Immunodiagnostics Lecture Notes 20-21 PDF

Immunodiagnostics Suggested Reading for Lectures 20-21: Online sources for specific tests Cellular & Molecular Immunology, Appendix III Kuby Immunology, Chapter 6 MICRG 1553 Immunology Kathryn Leyva, Ph.D. Learning Objectives ⚫ Understand the difference b...

Immunodiagnostics Suggested Reading for Lectures 20-21: Online sources for specific tests Cellular & Molecular Immunology, Appendix III Kuby Immunology, Chapter 6 MICRG 1553 Immunology Kathryn Leyva, Ph.D. Learning Objectives ⚫ Understand the difference between qualitative and quantitative PCR reactions ⚫ Compare/contrast the procedures for each molecular assay ⚫ Interpret results given ⚫ Understand the meaning of a CT value ⚫ Define: sensitivity, specificity, titer, serology ⚫ Calculate sensitivity and specificity ⚫ Determine titers ⚫ Describe the general principle & basic technique of each immunoassay discussed ⚫ Compare/contrast precipitation & agglutination reactions ⚫ Compare/contrast each labeled immunoassay ⚫ Understand the differences between direct and indirect immunoassays ⚫ Identify primary and secondary antibodies in these immunoassays ⚫ Interpret results for each assay covered: ⚫ Double immunodiffusion ⚫ Latex agglutination, hemagglutination, & hemagglutination inhibition ⚫ ELISAs, lateral flow assays, & Western blots (Immunoblot) ⚫ Immunohistochemistry, immunofluorescence assays & flow cytometry 2  Introduction: Diagnostics ⚫ Diagnosis = the process of identifying a disease, condition, or injury based on a patient’s signs and symptoms ⚫ Diagnostic assays are often used to help make a diagnosis ⚫ They are often divided into whether they are direct or indirect assays: ⚫ Direct ⚫ Indirect assays: assays: detects the detects the pathogen or immune pathogen response components (typically) to a pathogen or other antigen https://www.nature.com/articles/nrmicro2459 3 Choosing the Appropriate Diagnostic Assay ⚫ As a general rule, the choice of assay will depend on the stage of disease ⚫ Example: diagnosis of COVID-19 ⚫ Antigen-based tests to detect SARS-CoV-2 are typically positive earlier in the course of infection ⚫ Virus is actively replicating ⚫ Later in the disease process and/or as the patient recovers, serology to detect antibodies to SARS-CoV-2 will be detected if the patient was infected ⚫ At this point, antigen tests will likely be negative as the virus is being/has been eliminated by https://www.siemens-healthineers.com/laboratory-diagnostics/assays-by-diseases-conditions/infectious-disease-assays/serology-testing-for-covid-19 the immune system 4 Sensitivity & Specificity of Diagnostic Tests ⚫ Sensitivity and specificity are two performance measures for many diagnostic tests ⚫ Sensitivity = percentage of people who have the disease and test positive ⚫ A test with a high sensitivity will yield very few false negatives ⚫ Specificity = percentage of people who do not have the disease and test negative ⚫ A test with a high specificity will yield very few false positives Sensitivity Specificity Definition % of pts with a % of pts without disease that test disease that test positive negative Value of 100% Test correctly Test correctly identifies every identifies every pt with the pt without the disease disease Outcome True Positive True Negative https://www.osmosis.org/learn/Sensitivity_and_specificity 5 Sensitivity & Specificity of Diagnostic Tests ⚫ Calculating sensitivity & specificity of a diagnostic test: ⚫ Sensitivity = true positives over the total number of sick individuals (true positives + false negatives) ⚫ Specificity = true negatives over the total number of healthy individuals (true negatives + false positives) The reported sensitivity and specificity of this COVID-19 antigen test varies based on several factors: Age of patient Presence or absence of symptoms Day test is taken after symptom onset https://www.medwave.cl/revisiones/metodinvestreport/8432.html?lang=en 6 Sensitivity & Specificity of Diagnostic Tests ⚫ Sensitivity and specificity are inversely proportional to one another ⚫ To increase the sensitivity of a test, often its specificity will be reduced (and vice versa) ⚫ This is illustrated in this figure: C = cut-off value for a given diagnostic test CL = 100% sensitivity CU = 100% specificity https://hdsr.mitpress.mit.edu/pub/fx7g21em/release/2 7 Diagnostic Assays ⚫ There are a wide variety of diagnostic assays available for screening a population for the presence of antibodies, identification of infectious agents, evaluating the course of an infection, and diagnosis of various diseases ⚫ In this course, we will focus on: ⚫ Molecular assays: detection of genetic material from a pathogen ⚫ Immunoassays: detection of pathogen-specific antibodies or antigens ⚫ These tests are often referred to as serological assays or antigen-antibody assays https://incacare.live/innovations-infectious-disease-testing-technologies/ https://peerj.com/articles/10180/ 8 Molecular Assays ⚫ Many molecular assays used in the diagnostic laboratory are based on polymerase chain reaction (PCR) amplification of DNA from a pathogen ⚫ PCR can be performed directly from patient specimens (or from isolates) ⚫ The goal is to amplify the nucleic acid of interest to a detectable limit ⚫ There are different PCR methods to amplify DNA, depending on the desired information: ⚫ Qualitative: detection of pathogen DNA or RNA - a “yes” or “no” result ⚫ Conventional PCR: Amplification of DNA ⚫ Reverse-transcriptase PCR (RT-PCR): Amplification of DNA after converting RNA to DNA ⚫ Quantitative: determination of the amount of DNA or RNA from the pathogen ⚫ Quantitative PCR (qPCR): Amplification and quantitation of the amount of DNA ▪ Note: Quantitative PCR is also called “real-time” PCR, not to be confused with reverse-transcriptase PCR!! ⚫ Quantitative, reverse-transcriptase PCR (qRT-PCR): Amplification and quantitation of the amount of DNA after converting RNA to DNA 9 Qualitative PCR Reactions https://www.omegafilters.com/life_science/ PCR_and_RT-PCR_overview ⚫ PCR involves the Pathogen (Pathogen) amplification of DNA or extracted from a pathogen (or cells) ⚫ If the pathogen contains RNA as its genetic material, an additional step to convert the RNA into cDNA = complementary DNA using a reverse transcription process occurs ⚫ Specific primers bind to a ⚫ The DNA or cDNA is heated DNA sequence on the to separate the double- pathogen, and a DNA stranded DNA into single- polymerase enzyme stranded DNA synthesizes dsDNA 10  https://youtu.be/2KoLnIwoZKU Qualitative PCR Reactions Step 1: Denaturation of dsDNA Step 2: Primer annealing After 30-40 cycles, Step 3: DNA millions of synthesis DNA copies of the gene region of interest are generated! https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553/ 11 Clinical Application: RT-PCR to Detect SARS-CoV-2 ⚫ PCR amplification of SARS-CoV-2 spike protein gene ⚫ SARS-CoV-2 is an RNA virus, so the viral RNA is first converted to cDNA ⚫ Primers specific for the SARS-CoV-2 spike protein are used ⚫ Amplification of the spike protein gene generates millions of copies ⚫ Results are analyzed using agarose gel electrophoresis Primers used in this experiment amplified a 245 bp region of the SARS-CoV-2 spike protein gene; all four samples (1-4) show primer-specific amplification of https://asm.org/articles/2020/april/covid-19-testing-faqs the target gene sequence 12  Quantitative PCR Reactions ⚫ Quantitative PCR (qPCR) involves the amplification & quantification of DNA ⚫ Again, for RNA pathogens, RNA will be converted to cDNA by reverse transcription ⚫ PCR amplification of DNA occurs in a similar manner, except during each cycle, a fluorescent dye binds to dsDNA to measure DNA amplification The more dsDNA copies there are, the higher the fluorescent intensity; the fluorescent signal increases after each cycle! https://www.aatbio.com/catalog/real-time-pcr-qpcr Note: there are fluorescent probes that can be used instead 13 of a fluorescent dye, but we won’t discuss this variation  https://youtu.be/1kvy17ugI4w Quantitative PCR Reactions ⚫ Just as for PCR, 30-40 cycles of DNA amplification, and the amount of fluorescence after each cycle is measured & plotted = amplification curve ⚫ There is always a “baseline” level of fluorescence with these assays, even when running the reaction without any DNA = no template control ⚫ After several cycles, the amount of Amplification fluorescence detected starts to rise above baseline in an exponential manner ⚫ The threshold is the level of fluorescence that reflects a statistically significant increase over the baseline signal No amplification ⚫ As the number of cycles increase, eventually the fluorescent signal detected crosses the threshold value ▪ CT value = cycle number at which the fluorescent signal crosses the threshold https://theory.labster.com/sybr-qpcr-analysis/ CT = cycle threshold (also known as Cq = quantification cycle) 14 Clinical Application: qRT-PCR to Detect & Quantify SARS-CoV-2 Levels ⚫ qPCR amplification and quantification of SARS-CoV-2 gene(s) ⚫ Samples from patients are collected, viral RNA is converted to cDNA ⚫ Amplification of 1+ SARS-CoV-2 genes using target-specific primers; the amount of fluorescence is measured & plotted after each cycle ⚫ CT value is determined for each sample; the lower the CT value, the greater the amount of SARS- CoV-2 RNA is present in the patient sample Dutta et al. (2022) Diagnostics (Basel) 12(6): 1503 15 Immunoassays (Antigen-Antibody Assays) ⚫ Immunoassays are used to detect the presence, and often amount, of either antibody or antigen ⚫ Each test is designed to detect either an antigen (Ag) or an antibody (Ab) in a patient sample ⚫ There are many different formats and variations of these assays; many are described as direct or indirect assays: ⚫ Direct assays: ⚫ Often used for the detection of patient antigen in a sample; keep in mind that antibodies can be antigens too! ⚫ Indirect assays: ⚫ Often used for the detection of patient antibodies in a sample ⚫ Can be used to amplify the signal during detection of patient antigen in a sample https://azurebiosystems.com/blog/evolution-elisa-immunoassays/ 16 Immunoassays (Antigen-Antibody Assays) ⚫ There are many immunoassays to detect the presence of, and often quantify, either an antigen or an antibody in a patient sample. We will focus on: ⚫ Precipitation reactions – soluble antigen and antibody react, become insoluble and “fall out” of solution (precipitate), allowing for visualization ⚫ Agglutination reactions – antigen-antibody binding “clumps” together (agglutinates) an insoluble particle or cell, allowing for visualization ⚫ Labeled immunoassays – using a label that can emit a measurable signal (radioactivity, color change, etc.) is attached to an antibody or antigen, allowing visualization of the antigen-antibody interaction; labels include: ⚫ Radioactive nuclides ⚫ Enzymes ⚫ Fluorophores = fluorescent compounds 17 Precipitation Reactions Lattice formation ⚫ The reaction between soluble antigens and antibodies may result in precipitation due to lattice formation = large immune complexes ⚫ Many antigens are polyvalent, so polyclonal antibodies (antiserum) are often used ⚫ For precipitation to occur, there needs to be an optimal concentration of both antigen & antibody = zone of equivalence Kuby Immunology ⚫ Antibody or antigen excess results in small/tiny immune complexes that do not precipitate (or not very well!) ⚫ There are various ways to set up these assays; we will just focus on one: double immunodiffusion https://basicmedicalkey.com/antigen-antibody-reactions-in-the-laboratory/ 18 Double (Ouchterlony) Immunodiffusion Precipitin line forms here ⚫ Immunodiffusion assays can be used to detect either patient antigen or antibody ⚫ Antigen and antibody are placed in separate wells ⚫ Each sample diffuses; a precipitin line forms at the zone of equivalence between the antigen and antibody well, if positive https://basicmedicalkey.com/antigen-antibody-reactions-in-the-laboratory/ Patient Patient sample sample containing Ag containing Ag 1&3 1 only Reagent antibody specific for Ag 1 4 Roitt et al., Immunology 5th ed Fig 29-2 C Fig. 6-6 Kuby Immunology 19 Clinical Application: Diagnosis of Valley Fever ⚫ A double immunodiffusion assay can be used to detect the presence of antibodies in a patient sample ⚫ Example: Detection of antibodies against the fungal pathogen, Coccidioides, the causative agent of Valley Fever ⚫ This fungus is often hard to diagnose from respiratory secretions, so serology is often used; helps to avoid more invasive procedures! The patient’s serum is Fungal Fungal placed in the center well Control anti-fungal antibodies and known fungal antigens are added into the respective wells (see fig) After incubation, a precipitin line forms if the 4 patient has antibodies to the fungal antigen(s) https://health.ucdavis.edu/valley-fever/about-valley-fever/coccidioides-diagnostic-testing/index.html C 20 Agglutination Reactions ⚫ When antibodies crosslink the surface antigen of 2+ target cells or antigen-coated particles, agglutination can be observed ⚫ IgM antibodies are the best agglutinins; they are pentamers! ⚫ Agglutination reactions occur when the immune complex is formed with an antigen OR antibody that is insoluble ⚫ Agglutination of cells or latex beads ⚫ Reactions involving RBCs: hemagglutination Red Blood Cells Antibodies Hemagglutination https://courses.lumenlearning.com/suny-microbiology/chapter/agglutination-assays/ 21 Latex Agglutination ⚫ Latex agglutination is designed to detect the presence of antigen or antibodies in a patient sample, depending on the assay ⚫ Latex beads are bound to either an antigen or an antibody ⚫ If the corresponding antibody or antigen is present in the patient sample, visible agglutination of the latex beads can be seen on the test card Clinical Application: Meningitis Latex agglutination assays are available for the detection of meningitis-causing bacteria o Known antibodies are coated with latex beads and mixed with bacterial antigens extracted from a CSF sample o If the antigen is present in the patient CSF sample, agglutination occurs https://www.thermofisher.com/order/catalog/product/R30859502#/R30859502 22 Hemagglutination (HA) Assays ⚫ Blood typing: ⚫ Performed to type RBCs for ABO & Rh antigens ⚫ RBCs are mixed with anti-A, anti-B, or anti-Rh (D) on a slide, card, or wells (microtiter plate) ⚫ Agglutination = positive result ⚫ No agglutination = negative result Note: agglutination looks different when performed in wells vs on a slide/card! No agglutination agglutination https://medicaltechnology100.weebly.com/blood-typing.html 23  Hemagglutination (HA) Assays ⚫ Coombs/Antiglobulin tests ⚫ Coombs tests are used to detect the presence of serum antibodies against RBCs or against pre-coated RBCs (assay dependent). 1. Direct Coombs test ▪ Isolate RBCs from patient ▪ Add Coombs reagent = anti-human Ig ▪ Agglutination occurs if the patient has antibodies bound to their own RBCs 2. Indirect Coombs test ▪ Obtain serum from patient ▪ Mix donor RBCs with patient serum; antibodies (if present) bind RBCs ▪ Add Coombs reagent = anti-human Ig ▪ Agglutination occurs if the patient’s serum contains antibodies against the donor’s RBCs Ig = immunoglobulin 24 Immunoassays: Titers ⚫ Results of immunoassays can be recorded as positive or negative, but some assays are designed to allow quantification of the amount of antibody or antigen ⚫ Quantification can be an actual concentration or can be a relative concentration = titer ⚫ Titer is determined by serial dilution of a patient sample ⚫ Titer = inverse of the greatest dilution in which the sample still yields a positive test result ⚫ Important note: Titers can be determined for both antibodies or antigens, depending on the immunoassay; the above figure illustrates how to determine an antibody titer 25 Determination of Hemagglutination (HA) Titers ⚫ HA can be used to determine the titer of antibody in a patient sample ⚫ Serial dilution of patient sample ⚫ Mix with RBCs and observe reaction in each well ⚫ Titer = inverse of the last dilution in the series that yields a positive result 26  https://virologyresearch services.com/2023/04/ Hemagglutination Inhibition (HI) 07/understanding-the- hai-assay/ ⚫ HI is used to determine the presence, and often titer, of antibodies against pathogens that naturally agglutinate RBCs 1. Serial dilution of patient sample containing suspected antibodies 2. Add virus specific for suspected antibodies 3. Add RBCs that the virus would naturally agglutinate Interpretation of results: o If antibodies are NOT present in the patient sample (B): viruses bind and agglutinate the Control RBCs = hemagglutination = negative test result o If antibodies ARE present in the patient Negative test sample (C): viruses bind patient antibodies result and are unable to agglutinate the RBCs = inhibition of hemagglutination = positive Positive test result test result https://www.cdc.gov/flu/about/professionals/antigenic.htm 27  Positive HI reaction: Clinical Application: HI for Influenza ⚫ Hemagglutination inhibition (HI) is a common assay performed to diagnose influenza virus infections ⚫ Influenza viruses have hemagglutinin, an outer envelope protein, that binds to sialic acid receptors on host epithelial cells, but also binds to and naturally agglutinates RBCs https://link.springer.com/protocol/ 10.1007/978-1-0716-0346-8_2 HI Assay for H1N1 What is the patient’s https://micro.magnet. fsu.edu/cells/viruses/ antibody titer at day 10? influenzavirus.html At day 24? At day 42? Days after infection Is the titer increasing or decreasing over time? ⚫ This HI assay is used to determine an antibody titer against the influenza virus Modified from: https://wwwnc.cdc.gov/ ⚫ A positive test result = inhibition of hemagglutination eid/article/16/2/09-1733-f1 Titer 28 Labeled Immunoassays ⚫ These assays use a detection label to detect either patient antigen or antibody, depending on how the assay is set up ⚫ There are commonalities in how these labeled assays are performed; what is different is the label and the method of detection. Common labels: ⚫ Enzymes (e.g., horseradish peroxidase, alkaline phosphatase) ⚫ Fluorophores (e.g., fluorescein isothiocyanate (FITC), rhodamine) ⚫ Radioactive nuclides (e.g., 125I, 3H) ⚫ Labeling an antibody or antigen allows for Antigen visualization or quantification of the antigen- Primary antibody reaction with the use of proper antibody instruments Secondary antibody ⚫ Direct assays: labeled primary antibody Label ⚫ Indirect assays: labeled secondary antibody https://www.abcam.com/secondary-antibodies/direct-vs-indirect-immunofluorescence 29 ELISA: Enzyme-Linked Immunosorbent Assays ⚫ ELISAs utilize enzyme-labeled antibodies to detect & quantify antigens or antibodies in a patient sample; serial dilutions can be done to determine titer(s) ⚫ Direct ELISA: Detection of patient antigen; labeled primary antibody ⚫ Indirect ELISA: Detection of patient antibody; labeled secondary antibody ⚫ Sandwich ELISAs: Can be set up as a direct or indirect assay ⚫ The enzyme and/or substrate can vary, resulting in either a color change or emittance of light that is measured; we will focus on color change 30 Direct ELISA ⚫ Steps of a Direct ELISA – detection of patient antigen: 1. Wells are coated with a patient sample; if antigen is present, it binds to the well 2. Add an enzyme-linked primary antibody that binds the patient antigen 3. Add a substrate for the enzyme; cleavage of the substrate is indicated by a color change ⚫ The intensity of the color is proportional to the amount of patient antibody ⚫ Direct ELISA is not often used in diagnostics as they are more expensive, less sensitive, and not as flexible than indirect ELISA or direct/indirect sandwich ELISAs https://www.aatbio.com/resources/faq-frequently-asked-questions/What-is-a-Direct-ELISA 31 Indirect ELISA ⚫ Steps of an indirect ELISA – detection of patient antibody: 1. Wells are coated with a known antigen 2. Add patient sample to the wells; if antibodies to the antigen are present, they will bind These are the unlabeled primary antibodies in the figure 3. Add an enzyme-linked secondary antibody that binds to the patient’s (primary) antibody 4. Add a substrate for the enzyme; cleavage of the substrate is indicated by a color change ⚫ The intensity of the color is proportional to the amount of patient antibody https://www.aatbio.com/resources/faq-frequently-asked-questions/What-is-an-Indirect-ELISA 32 Clinical Application: Lyme Disease Diagnosis ⚫ The initial test performed to diagnose Lyme disease, due to infection with Borrelia burgdorferi is often an indirect ELISA to detect anti-Borrelia antibodies in a patient Bacterium Surface proteins extracted from Patient Borrelia antibody These two figures are FYI – no need to memorize! https://www.integra-biosciences.com/china/en/blog/article/types-of-elisa-tests https://thenativeantigencompany.com/lyme-disease-a-rapidly-emerging-bacterial-infection-in-need-of-new-diagnostics/ 33 Direct & Indirect Sandwich ELISAs ⚫ Steps of Sandwich ELISAs – detection of patient antigen or antibody: 1. Wells are coated with a capture antibody https://www.antibody-creativebiolabs.com/sandwich-elisa-with-direct-detection.htm 2. Add antigen specific for the capture antibody ▪ Direct: Antigen from a patient sample ▪ Indirect: Either antigen from a patient sample or Secondary antibody known antigen provided in the test kit conjugate Primary antibody 3. Add antibody/antibodies: conjugate Primary detection ▪ Direct: Enzyme-labeled primary antibody antibody ▪ Indirect: Non-labeled primary detection antibody plus an enzyme-labeled secondary antibody ▪ Primary detection antibody could be from the patient sample or provided in the kit 4. Add a substrate for the enzyme; cleavage of the substrate is indicated by a color change ⚫ The intensity of the color is proportional to the amount of patient antibody 34  Fig 20-9 Kuby Immunology Clinical Application: HIV Testing HIV Anti-p24 proteins antibodies HIV p24 Anti-HIV antigen in Human Immunodeficiency Virus (HIV) antibodies in patient’s patient’s blood blood ⚫ Sandwich ELISAs Labeled are used to detect Labeled HIV anti-HIV antibodies both HIV antibodies antigens & HIV-specific proteins from a blood sample during HIV https://primeglobalpeople.com/2017/06/27/principles-science-behind-hiv-testing/ testing 35 Clinical Application: Lateral Flow Assays ⚫ Lateral flow assays are modified ELISAs that are designed as rapid, easy to use, point of care screening assays to detect patient antigen or antibody ⚫ Procedure (see figure; illustrates a rapid COVID-19 antigen test): Numerous examples! Just a few: Rapid strep test Pregnancy test HIV antigen or antibody tests COVID-19 antigen test (pictured) https://lateralflows.com/lateral-flow-assays/ https://www.nanoditech.com/products-Nano-Check-TM-COVID-19-Antigen-Test/31 36 Immunoblot = Western Blot In the lab, immunoblots are routinely used to identify individual proteins Clinically, immunoblots are often used to detect the presence of patient antibodies to pathogen protein(s) Basics of the procedure: = patient antibody Proteins (antigens) are obtained from a pathogen or other cells; the target protein is specific to the patient antibody you are looking for https://www.mblbio.com/bio/g/support/method/westernblotting.html 37 Clinical Application: WB in Lyme Diagnosis ⚫ An immunoblot (Western blot) is one confirmatory test for Lyme disease ⚫ Detect specific IgM and/or IgG antibodies against several proteins purified from Borrelia burgdorferi, the organism that causes Lyme disease (see slide 33) Each band visualized indicates that the patient has antibodies to that specific bacterial protein FYI: 5+ IgG bands is considered positive These figures are FYI – no need to memorize! https://thenativeantigencompany.com/lyme-disease-a-rapidly-emerging-bacterial-infection-in-need-of-new-diagnostics/ FYI: The numbers are the molecular weights of the bacterial proteins 38 Immunohistochemistry (IHC) Assays ⚫ IHC identifies antigens expressed by cells, often in tissue sections ⚫ The label is either an enzyme or a fluorophore ⚫ IHC assays are typically set up as indirect assays: ⚫ A primary antibody against the tissue antigen is added, followed by an enzyme-labeled secondary antibody ⚫ Add enzyme substrate, which is cleaved to generate a colored product visible under the light microscope https://www.biorender.com/template/ immunohistochemistry-ihc https://www.biomol.com/resources/applications/immunohistochemistry/ 39 Clinical Application: Diagnosis of Her2+ Cancer Cells ⚫ The HercepTestTM is an indirect IHC assay used to detect Her2 protein overexpression in some types of cancer (e.g., breast cancer) ⚫ Her2 = growth factor receptor ⚫ Tissue sections prepared from biopsied tissues are fixed and stained using an anti-Her2 primary antibody followed by an enzyme-conjugated secondary antibody https://www.uspharmacist.com/article/herceptest-for-the-detection-of-her2- protein-overexpression-in-breast-and-gastric-cancers https://blog.dana-farber.org/insight/2016/03/what-is-her2-positive-breast-cancer/ 40 Immunofluorescence (IF) Assays ⚫ Direct & indirect IF assays use fluorophore-labeled antibodies to detect & quantify antigens or antibodies in a patient sample ⚫ Direct IF assays: Detection of patient antigen; labeled primary antibody ⚫ Indirect IF assays: Detection of patient (primary) antibody; labeled secondary antibody ⚫ FYI: Also can be set up to detect patient antigen (similar to IHC); amplifies the signal https://www.ptglab.com/news/blog/want-to-upgrade-your-immunofluorescence-workflow-go-direct/ 41 Clinical Application: Diagnosis of Parasitic Infections ⚫ A direct IF assay can be used to detect the presence of Direct IF parasites within the stool ⚫ Fluorescently-labeled primary antibodies against Giardia (red arrows) and Cryptosporidium (yellow arrows) were added; visualize under a fluorescent microscope https://www.cdc.gov/dpdx/cryptosporidiosis/index.html https://di.uq.edu.au 42 Clinical Application: Detection of Anti-Nuclear Antibodies ⚫ The antinuclear antibody test (ANA) is an indirect IF assay used to aid diagnosis of some autoimmune diseases https://www.nature.com/articles/nrrheum.2017.74 ⚫ Patient serum is added to cells on a slide; if the patient has anti-nuclear antibodies, they will bind to the nuclear antigens ⚫ Add fluorescently-labeled secondary anti-IgG antibodies ⚫ Visualize under a fluorescent microscope ⚫ FYI: The pattern of nuclear staining can aid in diagnosis http://www.cyberounds.com/cmecontent/art380.html?pf=yes 43 Flow Cytometry ⚫ Cells are bound (stained) with fluorescent-labeled antibodies ⚫ Antibodies used are specific for (bind to) cell- specific surface markers, such as CD3, CD4, CD8, CD19, CD56, etc ⚫ Each antibody is labeled with a different fluorophore ⚫ Allows for identification of different cell types ⚫ Cells pass through a laser that reads the fluorescent signal, and each cell is counted and plotted on a graph ⚫ X & Y axes = fluorescence intensity of each fluorophore (see next slide) ⚫ If cells are also separated, the procedure is known as fluorescence-activated cell sorting (FACS) https://www.mdpi.com/2227-9059/9/11/1613 44 Flow Cytometry ⚫ The computer generates a dot plot where each dot represents a single cell ⚫ In this example, FITC-labeled (yellow) and PE- labeled (pink) antibodies were added to a mixture Antibody to marker A of cells, then the cells were analyzed using flow cytometry ⚫ Each antibody is specific for a different cell surface marker ⚫ As each cell passes by the laser, the fluorescence color and intensity on each cell is detected and plotted within one of 4 quadrants (see fig) Antibody to marker B ⚫ LL: cells negative for both markers ⚫ UL: cells positive for marker A (y-axis) ⚫ LR: cells positive for marker B (x-axis) ⚫ UR: cells positive for both markers https://www.miltenyibiotec.com/US-en/resources/macs-handbook/macs-technologies/flow- cytometry/flow-cytometry-basics.html#gref 45  Clinical Application: Analysis of T Cell Subsets ⚫ Flow cytometry can be used to analyze T cell subsets in different tissues ⚫ Lymphocytes were isolated from the blood (PBMCs) & the GI mucosa (MMCs) from HIV- positive patients and flow cytometry was performed using labeled anti-CD4 and anti-CD8 antibodies ⚫ Which tissue (blood or GI mucosa) is showing lower numbers of helper T cells? https://www.prn.org/index.php/progression/ article/hiv_1_gastrointestinal_galt_267 PBMCs = peripheral blood mononuclear cells MMCs = mucosal mononuclear cells 46 Summary ⚫ Titer = relative concentration; the reciprocal of the last dilution of antiserum (or antigen) causing a measurable effect in an immunoassay Know how to determine titers for patient samples No agglutination Agglutination Titer = inverse of the highest dilution, but you’ll sometimes see people reporting the titer Know how to interpret as 1:1000 instead of 1000! positive and negative test results for all assays 47 Summary Know how to interpret positive and negative test ⚫ Immunoassays: results for all assays ⚫ Precipitation reactions (know examples) ⚫ Soluble antigen + soluble antibody diffuse toward each other ⚫ Precipitation at zone of equivalence; gives a precipitin line or ring ⚫ Agglutination reactions (know examples) ⚫ Either antigen or antibody is insoluble (cell, latex bead, etc) causing agglutination ⚫ Assays can be set up to look for agglutination or inhibition of agglutination (often for RBCs) ⚫ Labeled immunoassays (know examples) ⚫ Use antibodies conjugated with a label for detection ▪ Enzymes for ELISAs, WB, and IHC ▪ Fluorophores for WB, IHC, IFA, DFA, and flow cytometry ⚫ Labels are on the primary or secondary antibodies, depending on the specifics of the assay ▪ Three main forms of ELISA: direct, indirect, and sandwich (direct and indirect) ▪ WB & IHC typically use enzyme-labeled or fluorophore-labeled antibodies ▪ Flow cytometry counts and plots different cell types; FACS also sorts cells 48  Midwestern Wellness Support Professional school can be difficult, we have resources Midwestern University Student Counseling Academic Live Care Please email to request an appointment: https://midwestern.myahpcare.com/telehealth [email protected] To register: Use your midwestern.edu email Service Key: ALC In the Moment Mental Health Counseling When making appointments, there will be a 24/7 live telephonic counseling when you need to speak to price listed. 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