Topic 5  Antigen -Antibody Interactions

Introduction

Antibodies constitute the humoral arm of acquired immunity that provide protection against infectious organisms and their toxic products.  Therefore, the interaction between antigen and antibody is of paramount importance.  We should remember from the previous lesson that when an antigen and its specific antibody combine they interact through the chemical groups on the surface of the epitope and the complementarity determining regions (CDRs) on the immunoglobulin.  In a classical chemical reaction molecules are assembled through the establishment of firm covalent bonds.  These bonds can be broken only by the input of a significant amount of energy.  By contrast, the formation of noncovalent bonds provides a rapid and reversible way of forming complexes and permits reuse of molecules in a way that covalent bonding would not allow.   In this section we will explore details of the nature of the antigen-antibody interactions.  We will learn about biological consequences of these interactions and their applications in various immunologic assays.

Objectives

On completion of this section and the required readings, you should be able to:

n  describe the forces that encourage primary antigen-antibody interactions;

n  distinguish between antibody affinity and avidity;

n  describe the strength of the primary antigen-Antibody interactions using equilibrium dialysis. Include the terms K and Ka;

n  compare and contrast RIA and ELISA;

n  describe direct and indirect fluorescent antibody methods;

n  describe a secondary antigen-antibody interaction in terms of lattice formation and antigen - antibody ratios;

n  explain zone phenomena;

n  construct a table to compare the various procedures used to determine the presence of soluble antigen or antibody in a fluid and in a gel;

n  assess the reasons for using the different gel precipitin reactions;

n  distinguish between agglutination and precipitin reactions and give the advantages and disadvantages of each.

Required Reading

Refer to the required readings in the textbook key

P Key Terms

P Key Concepts

n  The reaction between an antibody and an epitope does not involve covalent forces.  Consequently, for a significant interaction, the antibody combining site and the epitope require a close steric.

n  Only the reaction between a multivalent antigen and at least a divalent antibody can bring about secondary antigen-antibody reactions which depend upon cross-linking of antigen molecules by antibodies

n  The interaction of a soluble antigen and precipitating antibody forms an antigen-antibody complex that has a lattice structure and precipitates out of solution. 

n  The interaction between a soluble antibody and an insoluble particulate antigen results in agglutination.  Agglutination reactions are more sensitive then precipitin reactions.

DID YOU KNOW?

Radioimmunoasssay is a test that is based on competitive inhibition of nonlabeled and labeled antigen for antibody (in antibody deficiency).  It necessitates the separation of antibody-bound from nonbound labeled antigen.  Separation is usually achieved by the anti-immunoglobulin procedure.

ELISA.   This enzyme -linked immunosorbent assay is essentially a solid-phase immunoassay in which an enzyme is linked to the anti-immunoglobulin.  Quantitation of enzyme-linked anti-immunoglobulin is achieved by colorimetric evaluation, after addition of a substrate which changes colour upon the action of the enzyme.

Immunofluorescence.  In immunofluorescence, the antigen is detected by the use of fluorescence-labeled immunoglobulins.  In direct immunofluorescence, the antibody to the antigen in question carries a fluorescent label.  In indirect immunofluorescence, the antigen-specific antibody is not labeled; it is detected by the addition of fluorescently labeled anti-immunoglobulin

Origins of Radioimmunoasssay. 

We decided to include this story about developing of RIA because it illustrates, how the process of the discovery often occurs - unexpectedly.  Frequently in order to establish and prove a new theory, old beliefs must be overturned.  The ones who challenge the current status quo are often considered to be wrong and are not taken seriously.  New discoveries  were sometimes so outrageous that the science community had difficulties accepting them. The main idea we would like you to take from this review is that it is simply wrong to assume that we have all the answers yet, or that we have a monopoly on the truth.   We are barely “scratching the surface” of science, and as we learn more, we can see how much more there is still to be discovered.   However it is equally true that even if some discoveries happen “by accident” they are later recognized and appreciated only by “prepared minds.”  Therefore immunology has to be viewed as a dynamic, constantly changing field.  The example below shows how the new answers may sometimes come from completely unexpected places.

Measurements of the concentrations of many bioactive substances, such as hormones, are routinely accomplished by means of RIA.  These assays are performed fairly rapidly; they require only small amounts of sample, and large numbers of samples can be evaluated simultaneously.  However, this was not always the case.

Interest in measuring the levels of biologically relevant substances has a long history.  For substances that are present in milligram quantities, or greater, numerous chemical and biochemical techniques have been available.  However, for substances present in the submilligram range, techniques have been limited.  This is the case for many regulatory substances.

As recently as the early 1960s, bioassays were the method of choice and, in some instances, the only method available for assessing the concentration of many hormones, neurotransmitters, and other significant biologically active substances.  Bioassays can be extremely sensitive.  However, they require a biological test system and inherently are difficult to calibrate.  Moreover, they are cumbersome to perform.

The development of RIA, and their subsequent exploitation for a wide range of application, arose as an attempt to test a hypothesis concerning insulin degradation in maturity-onset diabetes and not as an attempt to generate a more sensitive tool for measuring insulin itself.

While attempting to study the metabolism of radioactively labeled insulin, Berson, Yalow, and their colleagues noted a curious phenomenon.  Individuals who had received insulin treatment, for a variety of reasons, displayed slower disappearance of the labeled insulin than did persons who had not been exposed to insulin injections.  They considered that this was the result of binding of the injected insulin by Abs produced by prior exposure to insulin.  They subsequently developed highly sensitive radio-isotopic techniques to detect the insulin-Ab complexes.  The culmination of their effort was the development of the RIA. 

In retrospect, it seems surprising that the original attempts to publish the results of the pioneering RIA experiments met with skepticism and resistance from immunologists.  Nevertheless, the validity and value of RIA has been more than amply demonstrated since that time, as evidenced by the extensive dependence upon this technique in both clinical and research laboratories.  In addition, the scientific community expressed its appreciation and acknowledged Rosalyn Yalow’s contribution in the development of the powerful tool by awarding her the Nobel Prize in 1977.

As appeared in Fundamental Immunology. Coleman, Lombard, Sicard, Wm. C. Brown Publishers, 1992

Rosalyn S. Yalow was born in New York in 1021. She was not an immunologist but an endocrinologist who was studying diabetes mellitus.  She required a method of measuring the very low levels of antibodies against insulin found in the serum of diabetics and developed the idea of labeling the insulin with a radioisotope.  This technique was eventually refined so that exquisitely small levels of molecules such as hormones could be readily measured.  The technique of radioimmunoassay is now used in many fields other than endocrinology.

I.R. Tizard. Immunology an Introduction, 4^th edition, Saunders College Publishing, Harcourt Brace College Publishers, 1995

Review Questions

1. Textbook Study Questions

Review questions at the end of the Chapter 6.  The answers with explanations are available at the end of the textbook.

2.  Multiple Choice Questions

1.  The major forces linking antigen to antibody are

A) hydrogen bonds

B)  van der Walls

C)  hydrophobic bonds

D) ionic bonds

E)  all of the above

2.  The complementarity determining regions of immunoglobulins

A) activate complement

B)  bind to cells

C)  mediate opsonization

D) bind to antigen

E)  make the molecule flexible

3.  The strength of binding between an antigen and the Fab of its antibody is called

A) avidity

B)  valency

C)  hydrophobicity

D) electrophoretic mobility

E)  affinity

4.  The ligand for the small protein avidin is

A) immunoglobulin

B)  biotin

C)  fluorescein

D)ferritin

E)  antiglobulin

5.  Immune complexes precipitates are formed in

A) antigen excess

B)  antibody excess

C)  the zone of equivalence of antigen and antibody

D) the presence of concentrated acid

E)  the absence of electrolyte

3. Short Answer/Definition Questions

1.  Cross reactivity of antibodies creates problems for their application in serology. Explain.

2.  Differentiate between a primary and a secondary antigen-antibody reaction. What are three important characteristics that distinguish the two reactions?

3.  What kinds of noncovalent interactions are important in antigen-antibody interactions? What aspect of these interactions is most important and why?

4.  How is equilibrium dialysis used to measure PRIMARY antigen-antibody reactions?

5.  Differentiate between avidity and affinity.

6.  Discuss the term lattice formation.

7.  What are the pros and cons of RIA?

8.  Describe two types of immunofluorescence tests.

9.  What are the advantages of the indirect procedure over the direct procedure?

10. What are some commonly used fluors?

11. What colour does each fluor emit?

12. What makes precipitin reactions visible?

13. What two factors are important in the development of precipitin reactions?

14. Three patterns can be observed in the Ouchterlony test. Draw and label diagrams to illustrate these patterns. What does each pattern show?

15. What is the major advantage of immunoelectrophoresis over immunodiffusion? What are the disadvantages?

16. How does agglutination differ from precipitation?

17. Why are agglutination tests more sensitive that precipitin tests? Differentiate between direct and indirect agglutination reactions.

18. What is a major advantage of indirect agglutination reaction over direct reactions?

Where to Go from Here

Once you have completed the review, take some time and complete the objectives. If you are having trouble with any of the concepts, contact your instructor.

It is important that you get clarification on any of the topics or concepts that you are having difficulty with as soon as they arise. Many of the concepts build on each other so it is vital that you not only keep up with the schedule, but clear up any questions or problems as they arise. Also, remember to regularly check your Instructor and Assignment Information for assignments and due dates for completing them.

When you are confident that you can complete the objectives, proceed to the next topic.