Isolation of Lymphocytes

The methods used to measure cellular immunity are experimentally complex. The first step is to isolate human lymphocytes from blood, which can be achieved using Ficoll density gradient centrifugation. Certain lymphocyte

— Basic Solid Phase Test Types -

Unknow

Conjugate

Unknown antigen a Direct test

Unkn Known

Known antibody

Unknown antigen

Known antibody

Unknown antigen

Conjugate (labeled antigen)

c Capture method

Unknown IgM antibody

Conjugate (labeled antigen)

Anti-IgM

b Sandwich method c Capture method

Known antibody

Strong signal

Labeled antigen d Competitive test

Known antibody

Known antibody

Known antibody

Strong signal

Labeled antigen

Labeled antigen

Labeled antigen d Competitive test

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130 2 Basic Principles of Immunology — Radioimmunosorbent Test (RIST) -

Plastic surface Anti-IgE

Plastic surface Anti-IgE

Plateau

(max. binding capacity)

Labeled IgE*

Amount of labeled IgE

Standard curve

Radioactivity in test tube

IgE in patient serum b

IgE from patient serum

Amount of unlabeled IgE in IgE* test solution

Fig. 2.28 RIST is a competitive radioimmunoassay (RIA) used for quantitative measurement of antibodies of any given Ig class (in this example total IgE) within patient serum. Anti-IgE antibodies are allowed to adsorb to the solid phase (plastic surface). In the first instance, defined concentrations of radiolabeled IgE (IgE*) are used to determine the maximum binding capacity of these antibodies (a). The actual test (b) is then performed using the IgE* concentration determined to result in 80% saturation of the fixed antibodies: The IgE* test solution is added to the fixed anti-IgE antibodies and the patient serum is then added by pipette. The more IgE the serum contains, the more IgE* will be displaced by the patients antibodies, and the lower the radioactivity level will be in the test tube. The IgE concentration in the patient serum is then calculated based on a standard curve established previously by progressively "diluting" the IgE* test solution with unlabeled IgE.

populations can be coated with magnetic beads, or sheep erythrocytes loaded with specific antibodies, then purified using a magnet or a Ficoll gradient. The fluorescence-activated cell sorter (FACS, Fig. 2.31, p. 133) is now regularly used for this purpose. In this assay, monoclonal antibodies labeled with various fluorochromes directed against cell surface antigens (such as CD4, CD8), or against intracellular cytokines (which involves the use of detergents to increase the permeability of the cell membrane), are incubated with the isolated blood lymphocytes. Alternatively, antigen-specific T lymphocytes can be labeled with MHC class I or II plus peptide tetramers (see below). Following incubation, and several washing steps, the equipment identifies and counts the antibody-loaded lymphocytes, employing magnetic pulse sorting as required.

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All rights reserved. Usage subject to terms and conditions of license.

— Radioallergosorbent Test (RAST)

- Cellulose plate (solid phase)

Antigen

-VgE

-Anti-lgE

Addition of antigen (solid phase)

Wash

Patient serum with IgE?

Wash

Addition of labeled Anti-IgE

Fig.2.29 This test isa highly sensitive detection method for the presence of specific IgE in patient serum. Antigen is bound covalently to a cellulose plate (solid phase). Any IgE in the serum that binds to the antigen is then detected using radiolabeled anti-IgE antibodies.

-VgE

Tetramer test for detection of specific T cells (Fig. 2.32, p. 134): recombinant MHC class I antigen coupled to biotin, labeled with avidin, and correctly folded together with peptide and ß2 microglobulin, forms tetramers; which are recognized by specific TCRs. Subsequent analysis of tetramer binding using FACS equipment is based on the color indicator of the avidin (fluores-cein, phycoerythrin, etc.). Tetramers specific for MHC class II antigens plus

Fig. 2.30 In the ELISPOT assay antigens, or specific anti-IL antibodies, are applied to the plastic surface. It is then possible to determine the number of immune cells releasing antibodies specific for the applied antigen, or releasing interleukins that are recognized by the applied anti-IL antibodies. Following incubation at 37 °C, the immune complexes which form around these cells can be visualized using a covering agarose layer which includes an enzyme-coupled antibody. These enzymes catalyze a color reaction, resulting in the formation of color spots, each of which will correspond to a single cell producing the specific antibody or interleukin.

Medical Microbiology

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All rights reserved. Usage subject to terms and conditions of license.

peptide can theoretically be used to assay specific CD4+ T cells, but are still difficult to manufacture. Using the tetramer test, specific T cells can be detected directly from blood or lymphoid organs. Histological applications are feasible, but still difficult.

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