Antigens, Phagocytosis, T-Cells, B-Cells, Monoclonal Antibodies, HIV & ELISA

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Antigens & Non-Specific Phagocytosis

Antigens are cell-surface proteins/glycoproteins with a specific 3D tertiary structure that trigger an immune response when recognised as non-self. In phagocytosis: 1. Phagocyte is attracted to pathogen by chemical products (chemotaxis). 2. Phagocyte engulfs pathogen by endocytosis to form a phagosome. 3. Lysosomes fuse with the phagosome (forming a phagolysosome) and release hydrolytic lysozymes that digest the pathogen. 4. Phagocyte displays pathogen antigens on MHC proteins on its surface, becoming an antigen-presenting cell (APC).

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Cell-Mediated Response (T-Lymphocytes)

T-lymphocytes mature in the thymus and respond to antigens presented on body cells (APCs, virus-infected cells, cancer cells). Helper T-cells (Tₕ) with complementary CD4/T-cell receptors bind presented antigens and divide by mitosis to secrete cytokines that: stimulate phagocytes, activate cytotoxic T-cells (T꜀), and stimulate specific B-cells. Cytotoxic T-cells release perforin, which forms pores in infected host cell membranes causing water/ion influx and cell lysis.

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Humoral Response (B-Lymphocytes & Clonal Selection)

B-lymphocytes mature in bone marrow and respond to soluble antigens in blood/tissue fluid. 1. A B-cell with a complementary membrane-bound antibody binds the foreign antigen and engulfs/displays it on its surface. 2. An activated Helper T-cell binds the presented antigen and secretes cytokines that stimulate the B-cell (clonal selection). 3. The B-cell divides rapidly by mitosis (clonal expansion) and differentiates into plasma cells (secrete specific monoclonal antibodies) and memory B-cells (provide rapid, high-concentration secondary response).

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Antibody Structure & Agglutination

Y-shaped quaternary proteins (immunoglobulins) composed of four polypeptide chains (two heavy chains and two light chains) held together by disulfide bridges. Each antibody has a constant region (binds receptors on phagocytes) and two identical variable regions formed by a specific amino acid sequence/tertiary structure that serves as the antigen-binding site. Having two binding sites allows antibodies to cross-link multiple pathogens together (agglutination) for efficient phagocytosis.

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Monoclonal Antibodies & ELISA (Enzyme-Linked Immunosorbent Assay)

Monoclonal antibodies are identical antibodies produced from a single clone of B-cell hybridomas, specific to one epitope. In an indirect ELISA (e.g. HIV testing): 1. HIV antigen is bound to well floor; patient serum is added so any anti-HIV antibodies bind. 2. Wash well to remove unbound antibodies. 3. Add secondary monoclonal antibody conjugated to an enzyme, specific to human antibodies. 4. Wash well thoroughly to remove unbound enzyme-linked antibody (preventing false positives). 5. Add colourless substrate; enzyme converts it into a coloured product.

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HIV Structure, Replication & AIDS

Human Immunodeficiency Virus consists of a lipid envelope embedded with attachment glycoproteins (gp120), surrounding a protein capsid containing two single-stranded RNA molecules and the enzymes reverse transcriptase and integrase. HIV binds CD4 receptors on Helper T-cells; viral RNA enters, reverse transcriptase synthesises viral cDNA from viral RNA, integrase inserts viral DNA into host genome, and host RNA polymerase/ribosomes assemble new virions that bud out and lyse Tₕ cells. When Tₕ counts fall below ~200 cells mm⁻³, B-cells and phagocytes cannot be activated (AIDS), leading to fatal opportunistic infections.

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1. Q1

   Describe how the presentation of a foreign antigen leads to the secretion of specific antibodies by plasma cells. (5 marks)

   Check Mark Scheme

   Click “Check Mark Scheme” to compare your answer, 1. A B-lymphocyte with membrane-bound surface antibodies complementary in shape to the foreign antigen binds the antigen, internalises it by endocytosis, and presents the antigen on its cell surface membrane [1 mark]. 2. A Helper T-cell (Tₕ) with a complementary receptor binds to the presented antigen on the B-cell and secretes cytokines [1 mark]. 3. The cytokines stimulate the selected B-cell to divide rapidly by mitosis (clonal expansion), producing a large clone of genetically identical B-cells [1 mark]. 4. These cloned B-cells differentiate into memory B-cells and plasma cells [1 mark]. 5. Plasma cells contain extensive rough endoplasmic reticulum and Golgi apparatus to synthesise and secrete large quantities of specific monoclonal antibodies by exocytosis [1 mark].

2. Q2

   Explain why the secondary immune response is much faster and produces a much higher concentration of antibodies than the primary immune response, and explain why a person can still catch influenza multiple times. (5 marks)

   Check Mark Scheme

   Click “Check Mark Scheme” to compare your answer, 1. During the primary immune response, clonal expansion produces long-lived memory B-cells and memory T-cells that remain circulating in the blood and lymph [1 mark]. 2. Upon re-infection with the same pathogen/antigen, memory B-cells recognise the antigen immediately without waiting for slow clonal selection from a tiny baseline pool [1 mark]. 3. Memory B-cells divide rapidly by mitosis and differentiate into a much larger number of plasma cells, secreting antibodies at a higher rate and destroying the pathogen before symptoms develop [1 mark]. 4. However, the influenza virus has a high mutation rate in the genes coding for its surface glycoproteins (haemagglutinin/neuraminidase), causing antigenic variability [1 mark]. 5. The new viral antigens have a different 3D tertiary structure that is no longer complementary to the receptors on existing memory cells or previously formed antibodies, requiring a new primary immune response [1 mark].

3. Q3

   Describe how a sandwich ELISA test is used to detect the presence of a specific malaria parasite antigen in a patient's blood sample. (4 marks)

   Check Mark Scheme

   Click “Check Mark Scheme” to compare your answer, 1. Immobilise primary monoclonal antibodies complementary to the malaria antigen onto the base of the test well, then add the patient's blood sample so any malaria antigens bind to form antigen-antibody complexes [1 mark]. 2. Wash the well to remove unbound blood components, then add a second monoclonal antibody that is complementary to a different epitope on the malaria antigen and has a reporter enzyme attached [1 mark]. 3. Wash the well thoroughly to remove any unbound enzyme-linked secondary antibodies so they do not cause a false-positive result [1 mark]. 4. Add a colourless substrate for the enzyme; if the malaria antigen is present, the bound enzyme catalyses a reaction producing a coloured product (whose intensity is proportional to antigen concentration) [1 mark].

4. Q4

   Explain how HIV replicates inside a human helper T-cell and how this ultimately leads to death from secondary infections in AIDS. (5 marks)

   Check Mark Scheme

   Click “Check Mark Scheme” to compare your answer, 1. The HIV attachment protein (gp120) binds specifically to the CD4 receptor on the cell surface membrane of a Helper T-cell, allowing the viral capsid to enter and release viral RNA and enzymes [1 mark]. 2. Viral reverse transcriptase uses the viral RNA as a template to synthesise complementary viral DNA (cDNA) from free host DNA nucleotides [1 mark]. 3. The double-stranded viral DNA is inserted into the host cell's nuclear DNA by the viral enzyme integrase, where host RNA polymerase transcribes it into viral mRNA and genomic RNA [1 mark]. 4. Host ribosomes translate viral mRNA into capsid and envelope proteins; new HIV particles assemble and bud out through the host cell membrane, lysing and destroying the Helper T-cell [1 mark]. 5. With extremely low Helper T-cell numbers, the patient cannot produce enough cytokines to activate B-cells (for antibody production) or cytotoxic T-cells/phagocytes, leaving them unable to destroy opportunistic pathogens (e.g. tuberculosis or pneumonia) [1 mark].

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