Transcription
Okay, so continuing with our review for immunoserology, let's now discuss the different concepts regarding antigen and body interactions.
First, we need to define avidity and affinity. The initial force of attraction or strength of the binding between a single combining site of an antibody and an epitope found on the antigen is what we refer to as affinity. The sum total of all these forces, which I think I emphasized earlier, is dependent on the valence of the antibody involved, and this is called avidity.
These interactions are made possible by non-covalent bonds, examples of which include electrostatic attraction, hydrogen bonding, hydrophobic interaction, and van der Waals forces. Since these are non-covalent bonds, it means that these antigen-antibody interactions or bindings can still be reversible. This is why, in blood banking, elution is possible; human antibodies are cells, and we can elute or remove them considering that the bonds are non-covalent.
Now, let's understand the concept of the zone of equivalence. This is important for us to see the visual reaction whenever we perform an agglutination or precipitation procedure. The zone of equivalence is achieved when the number of multivalent sites of antigen and antibody are approximately equal. This is when optimum precipitation or agglutination occurs.
Sometimes, we fail to achieve this zone of equivalence due to what we call the prozone and postzone phenomena. Remember that whenever there is a prozone or postzone phenomenon, this results in a false negative reaction. A false negative reaction occurs because the visual reaction can only be seen when we reach the zone of equivalence.
Now, what is the prozone phenomenon, and what causes it? Whenever there is a prozone phenomenon, it means there is an excess of antibodies. The remedy for this is to dilute the serum sample. Serum dilution is the remedy for a prozone phenomenon. In contrast, when there is an antigen excess, we have less antibody, and our remedy for this is to repeat the test after one to two weeks. The purpose of this is to allow the patient to develop more antibodies.
Again, the presence of prozone and postzone phenomena will result in a false negative reaction.
Now, let's differentiate precipitation from agglutination. The difference lies in the substances involved in these reactions. Precipitation refers to reactions between two soluble substances, where human antibodies are soluble and react with soluble antigens, resulting in insoluble complexes.
On the other hand, agglutination refers to the binding of soluble antibodies with particulate antigens. A concrete example of particulate antigens would be red blood cells, which form cellular aggregates. For both precipitation and agglutination reactions, our immunoglobulin M (IgM) is significant, considering it has a valence of 10, making it easy for IgM to bridge between antigens.
Now, let's talk about specific precipitation reactions. We have three classifications for that: precipitation in a fluid medium, precipitation by passive immunodiffusion, and precipitation by electrophoretic techniques.
First, we have precipitation in a fluid medium, which includes turbidimetry and nephelometry. In these methods, we measure the amount of light that is blocked by the solution. The lower the intensity of the light detected by our photodetector, the higher the concentration of the complexes present in our sample.
The difference between turbidimetry and nephelometry lies in the arrangement of the photodetector relative to the light source and the sample. In turbidimetry, the photodetector is positioned along the light path, while in nephelometry, the photodetector is positioned at a particular angle to measure the amount of light scattered by the solution. Nephelometry is more sensitive than turbidimetry and is often used to measure macromolecules, not just antigen-antibody complexes, but also large substances such as proteins.
Next, we go to precipitation by passive immunodiffusion. It is crucial to identify whether the direction of diffusion is one-way or if both the antigen and antibody are diffusing. We have single diffusion in one dimension, double diffusion in one dimension, single diffusion in two dimensions, and double diffusion in two dimensions.
In one dimension, the direction of diffusion is vertical, and we usually use a tube or a column. In single diffusion, only the antigens are diffusing while the antibodies are layered on top of the agar. This technique is also known as the Oudin's technique.
In double diffusion in one dimension, both the antigen and antibody are diffusing. The intervening column, which is just plain agar, allows the diffusing particles to go to an area of lower concentration, resulting in the formation of a precipitation band.
Next, we have single diffusion in two dimensions, where we use a petri dish or a slide, allowing diffusion in several directions. The positive result would be a precipitating ring, with the antibody incorporated in the agar and the antigen added to a pre-cut well.
There are two modifications of the single diffusion in two dimensions technique: the endpoint method (Mancini method) and the kinetic method. The endpoint method allows the antigen to fully diffuse, while the kinetic method does not allow complete diffusion.
Lastly, we have precipitation by immune diffusion, specifically the double diffusion in two dimensions technique, also known as the Ouchterlony technique. This technique helps identify whether two antigens are identical, non-identical, or partially identical based on the shape of the precipitating line.
Now, let's move on to agglutination reactions. There are two phases: sensitization and visible aggregates formation. I have included six types of agglutination reactions.
The first type involves antigens found naturally on a particle, such as red blood cells. This can be done to detect both antigen and antibody. For example, in blood typing, both forward and reverse typing are considered direct agglutination.
Next, we have passive agglutination, where particles are coated with antigens not normally found on their surface to detect antibodies from the patient sample.
In reverse passive agglutination, instead of coating the particles with antigens, we coat them with antibodies to detect antigens.
We also have agglutination using bacterial proteins as inner particles to which antibodies are attached. An example is Protein A from staphylococci, which binds to the Fc portion of IgG.
Next, we have reactions based on competition between particulate and soluble antigens for limited antibody combining sites. In this case, the absence of agglutination indicates a positive reaction, known as hemagglutination inhibition.
Lastly, we have anti-human globulin tests, which employ an anti-human globulin to detect antibody-coated cells. This is especially useful for detecting IgG antibodies.
Now, let's discuss diseases of the immune system, starting with hypersensitivity reactions. This is the overactivity of the immune system leading to tissue damage, classified into four types by Coombs and Gell.
Type 1 hypersensitivity, or anaphylaxis, involves cell-bound antibodies reacting with antigens to release physiologically active substances, with IgE as the mediator. Examples include anaphylaxis, hay fever, food allergies, bee stings, and bronchial asthma.
Type 2 hypersensitivity, known as cytotoxic hypersensitivity, involves free antibodies reacting with antigens associated with cell surfaces, such as red blood cells and white blood cells. Examples include transfusion reactions and hemolytic anemia.
Type 3 hypersensitivity, or immune complex hypersensitivity, occurs when antibodies react with soluble antigens, forming complexes that deposit in tissues, leading to damage. Examples include serum sickness and rheumatoid arthritis.
Type 4 hypersensitivity is the only type not mediated by antibodies. It is cell-mediated or delayed hypersensitivity, where T-cells release pro-inflammatory cytokines. Examples include contact dermatitis and the tuberculin skin test.
Next, we have complement deficiencies. Deficiencies in the classical pathway can lead to lupus-like symptoms. A deficiency in C2 and C3 can result in recurrent infections with gram-positive encapsulated organisms.
Deficiency of the membrane attack complex can lead to recurrent systemic infections. Deficiency of the C1 esterase inhibitor results in hereditary angioedema.
Now, let's proceed to immunodeficiency, which involves defects in the T and B cell systems. For example, Bruton's agammaglobulinemia is due to arrested differentiation at the pre-B cell stage, leading to the absence of B cells and immunoglobulins.
Selective IgA deficiency is characterized by a lack of IgA, while DiGeorge anomaly involves a developmental abnormality affecting T-cell development.
Wiskott-Aldrich syndrome is a rare X-linked recessive syndrome characterized by a triad of thrombocytopenia, eczema, and recurrent infections.
Ataxia-telangiectasia is a rare autosomal recessive syndrome characterized by chronic lung disease and progressive neurological degeneration.
Severe combined immunodeficiency affects both T and B cell functions, leading to severe infections that can be fatal within the first two years of life.
Now, let's discuss immunoproliferative disorders, starting with monoclonal gammopathies. These disorders are characterized by the abnormal proliferation of clonal cells, resulting in the production of proteins such as intact monoclonal immunoglobulin and free light and heavy chains.
Multiple myeloma develops in activated memory B cells or plasma blasts, often due to chromosomal translocation. Waldenström macroglobulinemia is a lymphoplasmacytic cancer producing large amounts of IgM.
Light chain amyloidosis involves a clonal population of plasma cells producing monoclonal light chains that misfold and deposit in tissues. X-linked lymphoproliferative disease results in a life-threatening reaction when individuals are infected with Epstein-Barr virus.
Now, let's move on to autoimmune diseases, which result from the loss of self-tolerance in the immune system. Autoimmune diseases can be systemic or organ-specific, affecting various body systems.
Examples include ankylosing spondylitis, autoimmune Addison's disease, Graves' disease, Hashimoto's thyroiditis, chronic active hepatitis, primary biliary cirrhosis, and rheumatoid arthritis.
In systemic lupus erythematosus, the hallmark autoantibodies are anti-double-stranded DNA and anti-Smith antibodies.
Now, let's discuss transplantation immunology. There are different categories of transplant donors: autografts, homografts, isografts, and xenografts.
Transplant rejection can occur in several forms: hyperacute rejection, acute rejection, chronic rejection, and graft-versus-host disease.
Lastly, let's cover tumor markers and their clinical utility. Tumor markers are used to check for recurrence of cancer, with examples including AFP for hepatocellular carcinoma, CA 125 for ovarian cancer, and PSA for prostate cancer.
Now, let's move on to infectious disease serology, starting with syphilis. Syphilis is caused by Treponema pallidum, and serological methods for diagnosis are divided into non-treponemal tests and treponemal tests.
Non-treponemal tests detect non-specific antibodies, while treponemal tests confirm the presence of antibodies directed against Treponema pallidum.
Next, we have Lyme disease, which is diagnosed using a two-tiered approach involving screening tests and confirmatory tests like Western blot.
For group A streptococcal infections, we measure anti-streptolysin O levels to assess recent infections.
Infectious mononucleosis is associated with Epstein-Barr virus, and we can identify serological responses based on the presence of specific antibodies.
Lastly, hepatitis A and B infections have specific markers for diagnosis, and HIV infection is monitored through various tests, including antibody detection and viral load assays.
That concludes our session for today. Thank you!