Immunology - Flow Cytometry and Enzyme Immunoassay - Lab Report Example

?Experiment on Sandwich ELISA and Flow Cytometry ELISA is a laboratory technique commonly used to measure the analyte concentration (normally antigens or antibodies) in solution. Enzyme immunoassay (EIA) or ELISA is different from other antibody-based assays due to the non-specific and specific interaction separation which takes place through serial binding to a solid surface, normally a polystyrene multiwell plate, and also it is because quantitative results can be attained. Flow Cytometry is a technique with automatic sorting devices that is able to sort successive streams of droplets into various fractions based on the type of fluorescence emitted by the droplet (Lee et al,1999).In this experiment, sandwich ELISA and Cytometry were employed; For sandwich ELISA, it was used to measures the antigen quantity between two antibody layers. On the other hand flow Cytometry was employed to analyze the biological material by detection of light-absorbing or a cell fraction that passes through a laser beam narrow stream. Introduction By definition an immunoassay is a biochemical test employed to measure the substance level in a biological liquid, such as urine or serum by means of antibody- antigen reaction (Borrebaeck, 2000). Monoclonal antibodies are used more often due to their binding ability to one unique site of the antigen molecule, thus providing a more specific and accurate test. (Borrebaeck ,2000). All ELISAs are based on specific interaction between an epitope, a three dimensional or a small linear ‘‘amino acids’’ sequence on an antigen and a matching antibody binding site. The antibodies employed in an ELISA can either be monoclonal (generated from hybridomas cells and have the ability to bind specifically to a unique epitope) or polyclonal (antibodies pool purified from animal sera that are able to bind to multiple epitopes).It is possible to use both polyclonal and monoclonal antibodies in an ELISA. Polyclonal antibodies are however typically used in indirect ELISAs for the secondary detection, while monoclonal antibodies are typically used for capture or primary antigen detection. (Borrebaeck CAK ,2000). ELISA is among the available sensitive immunoassays. The typical range of detection is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity based on a specific antibody –antigen interaction properties. Furthermore, some substrates like those which yield enhanced fluorescent or chemiluminescent signal can be employed to improve on the results. It is known that detection which is indirect can generate higher signal levels and therefore should be more sensitive. However, it can as well result to background signal that is higher hence reducing the net specific signal levels. With regards to Sandwich ELISA which was carried out in this experiment. Matched antibodies pairs were used, in which each antibody is specific for an antigen molecule with unique, non-overlapping epitopes. The antibody, referred to as capture antibody, is coated first to the polystyrene plate. Next, the sample or analyte is added to the wells. The detection antibody, a second antibody layer, is added next after this step. This is for measuring the analyte concentration. ‘‘Polyclonal antibodies’’ can as well be used for detection and capture in a sandwich ELISA only if the variability is present in the polyclonal antibodies to enable both detection and capturing the analyte through various epitopes. The assay is referred to as direct sandwich ELISA if there is conjugation of detection antibody to an enzyme. On the other hand, it can be referred to as indirect sandwich ELISA if the detection antibody is not labeled This assay type (sandwich ELISA) has advantages that include: It has a high specificity, because there are two antibodies employed. The antigen is captured and then detected. It is the most suitable essay for complex samples, consequently the antigen don’t need purification prior to measurement. The essay also is sensitivity and more flexibility, since both indirect and direct detection methods can be employed Flow Cytometry Fluidics System One of the flow cytometry fundamentals is it’s capacity to measure individual particles’ properties. Particles are distributed randomly in three-dimensional space following the injection of a sample in solution into a flow cytometer. Basing on this, a sample should be ordered into single particles streams which can be interrogated by the detection system of the machine. Fluidics system is the one responsible for the management of this process. The fluidics system essentially has a channel through which the sample is injected, enclosed by an outer sheath which has a faster flowing fluid. A massive drag effect is created on a narrowing central chamber as the sheath fluid moves. These results to the change of the velocity of central fluid in which its flow front turns to be parabolic with zero velocity at the wall and greatest velocity at its center (Altman et al, 1996).The impact creates a single particles’ file referred to as hydrodynamic focusing. The fluid in the central chamber under optimal conditions (laminar flow) cannot be mixed with the fluid of the sheath. In the absence of hydrodynamic focusing, the instrument nozzle would typically be blocked, and it would not be possible to analyze one cell at a time. (Altman et al, 1996) Figure 2; Hydrodynamic focusing generates a single Particles’ stream Optics and Detection Every particle passes through either several or one beam of light after hydrodynamic focusing. Fluorescence emission or light scattering gives information on the properties of the particle. With regards to current flow cytometry, the arc lamp and the laser are the most frequently used light sources (Amano et al, 1998). Lasers generate a laser line (wavelength light) at several or one discreet frequencies (coherent light). Arc lamps seem to be less expensive as compared to lasers. However, this gives incoherent unstable light of wavelengths mixture which requires subsequent optical filtering. Light which scatters up to 20o offset from the laser axis of the beam in the forward direction can be gathered by lens referred to as the forward scatter channel (FSC). Approximately, the FSC intensity is equated to the size of the particle and can as well be employed to distinguish between living cells and cellular debris. Light measured at around 90o angle to the line of excitation is referred to as side scatter. The side scatter channel (SSC) gives information on the granular content within a particle. Both SSC and FSC are known to be unique for every particle, and when the two are combined, they might be applied to distinguish various cell types in a sample that is heterogeneous.(Mitra et al,1999). Measurements of fluorescence at various wavelengths can give qualitative and quantitative data on cell surface receptors that are fluorochrome-labeled or intracellular molecules like cytokines and DNA. Separate fluorescence (FL-) channels is employed by flow cytometers to detect emitted light. The value of detectors varies based on the machine as well as its manufacturer. Detectors are usually photomultiplier tubes (PMTs) or silicon photodiodes. Usually, SP are employed to measure forward scatter when there are strong signal. PMTs are instruments that are more sensitive and are ideal for fluorescence and scatter readings. The detection specificity is regulated by optical filters, that block particular wavelengths while transmitting others. There are 3 main types of filters. ‘Long pass’ filters that permits light above a cut-off wavelength to pass through, ‘short pass’ permit light below a cut-off wavelength while ‘band pass’ transmit light within a specified narrow wavelengths range (referred to as band width). (Bigos et al, 1999) Signal Processing A small current is created when light hits a photo detector. Its voltage has amplitude that is proportional to light photons total number received by the detector. This voltage is later amplified through a series of logarithmic amplifiers to electrical signals large enough (5–10 volts) by analog to digital convertors (ADCs) to be graphically plotted. Normally, Log amplification is employed for fluorescence studies due to its capacity to compresses strong signals and expands weak signals, leading to a distribution that can be displayed easily on a histogram. Linear scaling is the most preferred in cases where such broad range signals are absent such as in DNA analysis. Each detector measurement is known as a ‘parameter’ e.g. side scatter, forward scatter, or fluorescence. (Crowley et al, 1999) Electrostatic Cell Sorting One of the main application of flow Cytometry is to separate cells basing on epitopes expression or subtype for further biological studies. This process is referred to as FACSTM analysis or cell sorting. Every particle is then probed with a beam of light after the sample is focused hydro-dynamically. The fluorescence and scatter signal is compared to the set instrument’s sort criteria. (Kantor and Roederer, 1997). The fluid stream is usually charged as it leaves the fluidics system nozzle if there is a match between the particle and the selection criteria. Actually, electrostatic charging takes place at a critical point referred to as the ‘break-off point’, which describes the point at which the droplet with the particle of interest is separated from the stream. To maintain consistency of the droplet sizes and to avoid a ‘‘break-off point’’ from occurring at random distances from the nozzle, the nozzle should be vibrated at a frequency that is high. Eventually the droplets will pass through a strong electrostatic field, and will be deflected to the right or left depending on their charge. (Kantor and Herzenberg, 1993). Sourcing Antibody Reagents 1- Mouse Anti-Human IL-18 Monoclonal Antibody Recombinant Human Interleukin-18 and IL-18 are the immungen and antigen of this antibody respectively. The host species of this antibody is the mouse. It is normally supplied in quantities of 0.5mg. It is unconjugated monoclonal antibody, IgG3 isotype in nature. It is commonly applicable in ELISA (0.5mg/ml). It is generated from a hybridoma cells by fusing of a mouse myeloma and a mouse B cells (SB116C clone). (Petricoin et al,2002) With regards to the application of this antibody in ELISA, it can be used at a concentration of 0.5mg/ml together with the required secondary reagents in the detection of IL-18. The application of this antibody product is limited to research studies only, and can’t be applied in medical treatment or diagnostic purposes. IL-18 Interleukin-18, also referred to as IFN? inducing factor, It is IL-1 family member with 18kD. This a pro-inflammatory cytokine produced primarily by antigen-presenting cells that includes macrophages or monocytes also mucosal and keratinocytes cell. After binding to targeted cells, it is able to induce the cell activity of natural killer (NK) cells, T lymphocytes proliferation and pro-inflammatory cytokines release. Furthermore, it can enhance expression of Fas Ligand in NK cells and promote Th1 cells’ cytotoxicity of Fas Ligand-mediated. IL-18 up-regulation has been associated to various inflammatory chronic conditions that include atherosclerosis, heart disease and rheumatoid arthritis. 2. Mouse Anti-Human CD43 Monoclonal Antibody CD43 is found on all leucocyte surfaces. It is an integral membrane protein which is expressed at high levels, except in resting B lymphocytes. Mouse Anti-Human CD43 Monoclonal Antibody is important in the identification of myeloid disorders and B-cell lymphomas of low-grade. MHCD4301 has the capacity to recognize human CD43 is found on every leukocyte (Petricoin et al,2002). In peripheral tissues, CD43 is expressed primarily on immature dendritic cells and in vitro on ‘immature myeloid monocyte-derived DC's’. CD43 is associated with initial interaction and dendritic cell (DC) migration between naive T lymphocytes and DC's. Applications Inspite of this antibody having not been tested for use in other given techniques, it doesn’t mean that it doesn’t qualify to be used in such given procedures. The proposed working dilutions are provided as a guide only. This product is known to suitable for flow Cytometry method. In regards to Flow Cytometry. It usually comes in 0.5ml.The concentration is on the label of the vial. 3. Fluorescein (FITC) – Conjugated Mouse Anti-Human CD8 Antibody This is a mouse monoclonal antibody that has the capacity to recognize CD8 antigen. Normally used Fluorescein conjugates relatively gives excellent, high absorptive fluorescence quantum yield, as well as good water solubility. It is an Ig Isotype Mouse IgG2a immunoglobulin and its clone is 3B5. It is a human CD8 conjugated to Fluorescein (FITC). CD8 is a 13 kDa single chain glycoprotein that serves as a core receptor of T-cell receptors. It binds MHC molecules. Application FITC is applicable in flow Cytometry, immunohistochemistry and immunoprecipitation. To ensure consistency, each product batch should be tested by flow Cytometry to ascertain whether it conforms to the standard reference reagent. 4. Polyclonal Phycoerythrin (PE or RPE)-Conjugated Sheep Anti-Mouse IgG Antibody It specifically reacts with mouse IgG together with its subclasses that includes IgG2a, IgG1, IgG3 and IgG2b. It has also indicated to have reactivity for mouse IgM and IgA but it never reacts with proteins in human serum. This is a Polyclonal Sheep Anti-Mouse IgG Antibody, RPE Conjugated with a mouse IgG immunogen, and a mouse antigen. It is reactive to mouse. It can be applied to flow Cytometry and affinity chromatography. It can also be used in immunocytofluorimetric analysis as a secondary antibody. Quantitative Sandwich ELISA for Mouse Interlekin-12 Interleukin 12 (IL-12) and Interleukin 23 (IL-23) are heterodimeric glycoprotein’s IL-12 cytokine family. This family shares a IL-12 disulfide-linked with p35 (35 kDa) subunit, a p40 (40 kDa) subunit, and with p19 (19 kDa) IL-23subunit.(Coakley et al,1996). This assay involves quantitative sandwich enzyme immunoassay. The microplates are pre-coated with monoclonal antibody specific to mouse IL-12 p40. Controls and sample standards are added to microwells. If there is present of any mouse IL-12 p40, it will bind specifically to the immobilized antibody. A polyclonal antibody conjugated to an enzyme, which is mouse IL-12 p40 specific is added into the microwells after washing off all unbound substances. After removing all unbound antibody-enzyme reagents by washing, the substrate solution is later added in the microwells. After addition of stop solution, the enzyme reaction will result to a blue product that will later turn yellow. The resulting color intensity will be measured and is directly proportional to the amount of the initially bound mouse IL-12 p40. Reagents and Materials Mouse IL-12 p40 pre-coated Microplates: These are pre-coated 96 well polystyrene microplates with mouse IL-12 p40 specific monoclonal antibody. A standard of Mouse IL-12 p40 (a dimer): Lyophilized recombinant mouse IL-12 p40 (1.5 ng/vial) in a buffered protein base with preservatives. Control of Mouse IL-12 p40 (dimer): A Lyophilized recombinant mouse IL-12 p40 in a Buffered Protein Base with Preservatives. The mouse IL-12 p40 concentration range is indicated on the label of the vial. Mouse IL-12 p40 Conjugate: polyclonal antibody (23 ml/vial) specific to mouse IL-12 p40-Horseradish Peroxidase. Assay Diluent RD1-18: Buffered protein solution (12.5 ml/vial) with preservatives. Calibrator Diluent RD5-4: buffered protein solution (21 ml/vial) with preservatives. Concentrate of Wash Buffer: concentrated solution (50 mL/vial 25-fold) of buffered surfactant with preservative. Stop Solution: solution of diluted hydrochloric acid (23 mL/vial). Color Reagent A: Stabilized hydrogen peroxide (12.5 ml/vial). Color Reagent B: stabilized chromogen (12.5 ml/vial). Plate Covers: Adhesive strips Materials and Methods 1. Human TNF-? High Sensitivity ELISA Human TNF-a Standard Preparation Concentrated standard of human TNF-a was diluted 25 times with diluent of the sample, by adding 960 ?l sample diluent in 40 ?l of the concentrated human TNF-a standard. The mixture was shook gently to mix. Biotin-Conjugate Preparation A solution of concentrated biotin-conjugate (0.03 ml) was diluted with the assay buffer (2.97 ml) 100 times in a clean plastic tube. Streptavidin-HRP Preparation A solution of concentrated streptavidin-HRP (0.015 ml) was diluted with the assay buffer (5.985 ml) 400 times in a clean plastic tube. Wash Buffer Preparation 25 ml of wash buffer was put into a clean 1000 ml cylinder, then the final volume, 1000 ml was attained by addition of deionized water, mixing was done gently to avoid foaming. Assay Buffer (1x) Preparation 2.5 ml of the assay buffer concentrate was put in a clean 100 ml cylinder, then the final volume, 100 ml was attained by addition of deionized water mixing was done gently to avoid foaming. Controls Preparation Controls preparation was by: Diluting lyophilized controls (10-30 minutes) with 800 ?l of distilled water, then mixed gently. The control was diluted 20 times by adding it to 50 ?l of control, 950 ?l of sample diluent. Amplification Diluent (1x) Preparation 3 ml of concentrated amplification diluent was diluted twice with distilled water (3 ml). Amplification Solution I Preparation 0.02 ml of amplification reagent I was diluted 300 times with amplification diluents (5.98 ml). Amplification Solution II Preparation 0.012 ml of amplification reagent II was diluted 500 times with assay buffer (5.988 ml). Sample diluent (100 ?l) was added to all the standard wells in duplicate. The prepared standard (100 ?l) of 40 pg/ml concentration was pipetted into A1 well in duplicate and the A1 and A2 wells’ contents were mixed by repeated aspiration and ejection (concentration standard 1, S1 = 20 pg/ml), then 100 ?l was transferred to B1 and B2 wells, respectively. This procedure was carried out 5 times, to make two human TNF-a rows of standard dilutions in the given range of 20.00 – 0.31 pg/ml. lastly, the contents (100 ?l) were discarded from G1and G2 last wells. A sample diluent (100 ?l) was added to the blank wells twice and 50 ?l of each sample was added twice to the wells of the samples. Biotin-conjugate (50 ?l) was added to all wells, and then the plate was later covered with adhesive film and incubated for two hours at room temperature (18 – 25°C) on a 100 rpm speed microplate shaker. Empty wells and adhesive film were removed then strips of microwell washed 6 times. Diluted streptavidin-HRP (100 ?l) was added to all wells, and then the plates were covered with adhesive film and incubated for one hour at room temperature (18 – 25°C) on a 100 rpm speed microplate shaker. Empty wells and Adhesive film were removed and strips of microwell washed 6 times. Amplification solution I (100 ?l) was added to all wells then covered the plate with adhesive film and incubated for 15 minutes at room temperature (18 – 25°C) on a 100 rpm speed microplate shaker. Empty wells and adhesive film were removed and strips of microwell washed 6 times. Amplification solution II (100 ?l) was added to all wells, then covered with adhesive film and incubated for 30 minutes at room temperature (18 – 25°C) on a 100 rpm speed microplate shaker. Empty wells and adhesive film were removed and microwell strips were washed 6 times. A solution of TMB substrate (100 ?l) was added to all wells, and then incubation done for 10-20 minutes at room temperature (18 – 25°C). Color development on the plate was monitored. The stop solution was added after the development of a dark blue color in the highest standard. Its substrate was spread evenly and quickly in the microwells to completely inactivate the enzyme. 2. Flow Cytometry Cells samples (whole blood) were prepared appropriately; the cell suspension was diluted in PBC to a 1?107 cell/ml concentration. It is recommended to use EDTA anti-coagulant, despite the use of acid-citrate dextrose or heparin which can produce accurate results. Cell suspension (100 ?l) was added to a given number of test tubes, then reagent A(100 ?l), which is fixation medium, was added and the mixture was incubated at room temperature for 15 minutes. PBC (3 ml) was added and centrifuged the mixture at 300?g, for 5 min and supernatant was discarded. Cells were then resuspended in reagent B (100 ?l), which is permeabilization medium, and conjugated antibody (positive FITC) was immediately added to tube 1 and unconjugated antibody (negative FITC) was added to tube 2. The wells were mixed and incubated at room temperature for 30 min. The mixture was later washed in PBC and supernatant discarded. Finally, the cells were re-suspended in sheath fluid to be analyzed immediately Sandwich ELISA Results The optical densities of the standard were obtained at different concentration as shown in table 1.The optical densities of the standard obtained were plotted against the amount of the sample then a standard curve was plotted. This standard curve was used to get the concentration of the unknown protein as shown in table 2.The concentration of the unknown was got first by extrapolating the unknown O.D then later the gradient was calculated. Human TNF-a Concentration (pg/ml) O.D at 450 nm 1000 0.957 500 0.766 250 0.516 125 0.273 62.2 0.188 31.25 0.143 15.6 0.161 0 0.144 Table 1: Concentration of the standard with corresponding optical density Unknown sample Concentration (pg/ml) of sample unknown Mean O.D. at 450 nm A 0.89 B 0.30 C 0.74 Table 2: concentration of the unknown obtained from the standard graph plotted (figure 1) Figure 1: Standard graph showing O.D against the concentration of Human TNF-a Graph slop= m=0.0011 A= 0.89 x0.0011=0.000979 x 2 = 0.002pg/ml B= 0.30 x 0.0011=0.00033 x2= 0.0007 pg/ml C=0.74 x 0.0011= 0.000814 x2= 0.0008 pg/ml Flow Cytometry Results On the density plot, each dot represents an individual cell that passed through the instrument as shown in figure 1. Flow Cytometry has the capacity to separate cells basing on epitopes expression or subtype for further biological studies. This process is referred to as FACSTM analysis or cell sorting. Every particle can be probed with a beam of light after the sample is focused hydro-dynamically. The fluorescence and scatter signal is compared to the set instrument’s sort criteria. Figure 1: Whole blood analysis using scatter and fluorescence Figure 2: logarithmic display graph showing the cell count(Y axis) against light scatter intensity(X axis) Discussion Basing on the Cytometry results, on the histogram, cells with minimal florescence could not be visualized hence could not be displayed on logarithmic scale. Consequently it was not possible to draw contours which are useful in identifying the type of cells. The cells were not properly presented because the sample used might have been too dilute (cells were too few) or most of the cells had minimal fluorescence and could not be visualized and displayed on logarithmic scale. Instead they could only be displayed well on logicle scale. This means that most cells in the sample had too low fluorescence. Low fluorescence of the cells can result from contamination of the cells and the biomarker (receptors) employed in the sorting of the cells by the instrument either was too few or not highly expressed in the cells. Because logarithmic scale is not able to display cells with negative values or zero values. They will pile up on the axis in the logarithm display. Hence it is recommended to use logicle scale when analyzing such type of sample. This will lead to all cells fluorescence in the sample to be visualized and displayed Flow Cytometry is a technique with automatic sorting devices that is able to sort successive streams of droplets into various fractions based on the type of fluorescence emitted by the droplet (Amano et al, 1998). Both SSC and FSC are unique for every particle, and when the two are combined, they can distinguish various cell types in a sample that is heterogeneous (figure 1 and 2). Measurements of fluorescence taken at various wavelengths gave qualitative and quantitative data on cell surface receptors which were fluorochrome-labeled. Separate fluorescence (FL-) channels was utilized by flow cytometers to detect light emitted. Detectors are either photomultiplier tubes (PMTs) or silicon photodiodes (Amano et al, 1998). In the experiment, light scattered up to 20o offset from the laser axis of the beam in the forward direction. It was collected by the forward scatter channel (FSC). The FSC intensity was equated to the size of the particle (Altman et al, 1996). This was also able to distinguish between living cells and cellular debris. On the other hand, light was measured at around 90o angle to the line of excitation referred to as side scatter. The side scatter channel (SSC) gave information on the granular content within a particle. Each particle was probed with a beam of light after the sample was focused hydro-dynamically. The fluorescence and scatter signal was compared to the set instrument’s sort criteria. With regards to sandwich ELISA carried out; ELISA as a technique is a benchmark for antigens quantification (Borrebaeck, 2000). Indeed several variations exist in this technique. ELISAs are more suitable to high-throughput screening due to the results are usually consistent, rapid and analyzed easily. Sandwich ELISA applied in this experiment, generated fairly good results, it utilized pre-matched, highly purified detector and capture antibodies. Though the results were not the best but they were sensible enough to be interpreted as seen in figure 1 and in table 1 and 2. The generated signal gave data that is highly specific and very sensitive. Matched antibodies pairs were used, in which each antibody is specific for an antigen molecule with unique, non-overlapping epitopes. The antibody, referred to as capture antibody, is first coated to the polystyrene plate. Next, the sample or analyte is added to the wells. The detection antibody, a second antibody layer, is added next after this step. This is for measuring the analyte concentration. The optical densities of the standard at different concentrations were not very close to the line of best fit. This could have resulted because of several factors. The test failure might be attributed to certain factors. Several of these errors might be prevented if the protocol was carefully read and properly comprehended prior to beginning the test. The expiration dates of reagents should be checked after identifying the test failure and it is always appropriate to make ensure the reagents such as the standard used in the experiment were stored as shown on the label of the product. After establishing this, the signs of deterioration or instability should be checked for in the standard solutions, (such as discoloration or precipitation). The substrate solutions need to be colorless. (Borrebaeck ,2000). During reagent storage and preparation, it is required to use clean containers, plastic disposable pipettes and tips. Kit reagents cross-contamination should be avoided by change of pipette tips between sample, reagent and standard addition. Lastly, the specified temperatures and incubation times should be strictly adhered to. The recommendation on accuracy improvement is that the standards and samples should be run in duplicate. Conclusion In conclusion, with regards to sandwich ELISA, the antigen is specifically captured and detected. It is the most suitable essay for complex samples, consequently the antigen don’t need purification prior to measurement. The essay also is sensitivity and more flexibility, since both indirect and direct detection methods can be employed. On the other hand in flow Cytometry, every particle passes through either several or one beam of light after hydrodynamic focusing. 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