Immunological techniques in helminthology.pptx 2023-MPHIL-2047.pptx

Techniques

1. Immunological techniques in helminthology Manahil riaz 2023-Mphil-2047 1 st semester Applied helmintholgy

2. Immunological techniques in helminthology Helminthology is the study of parasitic worms (helminths), and immunological techniques play a crucial role in understanding host-parasite interactions and developing diagnostic tools and treatments. Some key immunological techniques used in helminthology include: ELISA WESTERN BLOT IMMUNOPRECIPITATION INDIRECT FLOURESCENT ANTIBODY TEST POLYMERASE CHAIN REACTION (PCR) These techniques help researchers understand the immune response to helminth infections, identify potential diagnostic biomarkers, and develop effective treatments and vaccines.

3. Immunological techniques in helminthology Immunodiagnosis means diagnosis of diseases or organisms based on anti gen-antibody reactions in the body of diseased person. There is the requirements of various reagents for the immunodiagnosis of any disease: Antigen Antibody Label/ particle to visualize antigen-antibody reaction For thediagnosis of helminth: we require, parasite antigen, antibody against parasite antigen as major requirements.

4. Immunological techniques in helminthology The Enzyme-Linked Immunosorbent Assay (ELISA) is a commonly used diagnostic test for detecting various types of parasitic infections by detecting specific antibodies or antigens produced by the immune system in response to the parasite. How ELISA Works for Parasite Infections : Antigen or Antibody Capture : ELISA tests can detect either antigens (proteins produced by the parasite) or antibodies (immune proteins produced by the host in response to the parasite). Coating: A specific antigen or antibody is coated onto the surface of a microplate well. Incubation: The patient's serum or other bodily fluids containing antibodies or antigens is added to the wells and allowed to incubate. If the sample contains the target antigen or antibody, it will bind to the coated molecules on the plate.

5. Immunological techniques in helminthology Washing : The plate is washed to remove any unbound substances. Detection : An enzyme-linked secondary antibody or antigen is added, which binds to the captured antigen or antibody. Substrate Addition : A colorimetric substrate is added, which reacts with the enzyme to produce a color change. Measurement : The intensity of the color change is measured spectrophotometrically, and it correlates with the amount of antigen or antibody present in the sample.

6. Immunological techniques in helminthology Principle of ELISA : ELISA involves the following key steps: Coating: Antigens or antibodies are immobilized onto the surface of a microplate. Binding : The sample containing the target antigen or antibody is added to the wells of the microplate. If the target molecule is present, it binds to the immobilized antigen or antibody. Detection: A secondary antibody or antigen, labeled with an enzyme (such as horseradish peroxidase or alkaline phosphatase), is added. This enzyme reacts with a substrate to produce a detectable signal (e.g., color change). Measurement : The intensity of the signal is measured spectrophotometrically, and it correlates with the amount of target molecule present in the sample

7. Immunological techniques in helminthology Types of ELISA: Direct ELISA : Detects antigens directly using labeled antibodies. Indirect ELISA : Detects antibodies indirectly using labeled secondary antibodies. Sandwich ELISA : Detects antigens by sandwiching them between two antibodies. Competitive ELISA : Measures the concentration of an unlabeled antigen by competition with a labeled antigen for binding to a limited amount of antibody.

8. Immunological techniques in helminthology Polymerase Chain Reaction (PCR) is a powerful molecular biology technique used to amplify specific DNA sequences. PCR allows researchers to produce millions of copies of a particular DNA segment from a small initial sample, enabling various downstream applications such as DNA sequencing, cloning, and genetic analysis

9. Immunological techniques in helminthology Principle of PCR: Denaturation : The double-stranded DNA template is heated to around 94-98°C, causing the DNA strands to separate and denature into single strands. Annealing : The reaction temperature is lowered to around 50-65°C, allowing specific DNA primers to anneal to complementary sequences on the single-stranded DNA template. Extension : The temperature is raised to around 72°C, and a heat-stable DNA polymerase (such as Taq polymerase) synthesizes new DNA strands by extending from the primers. Repeat Cycles : The denaturation, annealing, and extension steps are repeated for multiple cycles (typically 20-40 cycles), resulting in exponential amplification of the target DNA sequence.

10. Immunological techniques in helminthology PCR Protocol : DNA Template : Start with a DNA template containing the target sequence to be amplified. Primers : Design and add specific oligonucleotide primers that flank the target sequence. Nucleotide s: Add deoxynucleotide triphosphates (dNTPs) as building blocks for DNA synthesis. DNA Polymerase : Include a heat-stable DNA polymerase (e.g., Taq polymerase) that can withstand the high temperatures required for denaturation. Buffer : Provide an appropriate buffer solution to maintain the optimal pH and ionic conditions for the PCR reaction. Thermal Cycler : Perform PCR in a thermal cycler that can precisely control the temperature cycling required for denaturation, annealing, and extension

11. Immunological techniques in helminthology Types of PCR : Conventional PCR : Standard PCR method involving denaturation, annealing, and extension cycles. Used for general amplification and cloning purposes. Real-Time PCR (qPCR): Allows real-time monitoring of DNA amplification using fluorescent dyes or probes. Enables quantification of initial DNA concentration and gene expression analysis. Reverse Transcription PCR (RT-PCR): Converts RNA into complementary DNA (cDNA) using reverse transcriptase enzyme before PCR amplification. Used for gene expression analysis and detecting RNA viruses. Nested PCR: Involves two rounds of PCR amplification using two sets of primers. Enhances specificity and sensitivity, useful for detecting low-abundance targets. Multiplex PCR : Amplifies multiple target sequences in a single reaction using multiple primer sets. Used for simultaneous detection of multiple pathogens or genetic markers. Digital PCR ( dPCR ): Divides the PCR reaction into thousands of separate partitions, allowing absolute quantification of DNA targets without standard curves

12. Immunological techniques in helminthology Western blot is a widely used laboratory technique in molecular biology and biochemistry that is employed to detect specific proteins in a sample Sample Preparation : The first step involves preparing a protein sample from tissues or cells. The sample is usually lysed to break open the cells and release the proteins. Gel Electrophoresis : The proteins in the sample are then separated based on their size using gel electrophoresis. Typically, a polyacrylamide gel is used in a process called SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). SDS is a detergent that denatures the proteins and gives them a uniform negative charge, allowing them to be separated by size when an electric current is applied.

13. Immunological techniques in helminthology Transfer to Membrane : After separation, the proteins are transferred from the gel onto a membrane (usually made of nitrocellulose or polyvinylidene difluoride (PVDF)). This step is done because the membrane provides a solid support that can be easily handled and probed. Blocking : To prevent non-specific binding of antibodies to the membrane, it is incubated with a blocking solution (such as non-fat dry milk or bovine serum albumin) that covers the surface of the membrane. Antibody Incubation: Primary Antibody : The membrane is incubated with a primary antibody that specifically binds to the target protein. Secondary Antibody : After washing away unbound primary antibodies, the membrane is incubated with a secondary antibody that recognizes and binds to the primary antibody. This secondary antibody is typically conjugated to an enzyme, such as horseradish peroxidase (HRP), or a fluorescent dye, which allows for detection

14. Immunological techniques in helminthology Detection : The target protein is visualized through a detection method appropriate for the secondary antibody. If the secondary antibody is enzyme-conjugated, a substrate is added that produces a detectable signal (such as a chemiluminescent or colorimetric reaction). For fluorescently labeled antibodies, the membrane is scanned using a fluorescence imaging system. Analysis : The signal detected corresponds to the presence and abundance of the target protein. The intensity of the bands on the blot can be quantified using software for more detailed analysis.

15. Immunological techniques in helminthology The Indirect Fluorescent Antibody Test (IFAT) is a serological assay used to detect antibodies against specific parasites in patient serum or other bodily fluids. IFAT is a valuable tool in diagnosing parasitic infections and assessing the immune response to these pathogens.

16. Immunological techniques in helminthology IFAT can be used to detect antibodies against a wide range of parasitic pathogens, including: Protozoa : Such as Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp., and Plasmodium spp. (malaria parasites). Helminths : Including various species of helminths like Schistosoma spp. (blood flukes), Fasciola spp. (liver flukes), and Echinococcus spp. (tapeworms). Arthropods : Some IFAT tests can also detect antibodies against arthropod-borne parasites, such as Leishmania spp. transmitted by sandflies.

17. Immunological techniques in helminthology References Bowman, D. D. (2020). " Georgis ' Parasitology for Veterinarians." Elsevier Zajac, A. M., & Conboy, G. A. (2012). "Veterinary Clinical Parasitology." John Wiley & Sons Jacobs, D. E., Fox, M. T., & Gibbons, L. M. (2007). "Principles of Veterinary Parasitology." Wiley-Blackwell. Soulsby , E. J. L. (1982). "Helminths, Arthropods and Protozoa of Domesticated Animals." Baillière Tindall. Harvey, J. W. (2012). "Veterinary Hematology: A Diagnostic Guide and Color Atlas." Elsevier Health Sciences. Taylor, M. A., Coop, R. L., & Wall, R. L. (2015). "Veterinary Parasitology." John Wiley & Sons.

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19. Immunological techniques in helminthology