Diagnostic Applications


            Serology--study of antigen-antibody reactions involving the measurement of

     antibodies and antigens in serum

Serological tests most widely used:  agglutination, precipitation, and

     complement-fixation reactions

                        Agglutination reactions--particulate antigens (bacteria and RBC’s) react

with their homologous antibodies to form agglutinated aggregates

                        Precipitin reactions--soluble antigens (bacterial cell extracts and serum

proteins) react with homologous antibodies to form precipitates

                        Complement-fixationreactions--immune cytolysis with two distinct

components, the test system and the indicator system

Agglutination tests

            Soluble antigens are adsorbed onto the surface of a particle or cell

            Antibodies act as a bridge to form a lattice network

            Bacterial Agglutination tests

                        Performed on or in slides and culture tubes

                        Typical serial dilutions of antiserum (antibody) to which a constant

     amount of antigen is added

                        Titer--relative value expressed as a reciprocal of the highest dilution that

     has clumping of cells and antibodies

                                    high titers::greater antibody concentration

                        Widal test--Salmonella typhii heat killed cultures

                        Weil-Felix test--rickettsial infections

                                    heterophile antigens--selective agglutination of Proteus spp.

            ABO blood-group-typing

                        Incompatible transfusions result in clumping and lysis of transferred cells

     by recipient’s isoantibodies and serum complement

                                    fever, prostration, renal insufficiency

                        Universal donor (O) and universal recipient (AB)


                                    Major--donor’s cells are tested against recipient’s serum

                                    Minor--donor’s serum is tested against recipient’s cells

            Rh blood type

                        Isoantibodies to Rh antigens are not present

                                    synthesis of antibodies occurs when Rh factor is introduced

                                                Rh positive--Rh factor D (27 specificities)

                                                            only D factor elicits hemolytic antibody synthesis

                        Erythroblastosis fetalis

                                    Approximately 5% Rh negative mothers produce anti-Rh


                                    Minimization of isoimmune response

                                                primary antibody response suppressed by introducing

antibodies (anti-Rh) by post-parturition injection


Precipitin tests

            Visible precipitate found at interface of soluble antigen and homologous antibody

            Typically a constant amount of antibody is used with varying dilutions of antigens

            Precipitin curve

                        Zone of antibody excess--all antigen has reacted

                        Equivalence zone--insoluble complexes

                        Zone of antigen excess--all antibody has reacted--soluble complexes

            Ring test--antigen-antibody layering

            Agar-diffusion methods--reactants diffuse together in agar

                        Simple diffusion method (Oudin)

                                    antibody containing gel overlaid by antigen

                                    level of precipiatition (reaction) determined by molecular size and


                        Double diffusion methods

                                    One dimensional (Oakley and Fulthorpe)

                                                double diffusion through agar blank

                                                            separating antibody and antigen

                                    Two dimensional (Ouchterlony)

                                                Antigen-antibody compared

                                                Bands of precipitate form in area between wells of antigen

     and antibody

                                                Precipitate occurs at optimum concentration of reactants

            Immunoelectrophoresis--migration of molecules due to electric charge

                        Positive particles travel to cathode

                        Negative particles travel anode

                        Precipitin specificity provides a critical indicator of identity


                        Microtechnique for determining trace amounts of antigen

                        Two step process:

1)      competition between unlabeled variable concentration antigen

 and labeled calculated concentration antigen

      (competition for limited antibody)

2)      addition of anti-antibody for precipitation of antigen-antibody


                        Radioactivity of precipitate

                        Used for hepatitis B antigen, insulin, testosterone, estrodiol, and IgE


Complement fixation tests

            Based on presence of complement fixing antibodies in serum

            Test involves two systems:

1)      complement-fixing system

serum, antigen and complement are mixed

2)      hemolytic indicator system

no hemolysis if antigen-antibody complex formed

            Used when test antigen and antibody combination does not give a visible reaction

                        (i.e. agglutination or precipitation)

            Used for bacterial, viral, protozoan, and fungal infections

                        Wasserman test for syphilis


Special serological tests

            Fluorescent-antibody techniques

                        Use of dyes--fluorescein or rhodamine isothiocyanate

                                    Direct method--fluorescent dye is conjugated with the antibody

which specifies for the antigen

                                    Indirect--antibody is not initially labeled--labeled antiserum

against globulin of animal species of original antibody

            ELISA (enzyme linked immunosorbent assay)

                        Based on two observations (requirements)

1)      antigen must be attached to solid phase support and maintain immunological capabilities

2)      antigen and antibodies can be bound to enzymes and resulting complexes are functional

Double antibody sandwich procedure

Indirect microplate ELISA procedure

Used for simultaneous immunodiagnosis

            Intracutaneous diagnostic tests

1)      Detecting immunity

Schick test--diphtheria (Corynebacterium sp.)

Dick test--scarlet fever (Streptococcus sp.)

2)      Hypersensitivity (allergic reactions)

Immediate type--IgE--histamine and serotonin

            20-30 minutes--persist for hours

Delayed type--T lymphocytes--release cytotoxins

            18 hours--persist for days

            Tuberculin test (BCG)

                        Bacille Calmette Guerin vaccine

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