EXERCISE 4

ANTIGEN-ANTIBODY II

I. INTRODUCTION

D. AGGLUTINATION AND IMMUNE LYSIS

Agglutination is a two-step reaction. The antibody is in solution so the character of the reaction depends largely on the physical state of the antigen. Antigens in a particulate state (bacteria or erythrocytes) are clumped or agglutinatedby the soluble antibody (agglutinin).

The PRIMARY reaction is the binding of the Fab portion of antibody to antigenic determinants on the particulate antigen and so is not visible to the eye.

The SECONDARY reaction is the result of the cross-linking by multivalent antibodies, of the particles of multivalent antigen to form aggregates largeenough to be detected visually.

The foregoing two-component system can in certain instances be enhanced by the addition of a third component, complement (C'). This is exemplified by the reaction of immune hemolysis, in which the antibody " sensitizes" erythrocytes which are then lysed by complement. Other mammalian cells (e.g., leukocytes) and certain bacteria (e.g., Gram-negative organisms) can be lysed by this system.

The most obvious clinical application of agglutination is blood-typing; erythrocyte blood group surface antigens (e.g., the ABO blood group factors) react with antibody that is specific for these allo-antigens to produce agglutination. The cross- matching of blood depends upon this technique.

The surface antigens of bacteria also react with appropriate antibodies to produce agglutination. A patient's serum can be tested for the presence of antibody to known antigens of certain bacteria (e.g., Salmonella typhi), giving diagnostically significant information. Conversely, bacteria such as Shigella, Escherichia, or Proteus isolated from cultures of clinical specimens are identified most definitively using antibodies of known specificity (serotyping).

E. BACTERIAL OPSONIZATION

II. LAB WORK
A. BACTERIAL AGGLUTINATION

Materials supplied: (work in pairs)

• 1 tube of unknown ENTERIC BACTERIA with dye added (brown cap )
• 1 tube of SALINE (pink cap)
• 1 Agglutination slide
• 2 Pasteur pipets
• Salmonella antiserum in dropper bottles
• Shigella antiserum in dropper bottles

1. Label the three wells on the Agglutination slide: Salmonella, Shigella and Control.
   
2. Place a drop of Salmonella antiserum in one well, and a drop of Shigella antiserum in the other well. Use a Pasteur pipet to place a drop of saline in the third well as Antigen Control. Discard the pipet properly.
3. Use the other Pasteur pipet to add one drop of the unknown bacterial suspension to each of the three wells. Discard the pipet properly.
4. Rock the slide gently to achieve mixing of the cells and antisera. Let sit for about 2 minutes, mixing occasionally.
5. Read both experimental wells for agglutination: the suspension appears granular. Use the saline well as a comparison (Negative Control). Examine your slide with the dissecting scope (front table) to confirm your conclusions. Discard these slides in the specifically marked disposal bucket.
6. Record your results in the slide diagram on the Report Sheet, as + or - agglutination. Identify the unknown enteric bacteria.
7. Observe the demonstration on blood typing.

Materials supplied: (work in pairs)

• 1 metal rack with 10 tubes.
• 1 tube of complement fixation (CF) saline for making dilutions. (yellow cap)
• 1 tube of erythrocyte antigen (0.75% suspension of sheep RBC)
• 2 pipets, 5 ml
• 1 pipet, 1 ml
• PipetAids (blue/green, in lab drawer)

Note: Tube #1 is already numbered and contains exactly 0.5 ml serum (heat inactivated).

1. Be sure all tubes are numbered 1-10. Label tube 1 with your name.
2. Use the green PipetAid and one 5 ml pipet to deliver exactly 0.5 ml CF saline to each of the 10 tubes.Discard this pipet. Put tube 10 aside.
   
3. Use the 1 ml pipet and the blue PipetAid to mix the contents of tube 1 and transfer 0.5 ml to tube 2. Mix the contents of tube 2, withdraw 0.5 ml and transfer it to tube 3. Perform this same action on each of the tubes through tube 9. Mix the contents of tube 9, withdraw 0.5 ml and DISCARD BOTH in the pipet jar. Tube 10 will be the ANTIGEN CONTROL.
4. Resuspend the sheep RBC suspension and use the other 5 ml pipet and the green PipetAid to add exactly 0.5 ml of erythrocyte suspension to each tube 1-10.
5. .Shake the rack well to mix the reagents, and place in the 37C WATERBATH. Take care that water does not get into your or other tubes. After 1 hour, all racks will be removed by the instructor and stored in the refrigerator until the next lab period.

Do not shake or mix your tubes. Start with Tube 10 and look at the pattern the RBCs make on the tube bottom. This is called a " button" . Compare all of the other tubes to this Control tube.Determine the titer by finding the tube with the highest serum dilution (lowest antibody concentration) that has an agglutination pattern that is completely different from the Control.The reciprocal of this dilution is expressed as the TITER. Intermediate reactions appearing between the Control tube and this other tube can be graded as " +/-" or " partial" agglutination. (See example at front table)
6. Observe the demonstration of immune hemolysis at the front table. The antiserum was diluted out serially in the same manner as you performed in lab. Active complement (C' ) was added to tubes 1- 9, and the C' Control tube. The same RBC suspension used in lab was added, the solutions mixed and incubated for 30 minutes at 37C, then refrigerated overnight.
7. Notice that the titer obtained by the Complement assay is higher than that determined by your experiment.

Why is this, if the same serum was used in each assay?

Record the hemolytic and agglutination endpoints here and on your Report Sheet.

Hemagglutination Results

Bacterial Opsonization:

1. Observe the two slides of phagocytosis on demonstration at the front table. One shows pnemococci without serum, and the other shows pneumococci with specific anti-capsular antibody.
2. The specific anti-capsular antibody binds to the capsular material on the cell, becoming an Ag-Ab complex, activating complement that enhances the phagocytic capacity of macrophages and lymphocytes.Many more pneumococci can be observed within the cells in the sample with the opsonized bacteria.

1. The serodiagnosis of a viral infection is dependent upon the reaction of virus-specific antibody with either virus or virus antigen. In order to assure that the positive reaction seen between virus and antibody is relevant to the current disease (and not a long standing immunity), a serum sample must be obtained as early in the course of disease as possible (acute phase serum), and a second sample collected in the later stages (convalescent phase serum). The acute and convalescent sera can then be compared for an increase in the amount of specific antibody to the suspected virus. An increase in titer greater than fourfold is considered indicative of a specific immune response, and thus provides serodiagnosis. Fourfold increases are significant because dilution errors would not transcend more than one dilution tube (twofold).
2. In the example demonstrated at the front table, Influenza A virus by itself can agglutinate RBCs because it recognizes a receptor on the RBC surface. If the virus is first mixed with antibody to the virus, then the receptors will be blocked and the virus will be " neutralized" and will be unable to agglutinate the RBCs. This reaction is called hemaagglutination inhibition. The quantity of virus in this test is constant so one can quantitate by dilution, the amount of Influenza A antibody in the serum.
3. On display at the front tables are two racks of tubes, each representing the quantitation of Influenza A antibody in a serum sample. The serum samples differ only as to when in the course of the disease they were obtained.
4. Determine the titers of each serum sample in the same manner as you did with your own tubes, by comparing each tube with the controls. Notice that a lack of hemagglutination (i.e. a button) indicates a positive antigen-antibody reaction. Make sure you understand the difference between this test and the hemagglutination test you performed in lab.

Virus Neutralization Results

ANTIBODY:

A globular protein that is produced by lymphocytes and plasma cells in response to an antigenic stimulus and that reacts specifically with the eliciting antigen.

• IMMUNOGLOBULIN - A general term for all globulin fractions that contain antibody.
• ANTIGEN BINDING SITE - That portion of the antibody molecule that binds to the antigenic determinant on the antigen. It is composed of the variable amino acid sequences of both light and heavy chains. One light chain and one heavy chainare required to form a single antigen-combining site.
• HETEROGENEITY OF ANTIBODY - The concept that many different kinds of antibody can be produced in response to a single antigen. Antibodies to a single antigen can differ from one another with respect to their antigen binding site, as well as physical, chemical and biological properties.

ANTIGEN:

Any substance (usually foreign) that can initiate an immune response and on subsequent exposure can react specifically with the product(s) of that immune response.

• IMMUNOGEN -A more precise term to denote an antigen that in one physical or chemical form may produce a more pronounced immune response than inanother form.
• TOLEROGEN - An antigen that invokes a specific immunological unresponsiveness due to its physical or molecular form. A tolerogen can become an immunogen ifits molecular form is changed.
• HAPTEN - A simple chemical group of low molecular weight, that by itself cannot elicit an immune response, but can do so if combined with a protein carrier.However, the hapten alone can react with the antibody elicited by the hapten- carrier complex (in e.g., hapten inhibition).
• ANTIGENIC DETERMINANT -That portion of the whole antigen molecule that actually combines with antibody or sensitized " T" lymphocytes.

ANTIGEN-ANTIBODY REACTIONS:

Antibody in solution binds to its specific antigen. This can result in various reactions, depending on the physical state and natureof the antigen.

• PRECIPITATION - Soluble antigen and antibody in optimum proportion to one another can form insoluble complexes that precipitate out of solution.
• AGGLUTINATION - Antigen bound to (or an integral part of) the surface of an insoluble particle (e.g., mammalian or bacterial cell) can bind with its specific antibody to cause aggregation of the particles.
• NEUTRALIZATION - Antigens that have enzymatic or toxic properties can react with antibody and as a result be neutralized or rendered inactive. For example,the interaction of viruses and antibody will prevent the virus from attaching and entering the host' s cells, thus neutralizing viral infectivity.

CELL MEDIATED IMMUNITY (CMI):

A part of the immune response to an antigen that involves the production of specifically primed " T" lymphocytes. These cellsare involved in the rejection of tumors and transplanted tissue as well as in defense against certain bacterial, mycotic, parasitic, and viral infections.

COMPLEMENT:

A complex set of serum proteins that are activated when antibody binds to antigen, by soluble antigen-antibody complexes, or by a " by-pass" system (alternate pathway) in the absence of specific antibody/antigen interactions.

CYTOLYSIS:

The lysis of bacteria or tumor or red blood cells by insertion of the membrane attack complex derived from complement activation.

HISTOCOMPATIBLE:

Sharing transplantation antigens.

HYPERSENSITIVITY REACTIONS:

These occur in 4 types:

• ANTIBODY-MEDIATED HYPERSENSITIVITY
  • Type I - Anaphylactic (" Immediate" ): IgE antibody is induced by allergen and binds to mast cells and basophils. After encountering the antigen again, the fixed IgE becomes cross-linked, inducing degranulation and release of mediators, especially histamine.
  • Type II - Cytotoxic: Antigens on a cell surface combine with antibody, that leads to complement-mediated lysis or other cytotoxic membrane damage.
  • Type III - Immune complex: Antigen-antibody immune complexes are deposited in tissues, complement is activated, and PMNs are attracted to the side, causing tissue damage.
• CELL-MEDIATED HYPERSENSITIVITY
  • Type IV - Delayed: T lymphocytes, sensitized by an antigen, release lymphokines upon second contact with the same antigen. The lymphokines induce inflammation and activate macrophages.

IMMUNE RESPONSE:

The immunological response of a host exposed to a foreign substance (antigen); it can be humoral (antibody production) and/or cell- mediated (sensitized " T" lymphocytes).

• " T" LYMPHOCYTE - A " thymic dependent" small lymphocyte that is responsiblefor the cell-mediated immune response and that recognizes antigen and influences production of antibody by interaction with " B" lymphocytes.
• " B" LYMPHOCYTE - A " thymic independent" (bone marrow?) small lymphocyte characterized by surface immunoglobulin antigen-receptors. It is thought to differentiate into plasma cells that produce antibody.
• AUXILIARY CELL - Macrophage or blood monocyte that " presents" antigen to " T" and " B" lymphocytes in a nonspecific manner for the initiation of an immune response.
• PLASMA CELL - A highly specialized " end" cell that synthesizes large amounts of antibody.

IMMUNITY:

A classical state of increased protection against or reaction to the effects of a foreign substance or microorganism involving all parameters of the immune response.

:
• NATURAL IMMUNITY - Nonspecific resistance not acquired through contact with an antigen. It includes skin and mucous membrane barriers and a variety of nonspecific immunologic factors.
• ACQUIRED IMMUNITY - Protection acquired by natural or deliberateintroduction of an antigen into a responsive host. It is specific and is mediated by eitherantibody or lymphoid cells (or both).

MEMBRANE ATTACK COMPLEX:

The end product of the activation of the complement cascade. MAC makes holes in the membranes of gram-negative bacteria, killing them, and in red blood or other cells, causing them to lyse.

OPSONIZATION:

The coating of an antigen or particle by substances (called opsonins) such as antibodies, complement components, etc., that facilitate uptake of the foreign particle into a phagocytic cell.

PHAGOCYTOSIS:

The process of internalization of particulate matter by cells such as macrophages and polymorphonuclear leukocytes (PMNs).