Diagnostic Testing

Question 1

What are western blot assays used for?

Answer 1

Used to detect patient antibodies against particular proteins or antigens

Confirmation after positive ELISA screening test

Question 2

Why are monoclonal antibodies useful in diagnostic testing?

Answer 2

They can evaluate for the presence of a specific antigen by binding to it

Note: the “antigen” can be another antibody; monoclonal antibodies can also detect the presence or absence of specific antibodies

Question 3

Which test would you use to get a patients white blood cell count?

Answer 3

Flow cytometry

Question 4

Which assay would you use to evaluate your patient’s HIV progression?

Answer 4

Flow cytometry to count T-cells

Question 5

Which assays can be used to evaluate cell function?

Answer 5

• Functional flow cytometry assay
  
  • Evaluate for the presence of a fluorescent product
  • Ex: Chronic granulomatous disease = no oxidative burst to kill pathogens
    
    • Use permeable dye
    • Oxidative burst = cell will fluoresce
    • No oxidative burst = cell will not fluoresce

Question 6

What is immunofluorescence used for?

Answer 6

Evaluating for an autoimmune response, especially in evaluating kidney disease and autoimmunity

Question 7

What are two possible sources of error in tests that use antibodies?

Answer 7

Corss reactivity with very closely related antigens

Antibodies present in the patient’s blood can interfere with the assay

Question 8

Which diagnostic tests can quantify the amount of IgG, IgM, IgA, and IgE isotypes in a patient sample?

Answer 8

Nephelometry (Use if large amounts are present as in IgG, IgM, IgA)

ELISA (Use if less abundant; IgE, specific subgroups of an isotype)

Question 9

What is flow cytometry used for?

Answer 9

• White blood cell differential analysis
  
  • Can evaluate the presence and amounts of lymphocytes, monocytes, granulocytes
• Count the type of a specific cell
  
  • Ex: evaluate immune status of an HIV patient by counting T-cells
• Anything where you are looking at amounts/types of cells

Question 10

Suppose a flow cytometry assay confirms that a patient’s CD4+ helper T-cells express CD40L when stimulated.

Can you rule out hyper-IgM? Why or why not?

Answer 10

You cannot rule out hyper-IgM, especially if there is a high clinical suspicion

CD40L may be present, but nonfunctional

• It may fail to bind to CD40
• There may be a defect in cytokine production

The takeaway: Standard flow cytometry can evaluate the presence or absence of a protein, but not necessarily its function

(But there are functional flow cytometry assays that evaluate for the presence of a fluorescent product)

Question 11

Your 3-week old patient has a family history of hyper-IgM syndrome

What is an appropriate assay to evaluate for the serum immunoglobulin levels?

Answer 11

Nephelometry to measure levels of IgM, IgG

(Could also use ELISA)

Question 12

What is a monoclonal antibody?

Answer 12

An antibody that binds one specific epitope of one specific antigen

Epitope = site on the antigen

Question 13

What is ELISA used for?

Answer 13

To measure amounts of antigen or antibody present in a patient specimen

Can determine amounts of IgG, IgM, IgA, IgE etc

Can also determine amounts of specific subgroups of each isotype

Question 14

What is nephelometry?

Describe the process of using monoclonal antibodies in nephelometry:

Answer 14

Nephelometry measures the amount of light that gets scattered while passing through a substance

• Monoclonal antibodies specific for an antigen are put into solution with the patient sample
  
  • Note: antibodies can be antigens! Known monoclonal antibodies can be specific for human antibodies that you are looking for in the patient sample
• If the antigen is present, it will bind to the monoclonal antibody and form a precipitate
• The precepitate will cause the light to scatter
• More antibody:antigen bindign = more scatter

Question 15

In patients with hyper-IgM syndrome, which T-cells would you expect to be deficient?

Which protein is commonly deficient in these cells?

Answer 15

Th2 CD4+ Helper T-cells

These cells are required to form the germinal centers in which IgM would class-switch to other isotypes

Often, patients with IgM fail to express CD40L on the Th2 CD4+ Helper T-cells; without CD40L, the T-cell cannot form the CD40:CD40L interaction needed to produce cytokines and form the germinal center

Question 16

What is a westen blot assay?

Describe the process of using monoclonal antibodies in western blot assays

Answer 16

Western blot assays detect patient antibodies against specific antigens

• Protein is separated using gel electrophoresis
• Proteins are transferred to filter papter
• The paper is exposed to a patient sample
• If the patient has antibodies against the protiens, they will bind to the proteins on the filter paper. Wash
• Expose the fliter paper to detection antibodies: enzyme-linked anti-human isotype antibodies (ex: anti-human IgG). Wash
• Measure enzymatic signal from the detection antibodies

Question 17

Which assay would you use to evaluate the protein expression of lymphocyte subsets?

Answer 17

Flow cytometry

Question 18

What is nephelometry used for?

What kind of information can you find out

Answer 18

Nephelometry is used to detect antigens or antibodies in the serum.

It can quantify the amount of antibody isotopes (IgG, IgM, IgA) in a patient sample

Question 19

Which assays are most commonly used to detect antibodies in a patient sample?

Answer 19

Nephelometry

ELISA

Western Blot

Question 20

What is ELISA?

Describe the process of using monoclonal antibodies in ELISA

Answer 20

ELISA = Enzyme-Linked Immunosorbent Assay

To measure an antibody in the patient serum

• Known antigen is fixed on a well
• Add patient serum. If antibody for the antigen is present, it will bind (Wash)
• Monoclonal antibody attached to an enzyme is added. This monoclonal antibody is specific for the patient’s antigen that is bound to the known antibody (Wash)
• Add enzyme substrate and measure enzyme activity
  
  • Color change = the monoclonal antibody w/attached enzyme is present, which means the patient’s antibody is present
  • Can quantify activity to quantify amount of antibody

To measure an antigen in the patient serum

• Basically the same process, but you fix a known, monoclonal antibody on a well (instead of an antigen)
• The enzyme-linked monoclonal antibody is specific for a different epitope of the antigen in question
• Quantify binding by measuring enzyme activity (Wash steps are still important)

Question 21

What is immunofluorescence?

Describe the process of using monoclonal antibodies in immunofluorescence

Answer 21

Immunofluorescence is a method of using fluorescent probes bound to antibodies to detect the presense whatever that antibody is specific for (usually autoantibodies or complement bound to pt. tissue)

• Monoclonal antibodies specific for the autoantibody or complement protein are labled with a fluorescent probe
• The labeled antibodies are exposed to the patient sample. Wash
• Expose sample to lights (laser) that will cause the fluorescent probes to emit a light of known wavelength
• Visualize the fluorescence via microscope
  
  • The binding pattern will give information about the type of antibody or immune response that is present

Question 22

What is flow cytometry?

Describe the process of using monoclonal antibodies in flow cytometry

Answer 22

Flow cytometry is a method of evaluating cells as they pass one by one in a fluid stream through a laser beam

To evaluate intrinsic properties

• Cells pass through a laser
• Intrinsic properties (size, complexity, nuclear features) will cause a specific pattern of forward and side scatter that is unique to each cell type

To evaluate protein markers (ex, presence of CD3, CD4, CD8)

• Cells are reacted with monoclonal antibodies labeled with fluorescent probes
  
  • ​Can use different antibodies with different specificities labeled with their own probes at one time
• Solution is passed through the flow cytometer
• Each cell will give off a specific signal based on the combination of fluorescently-labeled antibodies that bind
  
  • Can use to identify very specific cell types (ex: CD4+ T-cells)