The document discusses two types of detectors used in spectrophotometric instruments: photomultiplier tubes and photodiode arrays. Photomultiplier tubes use a cascade of electrons to amplify light signals, making them very sensitive, while photodiode arrays use integrated circuits containing many photodiodes to detect light across a spectrum. The document also discusses applications of UV-VIS spectroscopy like quantitative measurements of concentration and qualitative analysis of substance structure from absorption spectra. Immunoassays are described using antibodies to detect antigens, with discussion of monoclonal vs polyclonal antibodies, test sensitivity and specificity, and qualitative vs quantitative testing methods like agglutination.

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- 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
◘ 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
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
◘ 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
◘ 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
◘ 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
◘ 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 .