ELISA and Lateral Flow Assays PDF

## ELISPOT vs ELISA - **ELISPOT PLATE contain PVDF membrane while ELISA don't.** - **ELSPOT end product is precipitate while its solution for ELISA.** - ELISPOT will yield information about number of cells secreting cytokine of interest (e.g. n/1 million cells). - ELISA will yield information o...

## ELISPOT vs ELISA - **ELISPOT PLATE contain PVDF membrane while ELISA don't.** - **ELSPOT end product is precipitate while its solution for ELISA.** - ELISPOT will yield information about number of cells secreting cytokine of interest (e.g. n/1 million cells). - ELISA will yield information on the Concentration of the cytokine of interest produced by all cells in the culture (e.g. pg/1 ml of supernatant). - ELISA gives you information about how much cytokine is produced and secreted at a given point of time, but no information about how many cells are secretors of the cytokine. - In ELISPOT there is possibility of detecting parallel secretion of 2 proteins by the same cell. - In ELISA it is not possible to detect secretion of 2 proteins by the same cell parallely. ## FACS This image depicts a flow cytometer (FACS). FACS separates cells based on their fluorescent properties. - The cells are first stained with fluorescent dye. - The cells are then passed through a laser beam. - The fluorescent light emitted by the cells is detected by a detector. - The signal from the detector is used to sort the cells into different populations. The image shows a diagram of a flow cytometer, with a funnel leading to a tube of fluid droplets with several cells. - To the left of the funnel is a large cell labelled “fluorescent dye”, and below a small cell labelled “electromagnets” with arrows pointing up to the fluid droplets at the bottom. - On the right side of the funnel is a cell labelled “detector” with a cell below labelled “fluid droplet”. - Between the funnel and the detector is a box labelled “Laser”, with a red line labelled “90°” stretching from the laser box to the fluid droplet above. - There are two cells labelled, “Detector” inside the box, one with the label “FSC” and the other with the label “SSC”. - The cells are sorted based on their size (FSC) and granularity (SSC). At the bottom are two tubes labelled, “Negative Sample” and “Positive Sample”. The “Negative Sample” tube has two dark cells at the bottom, while the “Positive Sample” tube has two light colored cells at the bottom. ## FACS DOT PLOT This image shows FACS dot plots. - The dot plots shown are two-dimensional plots that show the relationship between two parameters, such as side scatter (SSC) and forward scatter (FSC). - Different cell populations can be identified based on their location on the dot plot. - For example, the dot plot in (A) shows lymphocytes and monocytes. ## Lateral Flow ELISA - Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. - Instantaneous diagnosis directly to patients, results are displayed within 5-30 min. - For the qualitative and quantitative detection of specific antigens and antibodies, as well as products of gene amplification (HIV-1 RNA, MDR TB). - Further industries in which LFA-based tests are employed include: - Veterinary medicine (bovine antibody to Anaplasma marginale) - Quality control (detection of Schistosoma circulating anodic antigen) - Product safety in food production (detection of shiga toxins) - Environmental health and safety (detection of bisphenol A (BPA) in aqueous samples) - In these areas of utilization, rapid tests are used to screen for: - Animal diseases - Pathogens - Chemicals - Toxins - Water pollutants - A variety of biological samples can be tested using LFAs, including urine, saliva, sweat, serum, plasma, whole blood and other fluids. ## Types of Lateral Flow Assays Based on the type of recognition element, lateral flow assays can be categorized as: - **LFIA (lateral flow immunoassay):** Antibodies are exclusive recognition element. - **NALFA (nucleic acid lateral flow assay):** Recognition element includes nucleic acids. ## One Step hCG Urine Test This image shows a one-step hCG urine test. - The test uses a lateral flow assay to detect the presence of hCG in urine. - The test consists of a sample pad, a membrane, and a control line. - The sample pad contains the analyte (hCG), and the conjugate pad contains antibodies conjugated to gold nanoparticles (anti-anal (anti-analyte Ab). - The membrane contains a test line, and the control line contains a control antibody (anti-IgG Ab). - Urine is applied to the sample pad. - The hCG in the urine binds to the monoclonal antibodies conjugated to the gold nanoparticles, and the complex migrates along the membrane. - If hCG is present, it will bind to the antibodies in the test line, causing a red line to appear. - The control line will always appear, regardless of whether hCG is present, and is intended to indicate that the test has been performed correctly, i.e. that the test is working properly and the sample is correctly flowing through the membrane. - The test line can be interpreted as either positive, negative,or weak positive. - Positive (pregnancy) - a second red line in the test area indicates that the test has detected the presence of HCG in your urine. - Negative (no pregnancy) - only a single red line in the control area, no line in the test area indicates that no HCG was detected in your urine, therefore you’re not pregnant. - Weak positive (test should be repeated)- a faint line in the test area indicates that there may be a trace amount of hCG in your urine. This can sometimes happen if you are very early in your pregnancy, or if you have a medical condition that can affect hCG levels. You should repeat the test with a fresh urine sample a few days later to confirm the results. ## Detection Methods Used in Lateral Flow Assays This image shows the different detection methods used in lateral flow assays. - **Labels:** - **Color labels** - **Quantitative:** Optical strip readers or camera (with imaging software) for measurement of the intensity of colours produced at test and control line - **Semi-quantitative:** Visual inspection of ladder bar, where the number of coloured lines is an indication of analyte concentration - **Qualitative:** Visual inspection of line colours - **Fluorescent labels** - **Quantitative:** Fluorescent strip reader, recording fluorescence intensity - **Semi-quantitative:** - **Qualitative:** - **Other labels** - **Quantitative:** Magnetic strip readers; Electrochemical detectors; Chemiluminescence readers - **Semi-quantitative:** - **Qualitative:** ## Advantages and Disadvantages of Using Lateral Flow Assays This image shows the advantages and disadvantages of using lateral flow assays (LFAs). - **Advantages:** - One-step assay, no washing steps necessary - Fast and low cost, low sample volume - Qualitative (on/off) or semi-quantitative result - Relatively short development time brings applications faster to the market - Simple test procedure - Applications at point of care/need - Proteins, haptens, nucleic acids and amplicons can be detected - Individual tests can be used or an array format for the mid-throughput screening - Long shelf life, no need to refrigerate, batches can be prepared in advance - For fluid samples, pretreatment is often not necessary - **Disadvantages:** - One-step assay - Inaccurate sample volume reduces precision - Restriction on total volume in test gives a limit of sensitivity - No possibility to enhance the response by enzyme reaction - Good antibody preparation is obligatory - Analysis time is dependent on nature of sample (e.g., viscosity) - Obstruction of pores due to matrix components - Sample pretreatment is needed for non-fluid samples ## Lateral Flow ELISA This image shows an example of lateral flow ELISA. - The test line contains antibodies against the analyte, and the control line contains anti-IgG antibodies. - When a sample containing the analyte is applied to the sample pad, the analyte binds to the antibodies conjugated to gold nanoparticles. - The complex migrates along the membrane, and if the analyte is present, it will bind to the antibodies in the test line, causing a red line to appear. - The control line will always appear, regardless of whether the analyte is present, indicating that the test has been performed correctly. - The test can be interpreted as either positive, negative, or weak positive, based on the presence or absence of a line in the test area. - The image shows a sample that is positive for the analyte, as a second red line appears in the test area.