Spectrophotometric Instruments (Detector) and Application of UV – VIS spectroscopy and Immunoassays

1. 1. 1 Spectrophotometric Instruments 4- Detector : • The purpose of the detector is convert the transmitted radiant energy into an equivalent amount of electrical energy . • Two types of detectors are frequently used :- 1- Photomultiplier tube ( PMT ) : • Incident light strikes the coated cathode , emitting electrons . • The electrons are attracted to a series of anodes , known as dynodes , each having a Successively higher positive voltage . • Theses dynodes are of a material that gives off many secondary electrons when hit by single electrons . • Initial electron emission at the cathods triggers a multiple cascade of electrons within the PMT itself . • Because of this amplification , PMT are used in instruments designed to be extremely sensitive to very low light levels . • the accumulation of electrons striking the anode produces a current signal that can be fed into a meter or recorder .
2. 2. 2 2- Photodiode arrays ( PDA ) : • are new detectors being used in modern spectrometers . • photodiodes are composed of silicon crystals that are sensitive to light in the wavelength range 170-1100 nm . • Upon photon absorption by the doiode , a current is generated in the photodiode that is proportional to the number of photons . • Although photodiodes are not as sensitive as PMT because of the lack of internal amplification , their excellent linearity , speed , and small size make them useful in applications where light levels are adequate . • PDA detectors are available in integrated circuits containing 256 to 2,048 photodiodes in a linear arrangement . • Each photodiode responds to a specific wavelength, and as a result, a complete UV/visible spectrum can be obtained in less than 1 second .
3. 3. 3 ◘ Application of UV – VIS spectroscopy : • Although many different types of operations can be carries out on a spectrophotometer, all applications fall in one of two categories. 1- Measurement of absorbance at a fixed wavelength :- • Are most often used to obtain quantitative information , such as the concentration of a solute in solution or the absorption coefficient of a chromophore . • For fixed-wavelength measurements with a single-beam instrument, a cuvette containing solvent only is placed in the sample beam and the instrument is adjusted to read “Zero” absorbance . • A matched cuvette containing sample plus solvent is then placed in the sample chamber and the absorbance is read directly from the display . • The adjustment to zero absorbance with only solvent in the sample chamber allows the operator to obtain a direct reading of absorbance for the sample . • Fixed-wavelength measurements using a double-beam spectrophotometer are made by first zeroing the instrument with no cuvette in either the sample or reference holder . • Alternatively , the spectrophotometer can be balanced by placing matched cuvettes containing water or solvent in both sample chambers . • Then, a cuvette containing pure solvent is placed in the reference position and a matched cuvette containing solvent plus sample is set in the sample position . • The absorbance reading given by the instrument is that of the samples; that is, the absorbance due to solvent is subtracted by the instrument. 2- Absorbance measurement as a function of wavelength :- • It provides qualitative information that assists in solving the identity and structure of a pure substance by detecting characteristic grouping of atoms in a molecule.
4. 4. 4 Immunoassays ◘ General considerations : • Immunoassays are available for analysis of over 100 different analytes . • Most immunoassay methods used specimens without any pretreatment and used very small sample volumes ( 10 µl-50 µl ) . • Antibodies ( Abs ) are incorporated into many clinical laboratory tests . • They are useful because of their unique properties in recognizing and distinguishing among closely related antigens ( Ags ) . • Ab molecules is an immunoglobulin protein binds to a site in the Ag . • An Ag is relatively large and complex and usually has multiple sites that can bind to Abs with different specificities; each site on the Ag referred to as an antigenic determinant or epitope . • Some tests aid in diagnosis of infections through the detection of Abs to infectious agents in patient specimens; examples are HIV , influenza , hepatitis , and rubella tests . • Other laboratory tests use the Ag-Ab reaction to measure or detect a substance not a part of the immune system, such as using Abs to measure drug or hormone levels .
5. 5. 5 ◘ Monoclonal and polyclonal antibodies : • Abs used in immunological testes can be monoclonal or polyclonal . • Monoclonal Abs are of one class and one specificity ( react with only one epitope ) and are derived from one ( mono ) clone, or cell line . • Monoclonal Abs are produced in laboratories and used as reagents in many immunodiagnostic kits . • polyclonal Abs are mixtures of antibodies produced by more than one ( poly ) cell line . • For instance , one bacterial infection will stimulate many plasma cells ( poly clones) to respond, each producing and secreting antibodies to a different bacterial epitope . • this results in a mixture of Abs in plasma that taken together can react with multiple Ags .
6. 6. 6 ◘ Test sensitivity and specificity • Sensitivity refers to the lower limit of detection, or the lowest concentration capable of being detected by a test method . • Failure to detect small amounts of a substance in a test will result in a false- negative result. • Specificity refers to the ability to detect only the substance for which the test is designed . • Reaction with other substances ( cross-reactivity ) decreases the specificity of the test and can cause false positive results .
7. 7. 7 ◘ Qualitative, Semi-qualitative and quantitative tests :- • Many immunological procedures are reported only as negative or positive ; these are called qualitative tests . • Other procedures are semi-quantitative or quantitative . • Semi-quantitative procedures usually estimate concentrations of antibodies or sometimes antigen . • The antibodies concentration can be estimated by making serial dilutions and determining the maximum dilution still capable of causing a visible reaction in the test procedure . • Semi-quantitative estimates of antibody concentration can be expressed as a titer , the reciprocal of the highest dilution showing a reaction • Quantitative immunological tests are less frequently performed . • Examples of some quantitative tests are drug assays .
8. 8. 8 ◘ Principles of Ag-Ab tests :- ♠ Examples of tests that incorporate the Ag-Ab reaction include : 1- Agglutination : • It is the visible clumping or aggregation of cells or particles as a result of reaction with a specific antibody . • IgM is the antibody class that reacts best in agglutination reactions because of its large size and multivalent binding capacity . • Antigen-coated cells or particles , such as red blood cells or latex beads, become linked together and form visible clumps when reacted with sufficient antibody . • In most agglutination test designs, the presence of agglutination indicates a positive test. • Blood typing, bacterial identification , and the classic latex test for rheumatoid arthritis are tests based on the agglutination reaction . • Agglutination tests can be qualitative or semi-quantitative . • Semi-quantitative tests can performed on slides, in tubes, or in special microtiter plates having wells with rounded bottoms . • Serial dilutions of serum are made and tested to determine the maximum dilution capable of causing agglutination , which is reported as a titer . • When agglutination tests are performed in microtiter plates , a negative test indicated by the presence of non-agglutinated particles concentrated in a small dot in the bottom of the well . • A diffuse pattern of cells spread over the bottom of a round-bottomed well indicates a positive test .