additionally, monospecific antiserum raised against a single antigen of bacterium A is also cross-reactive, because one of the antibody-binding sites (epitopes) on this antigen is shared by an antigen of bacterium B. However, a monoclonal antibody against an epitope on this antigen was found to be unique to bacterium A and to have no cross- reactivity.

FIGURE 58-3 Direct ELISA serology for the detection of specific antibodies in patient serum.

Western Blot

Western blot Immunoblotting (“Western blot”) whereby defined antigens are placed on strips of nitrocellulose paper. Following incubation, the strip is treated with an enzyme-labeled Ab. Addition of the substrate for the enzyme allows detection of Ag-specific bound Ab by colorimetric reaction. Useful for Abs in HIV infection and Lyme disease.

Western blot analysis can detect your protein of interest from a mixture of a great number of proteins. Western blotting can give you information about the size of your protein (with comparison to a size marker or ladder in kDa), and also give you information on protein expression (with comparison to a control such as untreated sample or another cell type or tissue). Summary: Western Blot Gives You Information on the: Size of your Protein Expression Amount of your Protein Western blot analysis can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro. Western blot is dependent on the quality of antibody you use to probe for your protein of interest, and how specific it is for this protein. Antibodies are now easily obtainable from commercial sources, and you can purchase one for your protein of interest. If your protein is a novel protein, you must produce an antibody yourself or get a company to do it for you. In this case you will need at least a small amount of your protein either purified from cell extracts or made as a recombinant (ie in vitro or in a recombinant protein expression system). Antibodies specific to your protein are vital to western blotting as they are able to bind specifically to your protein of interest instead of the thousands of proteins on your western blot!recombinant protein expression system

How Does Western Blotting Work? See Diagram 1 below. First things first. Obtain a protein sample you want to analyze, such as cell samples. Lyse the cells to release protein contents. Run these on a gel which separates proteins on the basis of size. Then transfer these gel proteins onto a membrane using electricity. This membrane can then be used to probe for proteins of interest using a primary antibody.Diagram 1 What You Need to Western Blot: A Protein Sample A Good Antibody to Detect your Protein of Interest Western blot relies on the primary antibody to detect this protein from the thousands of proteins on your membrane and previously on your gel! (a cell can contain 30,000 different proteins - and these same proteins can even be altered giving you over 300,000 different proteins!). Using an antibody recognizes your primary antibody (a secondary antibody) you build up a protein-antibody-antibody sandwich! The secondary antibody has a horse radish peroxidase enzyme which converts a luminol substrate to a light releasing substance! This light is detected as a spot on film. From this spot you can determine how much protein is there relative to other spots, or the size of the protein relative to a size marker that is run also on the gel.

Diagram 2 shows a western blot example gel. Lane 1 is a protein size marker ladder which shows different known sizes of proteins, this can be purchased commercially and the sizes of all the spots are given in a pamphlet. Lane 3 is a cancer sample and lane 5 is a normal sample. As you can see the protein in lane 3 has a higher expression than the cancer sample in lane 5, which is interesting. Also, the protein spots in lanes 3 and 5 are the same size as the 2nd spot in the size ladder from lane 1. We can then look at the known protein size from our brochure which we received with the ladder. We then determine that the size of the protein is 80 kDa. Our protein of interest is also 80 kDa. So we know that the western blot worked and that the protein is highly expressed in a cancer sample!Diagram 2 To Detect your Protein: Buy an Antibody Against Your Primary Antibody Source Use an ECL - Chemiluminescence Kit and Film to Get the Results

PCR

FIGURE 58-8 Real-time PCR. A. In the TaqMan (Applied Biosystems) procedure for confirming and quantifying PCR products, the PCR vessel contains target DNA (pink and lavender), primers (dark purple), and a DNA detection probe that hybridizes specifically to the amplified sequence (blue). Conjugated to this detection probe is a fluorescent dye and a fluorescence quencher in close proximity to one another, such that the fluorescence from the dye is quenched and is undetectable. As DNA synthesis proceeds, the 5′-exonuclease activity of the polymerase digests the detection probe, releasing the fluorescent dye and the quencher. As they separate from one another, the fluorescent dye becomes active and emits a signal. With each round of PCR, more detection probes hybridize and are cleaved, increasing the fluorescent signal geometrically until all of the primers and probes are exhausted. B. Fluorescent signals obtained with real-time PCRs beginning with decreasing quantities of target DNA. As the number of copies of target DNA decrease, the fluorescence curve shifts to the right. For quantitative PCR a standard curve can be generated by determining how many cycles of PCR are required to exceed a predetermined threshold for fluorescence detection (purple horizontal line). By reference to these standards, the number of target nucleic acid molecules in a patient sample can be determined.

Microarrays are miniaturised biological devices consisting in molecules, for example DNA or protein, named the "probes", that are orderly arranged at a miscoscopic scale onto a solid support such as a membrane or a glass microscope slide. The array elements bind specifically to labelled molecules, the "targets", present in complex molecular mixtures, generating signals that reveal the identity and the concentration of the interacting labelled species. Microarray analysis has a broad range of applications that involve different types of probes and/or targets. The most common application of DNA/oligonucleotide microarray is gene expression analysis. In this technique, RNA isolated from two samples are labelled with two different fluorochromes (generally the green cyanine 3 and the red cyanine 5 (Cy3, Cy5)) before being hybridised to a microarray consisting of large numbers of cDNAs/oligonucleotides orderly arranged onto a glass microscope slide. After hybridisation under stringent conditions, a scanner records, after excitation of the two fluorochromes at given wavelengths, the intensity of the fluorescence emission signals that is proportional to transcript levels in the biological samples. The microarray data are analysed using specific softwares that enables clustering of genes with similar expression patterns, assuming that they share common biological functions.

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