Power point presentation on Serological Methods

Immunology Note: Antibodies types

Notes on Antibodies Properties

Cell Mediated Immunity

CELL-MEDIATED IMMUNITY: Antibody-independent immunity, mediated by T_H1 cells.
Examples: Type-IV Hypersensitivity Responses, mycobacterium tuberculosis and other intracellular pathogens.

• MACROPHAGE ACTIVATION: IFN-gamma, secreted by T_H1 cells, activates macrophages. Effects are generally anaphylactic:
• Increase expression of MHC-II molecules in macrophages, to enhance their roles as APC's.
• Increase expression of Fc-receptors and CR3-receptors, to enhance opsonization.
• Increase oxidative-burst ------> reactive O₂-intermediates and reactive N₂ intermediates (such as NO).
• Increase synthesis of eicosanoids: PGD₂, PGI₂, TXA₂, LTB₄
• CYTOTOXIC LYMPHOCYTE (CTL) ACTIVATION: IL-2 converts CTL-Precursors ------> Active CTL
• IL-2 is secreted by T_H1 cells.
• Antigen activation of a CD8 cell causes it to up-regulate its expression of IL-2 Receptors. Subsequent IL-2 activation (by T_H1 cells) causes CTL proliferation.
• After antigen activation, both CTL's and T_H-Cells are dependent on IL-2 for their proliferation.
• TERMINATION: After antigen clearance, the levels of IL-2 decline and CTL's die by apoptosis. This ensures that the immune response is limited and help to prevent excessive tissue damage.
• CTL-MEDIATED CYTOTOXICITY:
• CONJUGATE FORMATION: CTL binds to MHC-I of the target cell (which is expressing antigen and is therefore targeted for destruction).
• TCR-CD3 complex on CD8-Cell binds to HLA-I of target cell
• Additional binding by LFA-1 of CD8 cell with ICAM-1 of target cell.
• PERFORIN MONOMERS: After conjugate formation, the CD8 cell releases perforin monomers from storage granules.
• These monomers polymerize within the membrane to form perforin pores ------> target-cell membrane lysis.
• DEATH BY APOPTOSIS: Some evidence exists that the CTL cell sends a signal to target cell which causes the target-cell to die by apoptosis.
• TNF-beta has been a signal that is implicated in this apoptosis signal.
  
• NATURAL KILLER (NK) CELLS: Non-specific (natural) killing of tumors and some virally infected cells.
• They are believed to derive from null cells. Strange cell lineage.
• KILLING METHOD: Similar to CTL's.
• They degranulate perforin-like monomers to cause target-cell lysis.
• They kill cells by apoptosis (mediated by TNF-alpha)
• NK-CELL PROPERTIES:
• They do not express CD8, CD3 or any other CD lymphocyte markers on their membranes.
• They have no immunologic memory.
• ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC): Many cytotoxic cells express Fc-receptors on their membranes. They can then kill an antibody(IgG)-coated cell by a non-specific process.
• Cells involved: Most natural immunity cells have Fc-Receptors and can thus partake in ADCC. Neutrophils, Eosinophils, Macrophages, NK-Cells, Monocytes
• Cells coated with IgM are not subject to ADCC because IgM has no available Fc region! Primarily IgG is the antibody responsible for ADCC. 

SHORT NOTE: CYTOKINES

SHORT NOTE: CYTOKINES
Cytokines are bioactive hormones, normally glycoproteins, which exercise a wide variety of biological effects on those cells which express the appropriate receptors. Cytokines are designated by their cellular origin such that

Ø      Monokines include those interleukins produced by macrophages/ monocytes,

Ø      lymphokines include those interleukins produced by lymphocytes.

Ø      Interleukins is used for cytokines which mostly influence cellular interactions.

All cytokines are cyto-regulatory proteins with molecular weights under 60 kDa (in most cases under 25 kDa). They are produced locally, have very short half-lives (a matter of seconds to minutes), and are effective at picomolar concentrations. The effects of cytokines may be paracrine (acting on cells near the production locus), or autocrine (the same cell both produces, and reacts to, the cytokine). By way of interaction with highly specific cell surface receptors, cytokines can induce cell-specific or more general effects (including mediator release, expression of differentiation molecules and regulation of cell surface molecule expression). The functions of cytokines are usually pleiotropic, in that they display a number of effects of the same, or of a different, nature on one or more cell types. Below is a summary of cytokine functions:

·        Promotion of inflammation: IL-1, IL-6, TNFα, chemokines (e.g., IL-8).

·         iinhibition of inflammation: IL-10, TGFß.

·         Promotion of hematopoiesis: GM-CSF, IL-3, G-CSF, M-CSF, IL-5, IL-7.

·         Activating B cells: CD40L, IL-6, IL-3, IL-4.

·         Activating T cells: IL-2, IL-4, IL-10, IL-13, IL-15.

·         Anti-infectious: IFNα, IFNß, IFNγ, TNFα.

·         Anti-proliferative: IFNα, IFNß, TNFα, TGFß.

Immunology Note

General feature of antigen antibody reactions

1. The reaction is specific; an antigen combines only with its homologous antibody and vice versa. The specificity however is not absolute and cross reactions may occur due to antigenic similarity or related ness.
2. Entire molecules react and not fragment.
3. There is no denaturation of the antigen or the antibody during the reaction.
4. The combination occurs at the surface, therefore it is the surface antigens that are immunologically relevant.
5. The combination is firm and irreversible. The firmness of the union is influenced by the affinity and avidity of the reaction.
   Affinity refers to the intensity of attraction between the antigen and antibody molecules. It is a function of the closeness of fit between an epitope and the antigen combining region of its antibody
   Avidity is the strength of the bond after the formation of the antigen antibody complexes. It reflects the overall combining property of the various antibody molecules in an antiserum, possessing different affinity constants with the multiple epitopes of the antigen.
6. Antigens and antibodies can combine in varying proportions, unlike chemicals with fixed valencies. Both antigens and antibody are multivalent, antibodies are generally bivalent, though IgM molecules may have five or ten combining sites. Antigens may have valencies up to hundreds.

                

Prozone phenomenon

At high antibody concentrations, the number of antibody binding sites may greatly exceed the number of epitopes. As a results, most antibodies bind antigen only univalently instead of multivalently. Antibodies that bind univalently can not crosslink one antigen to another. Prozone effects are readily diagnosed by performing the assay at a variety of antibody ( or antigen) concentration. As one dilutes to an optimum antibody concentration, one sees higher levels of agglutination. When using polyclonal antibodies incomplete antibodies also causes prozone effect.

Agglutination reactions

The interaction between antibody and a particulate antigen results in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Better agglutination takes place with IgM antibody than with IgG antibodies. Excess of an antibody also inhibits agglutination reaction; this inhibition is called prozone phenomenon.

Ø      Agglutination is more sensitive than precipitation for the detection of antibodies.

Ø      Agglutination occurs optimally when antigens and antibodies react in equivalent proportions. The zone phenomenon may be seen when either an antibody or an antigen is in excess. Incomplete or monovalent antibodies do not cause agglutination, though they combine with the antigen. They may act as blocking antibodies, inhibiting agglutination by the complete antibody added subsequently

Types of agglutination

§         Slide agglutination: Serotyping.

§         Tube agglutination: Widal test.

§         Indirect (passive agglutination): where soluble antigens are coated on vehicle particle.

Slide agglutination.

§         When a drop of the appropriate antiserum is added to a smooth, uniform suspension of a particulate antigen in a drop of saline on a slide or a tile, agglutination takes place.

§         A positive result is indicated by the clumping together of the particles and the clearing of the drop. Depending up on the titre of the serum, agglutination may occur instantly or with in seconds.

§          Clumping occurring after a minute may be due to drying of the fluid and should be disregarded.

§          It is essential to have on the same slide a control consisting of the antigen suspension in saline, without the antiserum, to ensure that the antigen is not autoagglutinable.

§         Slide agglutination is a routine procedure for the identification of many bacterial isolates from clinical specimens. It is also the method used for blood grouping and cross matching.

FIG: slide agglutination

Tube agglutination

·        This is the standard quantitative method for the measurement of antibodies.

Fig. (a).Tube agglutination test for determining antibody titer.

·        When a fixed volume of a particulate antigen suspension is added to an equal volume of serial dilutions of an antiserum in test tubes, the agglutination titre of the serum can be estimated.

·        Tube agglutination is routinely employed for the serological diagnosis of typhoid, brucellosis and typhus fever ( weil- felix reaction).

Widal test

The procedure involves adding a suspension of dead typhoid bacterial cells to a series of tubes containing the patient’s serum, which has been diluted out to various concentrations. After the tubes have been incubated for 30 minutes at 37° C, they are centrifuged and examined to note the amount of agglutination that has occurred. The reciprocal of the highest dilution at which agglutination is seen is designated as the antibody titer of the patient’s serum. For example, if the highest dilution at which agglutination occurs is 1:320, the titer is 320 antibody units per milliliter of serum. Naturally, the higher the titer, the greater is the antibody response of the individual to the disease.

 Two types of antigens are used, the H or the flagellar antigen and the O or the somatic antigen of the typhoid bacillus.

Ø      The H antigen is a formolised suspension of the bacillus and on combination with the antibody forms large, loose, fluffy clumps resembling wisps of cottonwood. Conical dreyers  tubes are used for H agglutination.

Ø      The O antigen is prepared by treating the bacterial suspension with alcohol. It forms tight, compact deposits resembling chalk powder. Round bottomed felix tubes are used for agglutination. Agglutinated bacilli spread out in a disc like pattern at the bottom of the tubes.

The tube agglutination test for brucellosis may be complicated by the prozone phenomenon and the presence of blocking antibodies. Several dilution of the serum should be tested to prevent false negative results due to prozone.

The weil- felix reaction for serodiagnosis of typhus fever is a heterophile agglutination test and is based on the sharing of a common antigen between typhus rickettsiae and some strains of proteus bacilli. Another example of the heterophile agglutination test is the streptococcus MG agglutination test for the diagnosis of primary atypical pneumonia.

Examples of agglutination tests using red blood cells as antigens are the Paul Bunnel test and the cold agglutination test. The cold agglutination test is positive in primary atypical pneumonia. The patient's sera agglutinate human O group erythrocytes at 4⁰C, the agglutination being irreversible at 37⁰C.

Hemagglutination is used in blood typing

Agglutination reactions are routinely performed to type red blood cells. In typing for the ABO antigens, RBCs are mixed on a slide with antisera to the A or B blood group antigens. If the antigen is present on the cells, they agglutinate, forming a visible clump on the slide. Determination of which antigens are present on donor and recipient blood is the basis for matching blood types for transfusions.

Particle agglutination

Numerous procedures have been developed to detect antigen via the agglutination (clumping) of an artificial carrier particle such as a latex bead with antibody bound to its surface.

Latex agglutination

Ø      Antibody molecules can be bound in random alignment to the surface of latex (polystyrene) beads. Antigen present in a specimen being tested binds to the combining sites of the antibody exposed on the surfaces of the latex beads, forming cross- linked aggregates of latex beads and antigen.

Ø      The size of the latex bead (0.8µm or larger) enhances the ease with which the agglutination reaction is recognized.

Ø      Levels of bacterial polysaccharides detected by latex agglutination have been shown to be as low as 1.0 ng /ml.

Ø      because the PH, osmolarity and ionic concentration of the solution influence the amount of binding that occurs, conditions under which latex agglutination procedures are carried out must be carefully standardized.

Ø      Additionally, some constituents of body fluids such as rheumatoid factor, have been found to cause false- positive reactions in the latex agglutination systems available. To counteract this problem. It is recommended that all specimens be treated by boiling or with ethylenediaminetetraacetic acid( EDTA) before testing.

Ø      Commercial test systems are usually performed on cardboard cards or glass slides; manufacturers recommendations should be followed precisely to ensure accurate results.

Ø      Reactions are graded on a 1+ to 4+ scale, with 2+ usually the minimum amount of agglutination seen in a positive sample.

Ø      Control latex (coated with antibody form the same animal species from which the specific antibody was made) is tested alongside the latex. If the patient specimen or the culture isolate reacts with both the test and control latex, the test is considered non specific and therefore uninterpretable.

Ø      Latex tests are very popular in clinical laboratories to detect antigen to Cryptococcus neoformans in CSF or serum and to confirm the presence of beta- hemolytic streptococcus form the culture plates. Latex tests are also available to detect streptococcus agalactiae, clostridium difficile toxins A and B and rotavirus.

Coagglutination

In this case the particles are killed and treated S aureus organism ( cowan I strain), which contain a large amount of an antibody- binding protein, protein A, in their cell walls. In contrast to latex particles, these staphylococci bind only the base of the heavy chain portion (Fc) of the antibody, leaving both (Fab)antigen- binding ends free to form complexes with specific antigen.

 Several commercial suppliers have prepared coagglutination reagents for identification of streptococci, including Lancefield groups  A, B , C, D, F , G and N; Streptococcus pneumoniae; Neisseria meningitides; N gonorrhoeae; and Haemophilus influenzae types A to F grown in culture. The coagglutination reaction is highly specific but may not be as sensitive for detecting small quantities of antigen as latex agglutination. Thus, it is not usually used for direct antigen detection.

Latex agglutination inhibition test

Fig 2. Latex agglutination inhibition test
a) negative b) positive

The latex agglutination inhibition test relies on competition for the antibody between a latex- drug conjugate and any drug that may be present in the sample (mostly urine). A urine sample is placed in the mixing well of a slide containing antibody reagent, buffer and latex reagent.

a) If the drug is absent, the latex- drug conjugate binds to the antibody and forms large particles that agglutinate. Therefore agglutination is evidence for the absence of drugs in the urine specimen

b) If a drug is present in the urine sample, it competes with the latex conjugate for the small amount of available antibody. A sufficient quantity of the drug will prevent the formation of particles and agglutination and a positive urine sample does not change the appearance of the test mixture.

Coombs test

Direct coombs test

Detection of incomplete antibodies on patients RBCs

Antibodies attached on the surface of the RBCs ( patient RBCs)
                                        +

               Antihuman globulin = agglutination.

Indirect coombs test:

Detection of antibodies in patients sera.
rhesus positive RBC + Patient serum ( if contains incomplete circulating Abs coats the surface of the RBC)+ Antihuman globulin which makes the bridge = agglutination