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Chemiluminescent immunoassay is a variation of the standard enzyme immunoassay (EIA), which is a biochemical technique used in immunology. They can also be used as diagnosis tools in medicine, as well as being in used in several other different industries for various applications.

How does it work ?

During EIA the process uses enzyme labeled antibodies and antigens to detect the small biological molecules required. The technique makes use of the basic immunology concept that an antigen binds a specific antibody. Such antigen molecules, which can be identified in a fluid sample, include molecules such as peptides, hormones and proteins. The enzymes used in chemiluminescent immunoassay convert a substrate to a reaction product, which emits a photon of light instead of developing a particular colour. Luminescence means that light is emitted by a substance when it returns from an excited state to a ground state. There are different types of luminescence and the various forms differ in the way that they achieve the excited state. For chemiluminescence it is light produced by a chemical reaction. This chemiluminescent substance can be excited by an oxidation reaction forming an intermediate. It is when this immediate return to a stable ground state happens, that a photon is released and this is detected by the luminescent signal instrument. The particular luminescence indicates the presence of the antigen. The amount of the particular biological molecule, which is being looked for and is present in the sample, is based on the luminescence observed. There are different types of substrate that are used for chemiluminescence, with the most popular types being luminol or its derivatives.

Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries,in ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal, most commonly a colour change in a chemical substrate.

HPLC is an automated version of column chromatography, which involves use of a stationary phase in the form of a column, a mobile phase, complete with a pump and a detector. The sample is injected within the column and mixed with the mobile phase followed by being pumped under high pressure. The analytes in the sample mixture interact with the stationary phase within the column differently depending on their chemical nature. Some might be retained for a longer time as compared to others and will be hence eluted at a later stage. Finally, all the eluted components are recorded by the detector and expressed in the form of a chromatogram. This chromatogram depicts each analyte within the mixture in the form of a peak plotted against the retention time (RT). The area under the curve (AUC) is generally depictive of the concentration of the analyte.

The main advantage of this technique is the platform is open and can be exploited to develop and standardize any protocol of interest. The sensitivity is very high, and with an appropriate choice of mobile phase, column and detector, multiple analytes can be identified and quantified in a single assay run.

This technique involves separation of charged molecules from a mixture on the basis of their electrophoretic mobility on a stationary phase in the presence of buffer of definite pH and electric current. The pre-treated sample mixture is loaded in to the stationary phase, wherein under the influence of electric current, the components in the mixture migrate and are detected by a detector in the end. Conventional electrophoresis can be labor intensive, involving manual setting up of the gel, followed by sample pre-treatment and loading with staining in the end for viewing and analysis. Capillary electrophoresis is a technology which involves use of thin capillaries filled with stationary phase for electrophoresis. The dimension of the capillary is designed in a way to maximize the surface is to volume ratio and minimize heating due to continuous use of electric current. The separation occurs on the basis of size as well as charge, and hence is sensitive enough to differentiate between small molecules as well.

An ion-selective electrode (ISE), also known as a specific ion electrode (SIE), is a transducer (or sensor) that converts the activity of a specific ion dissolved in a solution into an electrical potential. The voltage is theoretically dependent on the logarithm of the ionic activity, according to the Nernst equation. Ion-selective electrodes are used in analytical chemistry and biochemical/biophysical research, where measurements of ionic concentration in an aqueous solution are required

Enzyme electrodes definitely are not true ion-selective electrodes but usually are considered within the ion-specific electrode topic. Such an electrode has a "double reaction" mechanism - an enzyme reacts with a specific substance, and the product of this reaction (usually H+ or OH−) is detected by a true ion-selective electrode, such as a pH-selective electrodes. All these reactions occur inside a special membrane which covers the true ion-selective electrode, which is why enzyme electrodes sometimes are considered as ion-selective.

Immunophenotyping is a technique used to study the protein expressed by cells. This technique is commonly used in basic science research and laboratory diagnostic purpose. This can be done on tissue section (fresh or fixed tissue), cell suspension, etc. An example is the detection of tumor marker, such as in the diagnosis of leukemia. It involves the labelling of white blood cells with antibodies directed against surface proteins on their membrane. By choosing appropriate antibodies, the differentiation of leukemic cells can be accurately determined. The labelled cells are processed in a flow cytometer, a laser-based instrument capable of analyzing thousands of cells per second. The whole procedure can be performed on cells from the blood, bone marrow or spinal fluid in a matter of a few hours.

An example of information provided through Immunophenotyping: "The flow cytometric immunophenotyping report indicated the malignant cells were positive for CD19, CD10, dimCD20, CD45, HLA-DR, and λ immunoglobulin light chain. There was no co expression of CD5 or CD23 by the monoclonal B-cell population."

Flow cytometry is a technology that is used to analyse the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths.

The fluorescence can be measured to determine various properties of single particles, which are usually cells. Up to thousands of particles per second can be analysed as they pass through the liquid stream. Examples of the properties measured include the particle’s relative granularity, size and fluorescence intensity as well as its internal complexity. An optical-to-electronic coupling system is used to record the way in which the particle emits fluorescence and scatters incident light from the laser.

Nephelometry is a technique used in immunology to determine the levels of several blood plasma proteins. For example the total levels of antibodies isotypes or classes: Immunoglobulin M, Immunoglobulin G, and Immunoglobulin A. It is important in quantification of free light chains in diseases such as multiple myeloma. Quantification is important for disease classification and for disease monitoring once a patient has been treated (increased skewing of the ratio between kappa and lambda light chains after a patient has been treated is an indication of disease recurrence).

It is performed by measuring the turbidity in a water sample by passing light through the sample being measured. In nephelometry the measurement is made by measuring the light passed through a sample at an angle.

This technique is widely used in clinical laboratories because it is relatively easily automated. It is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (usually at 30 and 90 degrees).

Antibody and the antigen are mixed in concentrations such that only small aggregates are formed that do not quickly settle to the bottom. The amount of light scatter is measured and compared to the amount of scatter from known mixtures. The amount of the unknown is determined from a standard curve.

Nephelometry can be used to detect either antigen or antibody, but it is usually run with antibody as the reagent and the patient antigen as the unknown. In the Immunology Medical Lab, two types of tests can be run: "end point nephelometry" and "kinetic (rate) nephelometry".

End point nephelometry tests are run by allowing the antibody/antigen reaction to run through to completion (until all of the present reagent antibodies and the present patient sample antigens that can aggregate have done so and no more complexes can form). However, the large particles will fall out of the solution and cause a false scatter reading, thus kinetic nephelometry was devised.

In kinetic nephelometry, the rate of scatter is measured right after the reagent is added. As long as the reagent is constant the rate of change can be seen as directly related to the amount of antigen present.

The basic principle of this technology involves measurement of quantity of light absorbing analyte in a solution. This can only be however applied to solutions which follow the Beer Lambert’s law. Analytes which have the tendency to absorb light, when exposed to a beam of incident light, will absorb some. This results in reflection of a light of lower intensity. The intensity of the reflected light is then considered inversely proportional to the concentration of the analyte of interest in the solution. The main advantages involve ease of operation and wide variety of parameters which can be covered by this assay.

Molecular diagnostics is a collection of techniques used to analyse biological markers in the genome and proteome—the individual's genetic code and how their cells express their genes as proteins—by applying molecular biology to medical testing. The technique is used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients.

By analysing the specifics of the patient and their disease, molecular diagnostics offers the prospect of personalised medicine.

These tests are useful in a range of medical specialisms, including infectious disease, oncology, human leucocyte antigen typing (which investigates and predicts immune function), coagulation, and pharmacogenomics—the genetic prediction of which drugs will work best. They overlap with clinical chemistry (medical tests on bodily fluids).

Fourier transform infrared spectroscopy (FTIR)is a technique which is used to obtain an infrared spectrum of absorption or emission of a solid, liquid or gas. An FTIR spectrometer simultaneously collects high spectral resolution data over a wide spectral range. This confers a significant advantage over a dispersive spectrometer which measures intensity over a narrow range of wavelengths at a time.

The term Fourier transform infrared spectroscopy originates from the fact that a Fourier transform (a mathematical process) is required to convert the raw data into the actual spectrum.

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths. This means that a small DNA molecule will travel a greater distance through the gel than will a larger DNA molecule.

As previously mentioned, gel electrophoresis involves an electrical field; in particular, this field is applied such that one end of the gel has a positive charge and the other end has a negative charge. Because DNA and RNA are negatively charged molecules, they will be pulled toward the positively charged end of the gel. Proteins, however, are not negatively charged; thus, when researchers want to separate proteins using gel electrophoresis, they must first mix the proteins with a detergent called sodium dodecyl sulfate. This treatment makes the proteins unfold into a linear shape and coats them with a negative charge, which allows them to migrate toward the positive end of the gel and be separated. Finally, after the DNA, RNA, or protein molecules have been separated using gel electrophoresis, bands representing molecules of different sizes can be detected.

Coagulation (also known as clotting) is the process by which blood changes from a liquid to a gel, forming a blood clot. It potentially results in hemostasis, the cessation of blood loss from a damaged vessel, followed by repair. The mechanism of coagulation involves activation, adhesion, and aggregation of platelets along with deposition and maturation of fibrin. Disorders of coagulation are disease states which can result in bleeding (hemorrhage or bruising) or obstructive clotting (thrombosis).

Coagulation is highly conserved throughout biology; in all mammals, coagulation involves both a cellular (platelet) and a protein (coagulation factor) component. The system in humans has been the most extensively researched and is the best understood.

Coagulation begins almost instantly after an injury to the blood vessel has damaged the endothelium lining the vessel. Leaking of blood through the endothelium initiates two processes: changes in platelets, and the exposure of subendothilial tissue factor to plasma Factor VII, which ultimately leads to fibrin formation. Platelets immediately form a plug at the site of injury; this is called primary hemostasis. Secondary hemostasis occurs simultaneously: Additional coagulation factors or clotting factors beyond Factor VII (listed below) respond in a complex cascade to form fibrin strands, which strengthen the platelet plug.

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