2.4.2 Enzyme-Linked Immunosorbent Assay

What is a monoclonal antibody?

• An antibody produced from genetically identical or cloned B plasma cells, so all have the same tertiary structure.

Outline the uses of monoclonal antibodies

• Medical diagnosis

• Therapeutic drug delivery

• Block antigens or receptors

• Detection

What property of monoclonal antibodies allows them to target specific cells?

• They have a specific tertiary structure, so the variable region is complementary to a receptor, protein, or antigen found only on a specific cell type, for example a cancer cell.

How are monoclonal antibodies used to deliver drugs to specific cells?

• A therapeutic drug is attached to the antibody.

• The antibody binds to the cell’s target receptor, protein, or antigen, forming an antigen-antibody complex and delivering the drug.

What is another way monoclonal antibodies are used in treatment?

• Some monoclonal antibodies are designed to block antigens or receptors on cells.

What property allows monoclonal antibodies to be used in medical diagnosis?

• They have a specific tertiary structure, so the variable region is complementary to a receptor, protein, or antigen found only on a specific cell type associated with the diagnosis.

How are monoclonal antibodies used to detect specific molecules?

• A dye, stain, or fluorescent marker is attached to the antibody.

• The antibody binds to the cell’s target receptor, protein, or antigen, forming an antigen-antibody complex that can be visualised.

Briefly outline the difference between direct, indirect and sandwich ELISA.

• Direct ELISA: antigen from sample attached to well; enzyme-linked antibody binds directly to antigen.

• Indirect ELISA: antigen attached to well; sample antibody binds to antigen; enzyme-linked secondary antibody binds to sample antibody.

• Sandwich ELISA: antibody attached to well; antigen from sample binds; enzyme-linked secondary antibody binds to antigen.

What is the first step in direct ELISA?

• Attach the sample with potential antigens to the well.

What is the second step in direct ELISA?

• Add complementary monoclonal antibodies with enzymes attached, which bind to the antigens if present.

What is the third step in direct ELISA?

• Wash the well to remove unbound antibodies, preventing false positives.

What is the fourth step in direct ELISA?

• Add substrate, which the enzymes act on to create products that cause a colour change, indicating a positive result.

What is the first step in sandwich ELISA?

• Attach specific monoclonal antibodies to the well.

What is the second step in sandwich ELISA?

• Add the sample with potential antigens, then wash the well.

What is the third step in sandwich ELISA?

• Add complementary monoclonal antibodies with enzymes attached, which bind to the antigens if present.

What is the fourth step in sandwich ELISA?

• Wash the well to remove unbound antibodies, preventing false positives.

What is the fifth step in sandwich ELISA?

• Add substrate, which the enzymes act on to create products that cause a colour change, indicating a positive result.

What is the first step in indirect ELISA?

• Attach specific antigens to the well.

What is the second step in indirect ELISA?

• Add the sample with potential antibodies, then wash the well.

What is the third step in indirect ELISA?

• Add complementary monoclonal antibodies with enzymes attached, which bind to the antibodies if present.

What is the fourth step in indirect ELISA?

• Wash the well to remove unbound antibodies, preventing false positives.

What is the fifth step in indirect ELISA?

• Add substrate, which the enzymes act on to create products that cause a colour change, indicating a positive result.

What is one purpose of a control well in the ELISA test?

• To compare to the test and show that only the enzyme causes the colour change.

What is another purpose of a control well in the ELISA test?

• To compare to the test and show that all unbound antibodies have been washed away.

Why can failure to thoroughly wash the well result in a false positive?

• Antibodies with enzymes attached remain and are not washed out, so the substrate is converted into a coloured product.

What is one ethical issue associated with pre-clinical testing of vaccines and monoclonal antibodies?

• Pre-clinical testing on animals causes potential stress, harm, or mistreatment.

• However, animals are not killed and it helps produce new drugs to reduce human suffering.

What is an ethical issue associated with clinical trials on humans?

• Potential harm or side-effects.

What is a potential ethical issue with vaccines?

• People may continue high-risk activities and still develop or pass on the pathogen.

What is an ethical issue associated with using drugs?

• Potentially dangerous side effects.

What question should be asked about sample size when evaluating methodology?

• Was the sample size large enough to be representative of the population?

What question should be asked about participant diversity?

• Were participants diverse in terms of age, sex, ethnicity, and health status?

What question should be asked about control groups?

• Were placebo or control groups used for comparison?

What question should be asked about study duration?

• Was the duration of the study long enough to show long-term effects?

What question should be asked about reducing bias?

• Was the trial double-blind, meaning neither the doctor nor the patient knew who was given the drug or placebo, to reduce bias?

What question should be asked about side effects?

• What side effects were observed, and how frequently did they occur?

What question should be asked about control variables?

• Were factors such as health and previous medications controlled to ensure validity?

What question should be asked about statistical analysis?

• Was a statistical test used to see if there was a significant difference between the start and final results?

What can a large standard deviation in final results indicate?

• Large standard deviation shows that some patients did not benefit from the treatment.

What does overlap between standard deviations of start and final results suggest?

• Overlap suggests there may not be a significant difference in start and final results.

What questions should be asked about dosage?

• What dosage was optimum?

• Does increasing the dose increase effectiveness enough to justify the extra cost?

What question should be asked about production and distribution?

• Was the cost of production and distribution low enough?

If two variables change together in a way that can be seen on a scatter diagram, what is this called?

• Correlation = a change in one variable is reflected by a change in another variable, which can be identified on a scatter diagram.

What term describes when a change in one variable directly causes a change in another variable?

• Causation = a change in one variable causes a change in another variable.

Why is it incorrect to assume that because two variables are correlated, one must cause the other?

• Correlation does not mean causation → there may be other factors involved.