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It has also found applications in the food industry in detecting potential food allergens , such as milk , peanuts , walnuts , almonds , and eggs [25] and as serological blood test for coeliac disease. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus, the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate.

A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and a negative result. A cut-off point may be determined by comparing it with a known standard. Unknowns that generate a stronger signal than the known sample are "positive.

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From Wikipedia, the free encyclopedia. Biology portal Technology portal Chemistry portal. The Journal of Immunology. Food Forensics and Toxicology. Humana Press. In van der Vliet, P. Dry Chemistry: Analysis with carrier-bound reagents. Laboratory Techniques in Biochemistry and Molecular Biology. Nielsen, S. Food Analysis. Handbook of Surface Plasmon Resonance 2nd ed. Royal Society of Chemistry.

Immunology: Understanding the Immune System. The Journal of Clinical Investigation.

Introduction: Guidelines for the use of flow cytometry in immunology

Clinical Chemistry. Biochimica et Biophysica Acta. FEBS Letters. Chimera Biotech. Nature Nanotechnology. Totowa, N. Berg" : Methods in Molecular Biology. Thermo Fisher Scientific - US. Retrieved January Retrieved August 20, The American Journal of Gastroenterology. Journal of Clinical Laboratory Analysis. Clinical and Vaccine Immunology.

Medical tests used in immunology and for inflammation CPT — Stoppers can have anywhere from 0 — 3 holes, allowing for connections to tubing or inserting thermometers and stirrers. A variation of the stopper is the septum, which can be used to seal glassware and allows for easy access with a syringe needle. The downside of most flexible stoppers is that they break down over time, though newer Teflon stoppers are more robust but lack the physical flexibility.

Ground glass stoppers are used to seal flasks that have ground glass fittings. While the seal is very good, glass-to-glass connections are known to seize, so joint grease vacuum, Krytox, etc. Rubber stoppers are sized by number, ranging from — 10, whereas glass stoppers are sized by the diameter and length of the sealing section.

Connections between pieces of glassware are made using a variety of ground glass joints including a standard taper, ball-and-socket, and O-ring. The standard taper is the most common fitting. Glass joints are sized to fit into one another and a variety of size adapters are available. Like all other glass joints, grease is required to prevent seizing.

While the joint may be sealed, it is not a mechanically strong connection and can fall apart. To prevent glass pieces from separating, connector clips are used, which are sometimes referred to as Keck clips. These clips are color-coded for the size of the joint. Alternatives to connector clips include springs and wire. Clamping and supporting glassware is a vital part of a successful experiment. While some pieces of glassware, like beakers and Erlenmeyer flasks, have flat bottoms that can sit flat on a hotplate, other pieces of glassware, like round-bottom flasks, need to be supported using clamps.

Even with flat-bottom glassware, it can be far too easy for something like a vacuum filtration flask to fall over. Metal clamps are connected to the neck of a piece of glassware using either a three-finger or a standard clamp. The other end of the clamp is then attached to a ring stand or retort stand. Other clamps exist for special purposes, like chain-style for large pieces or water-bath clamps for thermometers.

The lab jack uses a scissoring action to raise or lower a piece of glassware. This is very convenient for large or heavy items and, when used in conjunction with a cork ring, can also be used to move round-bottom flasks. Just like in the kitchen, soap and water are typically used to clean glassware in the lab. When that fails, organic solvents, like acetone, are sometimes employed to remove sticky and insoluble organic deposits. Even then, some compounds adhere to glassware so well that they are impossible to remove without some form of chemical etching.

In the case of organic carbon-containing deposits, glassware can be soaked in a base bath composed of an alcohol ethanol and a strong base sodium hydroxide.

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This bath etches thin molecular layers of glass from the vessel, taking the stubborn deposits with it. It is very important to never place volumetric glassware in a base bath, which could lead to etching and a change in volume. When a metal has plated or infused into a piece of glassware, an acid bath made with a dilute strong acid, like hydrochloric, is used. The amphoteric nature of glass and the general oxidation of metal in acid lead to its cleaning power.

Regardless of the bath type, 24—48 h is required for effective deposit removal. Because of these desirable traits, glass has been used to create a wide assortment of apparatuses. Being unfamiliar with this equipment could lead to confusion, misuse and disaster. Therefore, a solid understanding of glassware is necessary to ensure safety and success in the lab.

Laboratory glassware is manufactured with different compositions, each possessing unique properties that are useful in different experimental conditions. Equipment made from consumer-grade, or "soda-lime", glass is the least expensive, and is adequate for many applications. However, rapid temperature changes can cause this glass to crack. Borosilicate glass, which exhibits little thermal expansion, is preferred in thermally stressful conditions.

This glass is manufactured through the addition of small amounts of boron, and is often used in bakeware, such as Pyrex. However, both borosilicate and standard glass contain impurities, resulting in reduced optical quality. Therefore, a glass composed of purely silicon and oxygen is utilized in situations that require the glass to be transparent to UV light. This is known as fused silica or fused quartz. We will begin our survey with glassware used for qualitative analysis.

Any measurements, or graduations, on this equipment are approximate, and they are best used for procedures that do not require high levels of accuracy. First, the beaker, one of the most common pieces of glassware, is available in a range of sizes. Beakers are often used to hold, mix, and heat reagents. Most have a small lip for pouring liquids. Test tubes, which are relatively small cylindrical vessels, are also used to store, heat, and mix chemicals.


Their design allows for multiple samples to be easily manipulated, stored, and observed at once. Watch glasses are used when a large surface area is needed for a small volume of liquid. This is common for crystallizing and evaporating procedures. Watch glasses can also be used as covers for beakers.

The crystallization dish is similar to the watch glass, proving a large surface area for liquids. However, it is more commonly used as a container for bath processes. Lastly, the flask. Each type of flask is shaped for its purpose, but all are designed with wide bodies and narrow necks, allowing the contents to be mixed without spilling. They are also easily fitted with stoppers. The Erlenmeyer flask is the most common. The flat bottom allows it to be directly heated and used in simple boiling and condensation procedures.

Immunological Techniques Made Easy: A Practical Guide to Common Laboratory Procedures

Next, we will review glassware used for accurately measuring liquids. The graduated cylinder is used to measure semi-precise volumes, and deliver to another container. The surface of most liquids forms a concave meniscus in narrow glassware. Volume should be read at the bottom for accuracy. While the graduated cylinder is versatile, volumetric glassware is used when a higher level of accuracy is required. Volumetric glassware can be an order of magnitude more precise than a graduated cylinder.

Each piece is marked with either "TD" or "TC". If the equipment is calibrated to transport the measured volume, it is marked "TD" for "To deliver". Conversely, other pieces of volumetric glassware are only calibrated to be accurate while holding the measured volume, and are marked "TC" for "To Contain".

The volumetric flask is used to make and contain solutions of precise volumes. This is done by first dissolving the solute, and then adding solvent to the graduation to dilute to the intended volume. Unlike the apparatuses that are accurate only to contain, the volumetric pipette is used to deliver a specific volume with a high degree of accuracy. The function of the immune system is to recognize self antigens from non-self antigens and defend the body against non-self foreign agents. The first line of defense against infection is intact skin, mucosal membrane surfaces, and secretions that prevent pathogens from penetrating into the body.

When a foreign agent penetrates the first line of resistance, an immune reaction is elicited and immune cells are recruited into the site of infection to clear microorganisms and damaged cells by phagocytosis. If the inflammation remains aggravated, antibody-mediated immune reaction is activated and different types of immune cells are engaged to resolve the disease. The immune system is composed of cellular and humoral elements. The cellular component includes mast cells, neutrophils, macrophages, T and B lymphocytes, and plasma cells. The humoral component includes complement, lyzozyme, interferon, antibodies, and cytokines.

All work cooperatively to eliminate immunogenic foreign substances from the body. Immune complex : This is the complex formation of a specific antibody-antigen. To aid in the diagnosis of disease caused by infectious microorganisms, immunoassays have been developed. These biochemical and serological techniques are based on the detection and quantitation of antibodies generated against an infectious agent, a microbe, or non-microbial antigen.

Because antibodies can be produced against any type of macromolecule, antibody-based techniques are useful in identifying molecules in solution or in cells. A blood sample is collected from the patient during the acute phase of the disease when antibody levels are high. Serum is then isolated and the concentration of antibodies is measured through various methods.

Most assays rely on the formation of large immune complexes when an antibody binds to a specific antigen which can be detected in solution or in gels. Recent methods employ pure antibodies or antigens that have been immobilized on a platform and that can be measured using an indicator molecule. These methods provide high sensitivity and specificity and have become standard techniques in diagnostic immunology.

Antibodies, part of the humoral immune response, are involved in pathogen detection and neutralization. Differentiated plasma cells are crucial players in the humoral immunity response. The antibodies they secrete are particularly significant against extracellular pathogens and toxins. Once secreted, antibodies circulate freely and act independently of plasma cells. Sometimes, antibodies can be transferred from one individual to another. This phenomenon, called passive immunity, also occurs naturally during breastfeeding, which makes breastfed infants highly resistant to infections during the first few months of life.

Antibody neutralization can prevent pathogens from entering and infecting host cells, as opposed to the cytotoxic T-cell-mediated approach of killing cells that are already infected to prevent progression of an established infection. The neutralized antibody-coated pathogens can then be filtered by the spleen and eliminated in urine or feces. Mechanisms of antibody action : Antibodies may inhibit infection by a preventing the antigen from binding to its target, b tagging a pathogen for destruction by macrophages or neutrophils, or c activating the complement cascade.

Antibodies also mark pathogens for destruction by phagocytic cells, such as macrophages or neutrophils, because they are highly attracted to macromolecules complexed with antibodies. Phagocytic enhancement by antibodies is called opsonization. In another process, complement fixation, IgM and IgG in serum bind to antigens, providing docking sites onto which sequential complement proteins can bind. The combination of antibodies and complement enhances opsonization even further, promoting rapid clearing of pathogens.

Not all antibodies bind with the same strength, specificity, and stability. In fact, antibodies exhibit different affinities attraction depending on the molecular complementarity between antigen and antibody molecules. An antibody with a higher affinity for a particular antigen would bind more strongly and stably.

It would be expected to present a more challenging defense against the pathogen corresponding to the specific antigen. Antibody affinity, avidity, and cross reactivity : a Affinity refers to the strength of single interactions between antigen and antibody, while avidity refers to the strength of all interactions combined. The term avidity describes binding by antibody classes that are secreted as joined, multivalent structures such as IgM and IgA.

Although avidity measures the strength of binding, just as affinity does, the avidity is not simply the sum of the affinities of the antibodies in a multimeric structure. The avidity depends on the number of identical binding sites on the antigen being detected, as well as other physical and chemical factors. Typically, multimeric antibodies, such as pentameric IgM, are classified as having lower affinity than monomeric antibodies, but high avidity. Antibodies secreted after binding to one epitope on an antigen may exhibit cross reactivity for the same or similar epitopes on different antigens.

Cross reactivity occurs when an antibody binds not to the antigen that elicited its synthesis and secretion, but to a different antigen. Because an epitope corresponds to such a small region the surface area of about four to six amino acids , it is possible for different macromolecules to exhibit the same molecular identities and orientations over short regions. Cross reactivity can be beneficial if an individual develops immunity to several related pathogens despite having been exposed to or vaccinated against only one of them.

For instance, antibody cross reactivity may occur against the similar surface structures of various Gram-negative bacteria. Conversely, antibodies raised against pathogenic molecular components that resemble self molecules may incorrectly mark host cells for destruction, causing autoimmune damage. These antibodies may have been initially raised against the nucleic acid of microorganisms, but later cross-reacted with self-antigens. This phenomenon is also called molecular mimicry. Serology is the study of blood serum and other bodily fluids for the identification of antibodies.

Serology is the scientific study of blood serum and other bodily fluids. In practical immunological terms, serology is the diagnostic identification of antibodies in the serum. Serological tests are performed on blood serum, and body fluids such as semen and saliva. In practice, the term usually refers to the diagnostic identification of antibodies in the serum or the detection of antigens of infectious agents in serum. A primary immune response occurs when a B cell sees an antigen for the first time.

Antigen binding to the surface of the B cell stimulates the production of antibodies that are capable of binding directly to the antigen. Because this first recognition process takes time for antibody development, there is an initial delay for the body to fight the invading antigens. Immunoglobulin M IgM is an antibody produced during the primary immune response and plays a significant role fighting infection.

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When an antigen is introduced into the body for the first time, large quantities of IgM are produced. Once IgG is produced in quantity, the IgG plays a greater role in the removal of antigens from the body due to its ability to bind to the antigen molecules more tightly. Through the course of an infection, a rapid spike of circulating IgM can be seen in the bloodstream. This is followed by a decrease of IgM as the amount of IgG increases. Medical laboratory personnel can identify the course and duration of an infection by measuring the ratio of IgM to IgG in the bloodstream.

A ratio high in IgM indicates that an infection is in its early stages, while a ratio high in IgG indicates that the infection is in its later stage. Immune response : When B and T cells begin to replicate, some of the offspring that they produce will end up becoming long-lived memory cells. Precipitation reactions are serological assays for the detection of immunoglobulin levels from the serum of a patient with infection.

Describe how precipitation reactions can be used for the detection of immunoglobulin levels in the serum of a patient. Precipitation reactions are based on the interaction of antibodies and antigens.

Get e-book Immunological Techniques Made Easy: A Practical Guide to Common Laboratory Procedures

They are based on two soluble reactants that come together to make one insoluble product, the precipitate. These reactions depend on the formation of lattices cross-links when antigen and antibody exist in optimal proportions. Excess of either component reduces lattice formation and subsequent precipitation. Precipitation reactions differ from agglutination reactions in the size and solubility of the antigen and sensitivity. Antigens are soluble molecules and larger in size in precipitation reactions. There are several precipitation methods applied in clinical laboratory for the diagnosis of disease.

These can be performed in semisolid media such as agar or agarose, or non-gel support media such as cellulose acetate. Precipitation reaction : Difference in the visual appearance of an aggregate and a precipitate. Precipitation methods include double immunodiffusion qualitative gel technique that determines the relationship between antigen and antibody , radial immunodiffusion semi-quantitation of proteins by gel diffusion using antibody incorporated in agar , and electroimmunodiffusion variation of the double immunodiffusion method reaction that uses an electric current to enhance the mobility of the reactants toward each other.

Precipitation reactions are less sensitive than agglutination reactions but remain gold standard serological techniques. The most commonly used serologic precipitation reactions are the Ouchterlony test based on double immunodiffusion and named after the Swedish physician who invented it , and the Mancini method based on single radial immunodiffusion. In the double immunodiffusion technique, three basic reaction patterns result from the relationship of antigens and antibodies. These patterns are identity, non-identity, and partial identity.

The Mancini method results in precipitin ring formation on a thin agarose layer. The diameter of the ring correlates with the concentration of proteins in the precipitin. Agglutination reactions are used to assess the presence of antibodies in a specimen by mixing it with particulate antigens. Describe how agglutination reactions can be used to assess the presence of antibodies in a specimen. Agglutination is the visible expression of the aggregation of antigens and antibodies.

Agglutination reactions apply to particulate test antigens that have been conjugated to a carrier. The carrier could be artificial such as latex or charcoal particles or biological such as red blood cells. These conjugated particles are reacted with patient serum presumably containing antibodies. The endpoint of the test is the observation of clumps resulting from that antigen-antibody complex formation.

The quality of the result is determined by the time of incubation with the antibody source, amount and avidity of the antigen conjugated to the carrier, and conditions of the test environment e. Various methods of agglutination are used in diagnostic immunology and these incude latex agglutination, flocculation tests, direct bacterial agglutination, and hemagglutination.

In latex agglutination, many antibody molecules are bound to latex beads particles , which increases the number of antigen-binding sites. If an antigen is present in a test specimen, it will bind to the antibody and form visible, cross-linked aggregates. Latex agglutination can also be performed with the antigen conjugated to the beads for testing the presence of antibodies in a serum specimen.

Flocculation tests are designed for antibody detection and are based on the interaction of soluble antigens with antibodies, producing a precipitate of fine particles that can be seen with the naked eye.

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Direct bacterial agglutination uses whole pathogens as a source of antigen. It measures the antibody level produced by a host infected with that pathogen. The binding of antibodies to surface antigens on the bacteria results in visible clumps. Hemagglutination uses erythrocytes as the biological carriers of bacterial antigens, and purified polysaccharides or proteins for determining the presence of corresponding antibodies in a specimen. Agglutination tests are easy to perform and in some cases are the most sensitive tests currently available. These tests have a wide range of applications in the clinical diagnosis of non- infectious immune disorders and infectious disease. Describe how neutralizing antibodies serve to block viral attachment to cells thus inhibiting viral replication.

A neutralizing antibody defends a cell from an antigen or infectious body by inhibiting or neutralizing any effect it has biologically. The antibody response is crucial for preventing many viral infections and may also contribute to the resolution of an infection. When a vertebrate is infected with a virus, antibodies are produced against many epitopes of multiple virus proteins. A subset of these antibodies can block viral infection by a process called neutralization. This usually involves the formation of a virus-antibody complex. Neutralizing antibody : Antibody neutralizing an antigen and preventing its biological effect.

This virus-antibody complex can prevent viral infections in many ways. It may interfere with virion binding to receptors, block uptake into cells, prevent uncoating of the genomes in endosomes, or cause aggregation of virus particles. Many enveloped viruses are lysed when antiviral antibodies and serum complement disrupt membranes.