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Introduction to Immunology and

Serology Diagnostic Techniques

1. Key Term:
- Immunology: is both the study of the human
immune system and the field of medicine that treats
diseases of the immune system.
- Serology: is the branch of science concerned with
serum to measure either antigens or antibodies in sera.
- Immunodiagnostic: is a medical diagnostic based on
highly specific interaction between an antibody and an
antigen. The antibody is used to detect the presence of
the antigen.

- Antibodies: also known as immunoglobulins
(Igs). They are a group of serum proteins
(globulins), secreted in a “soluble” form by B
lymphocyte in response to antigenic stimuli.
These antibodies bind with the stimulating
antigen and inactivating it.
- Antigen-binding site: Hypervariable region of an
antibody molecule that is the location where
binding a specific antigen takes place.
- Epitope: A part of an antigen, also as an antigen

. Structure of Immunoglobulin: The immunoglobulin molecules has two distinct regions. one of which (Fab) contains an antigen binding site that bind to an antigen.2. whereas the other (Fc) contain receptor that interact with a complement or phagocytes.

The concept of specificity or “exact fit” of the two molecules has been compared to a “lock and key fit” where the “lock” refers to the antigen – site (antibody) and the “key” to the epitope on an antigen. Lack and Key concept Antigen-antibody binding sites are located within the hypervariable segments of the variable region (VL and VH) on Fab segment of an antibody molecule.3. Any change in the hypervariable regions of an antibody may alter its specificity . . Natural of Ab Reactions: A.Immunoglobulins: Ag-Ab reactions: 3-1.

Reversible Since Ag-Ab reaction occur via non-covalent bonds they are by their nature reversible.B. . These include hydrogen bonds. electrostatic bonds. C. Multiple bonding between the Ag and the Ab ensures that the Ag will be bound tightly to the Ab. Non-covalent Bonds The bonds that hold the antigen in the antibody combining site are all non-covalent in natural.

It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody. . Affinity: The term refers to an intrinsic forces of attraction or association between an antibody (antigen-binding site) and one epitope on corresponding antigen (univalent antigen).2 Affinity and Avidity 1.3.

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the avidity between an antigen and an antibody is the sum of the affinities involved (i. Avidity: When the antigen consist of several repeating and identical epitopes (multivalent antigen).. known as avidity) Affinity refer to the strength between a single antigenic determinate and an individual antibody combining site whereas avidity refer to overall strength of binding between multivalent Ag’s and Ab’s.2. . affinity between antibodies and multivalent antigens.e.

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B. Specificity Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or ability of population of antibody molecules to react with only one antigen. Cross reactivity Cross reactivity refers to ability of an individual antibody combining site to react with more than one antigenic determinant or ability of population of antibody molecules to react with more than one antigen.3 Specificity and Cross Reactivity A. .3.

.Cross reactivity arise because: .The cross reacting antigen shares an epitope in common with antigen.Because it has an epitope which is structurally similar to one antigen. .

Factors Affecting Measurement of Antigen – Antibody Reactions: The only way that one knows that an antigen-antibody reaction has occurred is to have some means of directly or indirectly detecting the complexes formed between the antigen and antibody. The ease with one can detecte antigen-antibody reaction will depend on a number of factors.4. .

Affinity The higher affinity of the antibody for the antigen. the more stable will be the interaction is enhanced. B. .A. The binding between an antibody and a multivalent antigen is much stronger (has stronger avidity) than binding between an antibody and single epitope. Avidity Reaction between multivalent antigens and multivalent antibodies are more stable and those easer to detect.

This is depicted in this figure. Ag-Ab ratio The ratio between the antigen and antibody influences the detection of Ag/Ab complexes because the size of the complexes formed in related to the concentration of the antigen and antibody. Ab excess Ag excess Equivalence – Lattice formation .C.

If the antigen is a particulate. If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble Ag/Ab complexes.D. Physical form of the antigen The physical form of the antigen influences how one detects its reaction with an antibody. one generally looks for agglutination of antigen by the antibody. .

5. . Formation of Monoclonal and Polyclonal Antibodies: 5.1 Monoclonal antibodies: Identical antibodies with unique specificity made by single B cell.

.1 Polyclonal antibodies: Different antibodies with different specificity made by different clone of B cell.5.

. * Isolating antibody-producing cells (B cells) from the spleen of the mouse.5.3 Researchers make monoclonal and antibody by: * Injecting specific antigen into a host animal (typically a mouse). * Isolating successful hybridomas (fused cells) that produce antibodies specific for the antigen of interest. * Fusing these B cells with a specific type of tumor cell that grows easily in culture and produces antibodies.

Quality Assurance In Serology And Immunology Laboratory .

* Quality Control Program.1. requirements established for clinical laboratories a provided in applicable federal law and regulations. means those quality control. Key Term * Quality Assurance means a comprehensive process used by the laboratory to prevent and control errors that may occur at any interval from the time a test is ordered until it is reported. * The purpose of quality assurance is to prevent as many errors as possible and to detect those that do occur and the procedures used to recognize and minimize them. .

. titer of ASO different from neonate and adult. They included: 2.1.g.1age depended variation: age depended changes of changes of concentration or activities occur in a number of hematological and immunological analytes. Hence laboratory results should be interpreted keeping in a view the age of the patient e.2.1 Pre-analytical Factors: The term “Pre-analytical Factors” include all steps that a test sample must undergo until the actual test is carried out .

1. 2.3 Prolonged transportation and storage: prolonged storage prior to processing of the specimen can affect the results especially since many organisms do not survive for long unless they are kept in an environment which is rapidly changing.2. .1.2 Incorrect specimen identification or mislabeling: An incomplete specimen identification will obviously give wrong information despite all the precautions taken for a proper analytical procedure.

2.1. .5 Selection of right test method: Selection of right test method is of paramount importance.2.4 Selection of appropriate sample or collection the right specimen: An appropriate sample should be collected in an appropriate way. collection the right specimen is of critical importance.1.

2.1. . A laboratory not doing a test and yet receiving the samples for analysis will cause inordinate delay as well as a decline in the quality of the sample.6 Sending the sample to the right laboratory: The sample for analysis of particular analyte should be send to the right laboratory undertaking that particular investigation.

1. It is always advisable to avoid factors which cause hemolysis such as centrifuge blood sample at high speed before clotting.7 Centrifugation: Hematolysed blood specimens are not suitable for serological studies.2. .

2.2 Analytical Controls: Analytical Quality controls depend upon the following: 2.2.4 Procedure reliability using standard operating manuals.2.1 Equipment reliability.2.2 Reagent stability.2. 2. precision and accuracy of the test of the high order.3 Adequate calibration.2. integrity and efficiency. 2.2. .5 Specificity. 2. 2.6 Proficiency personnel and continuous updating of their Knowledge.2.

after the proper analytical process is over.3 Post-analytical controls: 1. . as well as the normal range of the results.2. it is important to record the findings on the right requisition slip in a clear way. Any possible remarks on the result obtained should be entered. 2.

3. 4. The significance of the result obtained should be highlighted wherever required. . There should be frequent dialog between laboratory personnel and the physicians for appropriate use of the facilities and right interpretation of results obtained in the laboratory.

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Nada .Done by : Dr.