Principles of Immunoassays Part 2 PDF

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Summary

Immunoassay Labels- Something that allows us to detect immune complexes formed that cannot be seen macroscopically. - Serve as indicators that allow us to see the immune complexes in solution. - Labels improve the analytical sensitivity of the immunoassay. Labels are not normally found in the patien...

Description

MLSC-3111, Clinical Biochemistry II Immunoassay Labels -

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Something that allows us to detect immune complexes formed that cannot be seen macroscopically. Serve as indicators that allow us to see the immune complexes in solution. Labels improve the analytical sensitivity of the immunoassay. Labels are not normally found in the patient samples to avoid interferences. Direct, the assay allows us to visually detect the complex formation. Used if the Ab-Ag reaction is visible with the eye. Does not require a label (nephelometry, turbidimetry). Indirect, the immunoassay complexes are detected indirectly by detecting the labels that have been coupled to the Ab or Ag. Used if the Ab-Ag reaction is not visible with the eye. Requires a label. The label itself does not distinguish between free and bound antigens. Labels should be o Easily attached to Ag/Ab o Easily measured with high sensitivity o Non-interferent with the Ab/Ag reaction o Inexpensive and non-toxic

Enzyme Labels -

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Catalysts that convert substrate to product. The enzyme is attached to the reagent Ab. Then, the substrate is added after immune complex immune complexes are formed. Once added, the substrate is catalyzed by the enzyme to produce a product. The product is measured to indirectly measure the AB-Ag complex. Most common enzymes used are o Horseradish Peroxidase o Alkaline Phosphatase o G6PD o Beta-Galactosidase The assay may measure either the enzyme product or the effect of the product on the solution (fluorescence, luminescence). The increase in product or decrease in substrate can also be measured. Enzyme Immunoassay, any assay that uses enzymes as the label. Most often they are competitive, heterogenous assays. Advantages of Enzyme Labels o High ES specific activity o Product is produced repetitively, and amplification improves sensitivity. o Stable with long shelf life. o Inexpensive o Safe o Easily automated.

MLSC-3111, Clinical Biochemistry II

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Disadvantages of Enzyme Labels o Still not as sensitive as other labels. May require combining with another label to increase sensitivity. o Environmental conditions that effects ES reactions must be tightly controlled (temperature, pH, [S] and time).

Fluorescent Labels -

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Compounds (fluorophores) that absorb light to emit light at a different wavelength. Common fluorescent labels include o Fluorescein isothiocyanate o Rhodamine o Umbelliferone o Europium o Phycobiliproteins Fluoroimmunoassay (FIA), any immunoassay that uses fluorophores as a label. Requires a fluorometer which is a detector. The intensity of light emitted by the compound is measured by the fluorometer at a 90-degree angle. Usually heterogenous, competitive assays. Advantages of Fluorescent Labels o Safe o Long shelf life o Easily automated o Specific Disadvantages of Fluorescent Labels o Still not the most sensitive label. May need more intense light from mercury/xenon arc lamps. o Quenching can occur to decrease sensitivity. o Limited selection of labels

Chemiluminescent Labels -

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Compounds that emit a photon of light as a result of oxidation. Requires oxidants for the reaction to occur. Common chemiluminescent labels are o Luminol o Acridinium ester Chemiluminescent Assays (CLIA), any immunoassay that uses chemiluminescent labels. The most widely used immunoassay in automated clinical biochemistry. These assays cannot be performed without a luminometer. Advantages of Chemiluminescent Labels o Long shelf life

MLSC-3111, Clinical Biochemistry II Safe Most sensitive label Easily automated Few interferences as other constituents in serum do not exhibit chemiluminescence. A lamp is not required since the light source is the reaction. The light produced in chemiluminescence will occur in a quick and short burst of light. Some chemiluminescent assays have been modified to give off an elongated light reaction. Known on the Vitros as an Enhanced Chemiluminescent Immunoassay (ECI). o o o o

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Radioactive Labels -

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Atoms with unstable nuclei that spontaneously emit beta or gamma radiation. The decay of a radionucleotide releases a specific spectrum of energy. Gamma-emitting nuclides are detected using crystal scintillation detectors (gamma counters). Iodine 125 (125I) is the most common radionuclide used in RIA. Radioimmunoassay, any immunoassay that uses a radionuclide as the label. Dated back to 1960 when it was introduced by Berson and Yalow as an assay for insulin in plasma. Represents the first time that hormone levels in the blood could be detected by an in vitro assay. Advantages of Radioimmunoassays o Sensitive for hormones and drugs Disadvantages of Radioimmunoassays o Exposure risk to staff o Requires license from the Atomic Energy Control Board o Expensive for use and disposal o Limited shelf life due to natural decay o Not easily automated o Requires a scintillation counter Biotin-Streptavidin, the affinity of streptavidin for biotin is the strongest non-covalent biological interaction known. It can bind four biotin molecules. Biotin is conjugated to the reagent Ab. Advantages, multiplies the signal which greatly increases sensitivity. Disadvantages, biotin from ingestion of multi-vitamins is interfering with many assays using the labelling system. This is a big problem as the interference can cause false positives or false negatives and is not easily identified. Solved by sending the sample out to another lab with a different assay.

Homogenous vs Heterogenous Methods

MLSC-3111, Clinical Biochemistry II -

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The sample Ag is in less concentration than the labeled Ab. Because of this, we must find a way to know how much of the labelled Ab is attached to an Ag. This is done through Immunoassay separation techniques. Homogenous Assay, an immunoassay that does not require a separation step to separate bound and free Ab before measuring complexes. RID, DID, Nephelometry and Turbidimetry do not require separation. These are designed for small Ag. Heterogenous Assays, bound and free antibody must be separated before label is measured. Done when we cannot tell if the label is bound or free in the reaction mixture. Separation must be rapid, reproducible, and independent from other substances in the assay/sample. Also cannot inhibit Ag-Ab binding and should minimize NSB.

Separation Techniques -

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Adsorption, free Ag adsorbed onto charcoal/dextran particles and removed by centrifugation. Precipitation, Ag/Ab precipitate out of solution to sperate bound and unbound Ag. Immune, a 2nd Ab is added to form a large complex which then precipitates out. Non-Immune, ETOH or PEG will denature protein bound complexes to make them precipitate out of solution. Requires centrifugation. Solid Phase, most common separation technique in the clinical lab. Uses various types of solid support to immobilize reagent Ab by adsorbing or covalently binding the Ab to the solid surface. Uses polystyrene surfaces or anything that the Ab will stick to. This prevents the release of the immobilized Ab or Ag. The sample is added to a tube coated in Ab stuck to the sides of the tube. Ag in the sample binds to Ab which is bound to the solid phase. Results in the Ag-Ab complex being stuck in place and not removed by washing. Washing removes only unbound Ag. Solid Phase Advantages o Easier to perform, no centrifugation. o Less time consuming o Easily automated Solid Phase Disadvantages o Requires an abundance of Ab or Ag to coat the inside solid phase. o Coating can be inconsistent o Requires more MLT skills to minimize intra-assay variability.

Competitive vs Non-Competitive Methods Non-Competitive Immunoassays -

All Ag molecules in the sample bind directly to the reagent Abs. Has two types o One-site, one Ag and one Ab form a complex.

MLSC-3111, Clinical Biochemistry II

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o Two-site, one Ag bound between two Abs. Two-site utilizes two Abs to sandwich the antibody between each other. One antibody (capture antibody) is immobilized to the solid surface while the other one carries the label. One-Step, involves a patient antigen and a labelled reagent Ab. Requires excess reagent Ab*. All Ag in the sample must be capable of binding to Abs in the reagent. When all the Ag binds, the excess unbound reagent Ab* is washed away. Bound Ab* is measured and all label detected is from sample Ag forming complex with Ab*. The amount of Ag in the sample is directly proportional to the intensity of the label signal on the Ab. Non-Competitive Sandwich IA, sample Ag added to unlabelled immobilized Ab. Reaction mixture then washed to remove unbound components of the sample. The second Ab is labelled and binds to all Ab-Ag complexes. Reaction mixture is washed again to wash away any excess unbound labelled Ab. The signal from the bound Ab* is measured. Non-competitive immunoassays are directly proportional to the sample AG concentration. The more Ag present, the more labelled Ab that will be bound. Sandwich assays are usually done with large antigens. Disadvantages, subject to the high dose hook effect. High Ag concentrations saturates all reagent Abs, causing it to go into the postzone and decrease the sample signal. Dilute the sample if this happens.

Competitive Immunoassays -

The sample Ag (analyte) is mixed with an excess amount of labelled reagent Ag. The labelled Ag is the same Ag as the analyte. The unlabelled and labelled Ags will then compete for binding on a limited amount of Abs. The sample will block the ability of the labelled Ag to bind to the Abs proportional to its concentration in solution. The unbound labelled Ag is washed away. Because of this, signal is inversely proportional to concentration of the analyte and the assays requires separation/washing (heterogenous).

Clinically Significant Immunoassays -

Immunoassays can be any combination of direct/indirect, competitive/non-competitive or homogenous/heterogenous.

ELISA -

Enzyme Linked Immunosorbent Assay. Uses an enzyme label and either an Ab/Ag is measured by absorbing into polystyrene microwells through hydrophobic interaction. Immunosorbent, an Ag/Ab that is used to remove an Ab/Ag form solution respectively.

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The solid phase used is the ELISA multi-well plate. The assay is heterogenous and separation is done through wash steps. After the final wash, the substrate is added to react with the enzyme to form colour in a positive result. Can be non-competitive or competitive or qualitative or quantitative. Direct Method, Ag immobilized in well of ELISA plate and then detected by one Ab directly conjugated to an enzyme. Indirect Method, Ag is adsorbed onto the ELISA plate. Detection is a two-step process. An unlabelled Ab is bound to the fixed Ag. Afterwards, an enzyme conjugated 2nd Ab direct against the 1st Ab is applied. The labelled 2nd will then produce a colour reaction when bound to the initial Ag-Ab complex in the ELISA solid phase. Used to detect type one diabetes (antibodies against pancreatic islets = positive reaction). Sandwich Method, labelled antibody binds to the initial Ag-Ab, but instead the labelled Ab binds to the Ag on the fixed Ab (sandwiches the Ag). Direct, Indirect and sandwich ELISAs are directly proportional assays. Competitive, both the sample Ag and Reagent Ag compete for a limited amount of binding sites in solution. Shows inverse proportionality.

IRMA -

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Immunoradiometric Assays are heterogenous assays that use radiolabelled antibodies to determine concentration of the analyte in the sample. Uses the same sandwich technique found in ELISA (non-competitive). Ab* bound in sandwiches is measure through a scintillation counter. Amount of bound Ab* is directly proportional to Ag concentration. Used to measure ferritin, calcitonin, HGH, CEA, AFP, PSA and other analytes.

MEIA -

EMIT

Non-competitive, heterogenous assay that detects immune complexes bound to latex microparticles which form on top of a glass fiber matrix. The sample combines with non-labelled reagent Ab on microparticles. Unbound reagent Ab flows through the glass fibre matrix and into the waste, while Ab-Ag gets stuck in the mesh. ALP-labelled conjugate (2nd Ab) added to stuck complexes. The 4-methylumbelliferyl phosphate substrate is catalyzed to fluorescent methylumbelliferone. There is a direct relationship between concentration of the analyte and the amount of MU produced. Advantageous in that it is fully automated and has a rapid TAT.

MLSC-3111, Clinical Biochemistry II -

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Competitive, homogenous immunoassay that does not require any separation or washing. Based on competitive binding between sample hapten and reagent enzyme labelled hapten. The enzyme labelled hapten bound to Ab loses label enzyme activity. Only free e-hapten reacts to create signal. Increased sample hapten = decreased bound e-hapten = increase free e-hapten (not blocked) = increased activity with substrate (more signal). The more sample hapten present, the less e-hapten will be bound to the Ab. There will be more free e-hapten. Measures the unbound reagent Ag.

FPIA -

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Competitive, homogenous assay that uses reagent Ag with a fluorescent fluorescein label that emits plane-polarized light upon excitation. Bound Sample Ag, has no label and is the patients antigen. Does not produce signal. Bound Reagent Ag-F, large, rotates slowly in solution and emits plane-polarized light at a longer wavelength. This is bound to the antibody and since it is large and slow, it will not move and emit the light when it rotates. Free Reagent Ag-F, a smaller antigen that rotates quickly in solution. This allows it to emit light in a different plane of space, resulting in little to no signal being detected. Ag concentration and signal are inversely related. A polarizing filter is used after the reaction vessel to detect polarized light from the bound reagent Ag-F....