The fine specificity of antibodies has made them powerful tools for visualizing specific intracellular tissue components expression that are typical of cell lineages, maturational stages, and a number of different types of leukemia. Most cancer centers are equipped with flow cy-tometers that are capable of performing and interpreting the multiparameter analyses necessary to provide useful pro files of surface markers on tumor cell populations. Flow cytometric determination of immuno-phenotypes allows:
Confirmation of diagnosis ■ Diagnosis when no clear judgment can be made based on morphology or patterns of cytochemical staining Identification of aberrant antigen profiles that can help identify the return of leukemia during remission Improved prediction of the course of the disease
An ALL of the pre-B lineage
(the most commonly occurring ALL)
CD19 (a B-cell coreceptor)
CD34 (marker of hematopoietic precursors)
ALL of the T lineage CD8
A B-lineage CLL
ALL of the T lineage CD8
(marker of some T cells, thymocytes and pluripotent hematopoietic cells)
Distribution of selected markers on some leukemic cell types. Shown are typical surface antigen profiles found on many, but not all, ALLs and CLLs.
by immunoelectron microscopy. In this technique, an electron-dense label is either conjugated to the Fc portion of a specific antibody for direct staining or conjugated to an antiimmunoglobulin reagent for indirect staining. A number of electron-dense labels have been employed, including ferritin and colloidal gold. Because the electron-dense label absorbs electrons, it can be visualized with the electron microscope as small black dots. In the case of immunogold labeling, different antibodies can be conjugated with gold particles of different sizes, allowing identification of several antigens within a cell by the different sizes of the electron-dense gold particles attached to the antibodies (Figure 6-16).
6 An immunoelectronmicrograph of the surface of a B-cell lymphoma was stained with two antibodies: one against class II MHC molecules labeled with 30-nm gold particles, and another against MHC class I molecules labeled with 15-nm gold particles. The density of class I molecules exceeds that of class II on this cell. Bar = 500 nm. [From A. Jenei et al., 1997, PNAS 94:7269-1214; courtesy of A. Jenei and S. Damjanovich, University Medical School of Debrecen, Hungary.]
■ Antigen-antibody interactions depend on four types of noncovalent interactions: hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions.
■ The affinity constant, which can be determined by Scatchard analysis, provides a quantitative measure of the strength of the interaction between an epitope of the antigen and a single binding site of an antibody. The avidity reflects the overall strength of the interactions between a multivalent antibody molecule and a multivalent antigen molecule at multiple sites.
■ The interaction of a soluble antigen and precipitating antibody in a liquid or gel medium forms an Ag-Ab precipitate. Electrophoresis can be combined with precipitation in gels in a technique called immunoelectrophoresis.
■ The interaction between a particulate antigen and agglutinating antibody (agglutinin) produces visible clumping, or agglutination that forms the basis of simple, rapid, and sensitive immunoassays.
■ Radioimmunoassay (RIA) is a highly sensitive and quantitative procedure that utilizes radioactively labeled antigen or antibody.
■ The enzyme-linked immunosorbent assay (ELISA) depends on an enzyme-substrate reaction that generates a colored reaction product. ELISA assays that employ chemiluminescence instead of a chromogenic reaction are the most sensitive immunoassays available.
■ In Western blotting, a protein mixture is separated by elec-trophoresis; then the protein bands are electrophoretically transferred onto nitrocellulose and identified with labeled antibody or labeled antigen.
■ Fluorescence microscopy using antibodies labeled with fluorescent molecules can be used to visualize antigen on or within cells.
■ Flow cytometry provides an unusually powerful technology for the quantitative analysis and sorting of cell populations labeled with one or more fluorescent antibodies.
Berzofsky, J. A., I. J. Berkower, and S. L. Epstein. 1991. Antigen-antibody interactions and monoclonal antibodies. In Fundamental Immunology, 3rd ed., W. E. Paul, ed. Raven Press, New York.
Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober. 1997. Current Protocols in Immunology. Wiley, New York.
Harlow, E., and D. Lane. 1999. Using Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press.
Herzenberg, L. A., ed. 1996. Weir's Handbook of Experimental Immunology, 5th ed. Oxford, Blackwell Scientific Publications.
Rose, N. R., E. C. de Macario, J. D. Folds, C. H. Lane, and R. M. Nakamura. 1997. Manual of Clinical Laboratory Immunology. American Society of Microbiology, Washington, D.C.
Stites, D. P., C. Rodgers, J. D. Folds, and J. Schmitz. 1997. Clinical laboratory detection of antigens and antibodies. In Medical Immunology, 9th ed., D. P. Stites, A. I. Terr, and T. G. Parslow, eds., Appelton and Lange, Stamford, CT.
Wild, D., ed. 2001. The Immunoassay Handbook. Nature Publishing Group, NY.
Explore the Pathology Laboratories of Arkansas to see what kinds of samples are taken from patients and what markers are used to evaluate lymphocyte populations by flow cy-tometry.
At the highly informative Australian Flow Cytometry Group Web site, one can find a carefully detailed and illustrated guide to the interpretation of flow cytometric analyses of clinical samples.
The Kirkegaard & Perry Laboratories Web site contains a subsite, http://www.kpl.com/support/immun/pds/50datasht/54-12-10.html, which allows one to follow a step-by-step procedure for using a chemiluminescent substrate in a sensitive immunoassay.
Clinical Focus Question Flow-cytometric analysis for the detection and measurement of subpopulations of leukocytes, including those of leukemia, is usually performed using monoclonal antibodies. Why is this the case?
1. Indicate whether each of the following statements is true or false. If you think a statement is false, explain why.
a. Indirect immunofluorescence is a more sensitive technique than direct immunofluorescence.
b. Most antigens induce a polyclonal response.
c. A papain digest of anti-SRBC antibodies can agglutinate sheep red blood cells (SRBCs).
d. A pepsin digest of anti-SRBC antibodies can agglutinate SRBCs.
e. Indirect immunofluorescence can be performed using a Fab fragment as the primary, nonlabeled antibody.
f. For precipitation to occur, both antigen and antibody must be multivalent.
g. Analysis of a cell population by flow cytometry can simultaneously provide information on both the size distribution and antigen profile of cell populations containing several different cell types.
h. ELISA tests using chemiluminescence are more sensitive than chromogenic ones and precipitation tests are more sensitive than agglutination tests.
i. Western blotting and immunoprecipitation assays are useful quantitative assays for measuring the levels of proteins in cells or tissues.
j. Assume antibody A and antibody B both react with an epitope C. Furthermore, assume that antibody A has a Ka 5 times greater than that of antibody B. The strength of the monovalent reaction of antibody A with epitope C will always be greater than the avidity of antibody B for an antigen with multiple copies of epitope C.
2. You have obtained a preparation of purified bovine serum albumin (BSA) from normal bovine serum. To determine whether any other serum proteins remain in this preparation of BSA, you decide to use immunoelectrophoresis.
a. What antigen would you use to prepare the antiserum needed to detect impurities in the BSA preparation?
b. Assuming that the BSA preparation is pure, draw the im-munoelectrophoretic pattern you would expect if the assay was performed with bovine serum in a well above a trough containing the antiserum you prepared in (a) and the BSA sample in a well below the trough as shown below:
3. The labels from four bottles (A, B, C, and D) of hapten-carrier conjugates were accidentally removed. However, it was known that each bottle contained either 1) hapten 1-carrier 1 (H1-C1), 2) hapten 1-carrier 2 (H1-C2), 3) hapten 2-carrier 1 (H2-C1), or 4) hapten 2-carrier 2 (H2-C2). Carrier 1 has a molecular weight of 60,000 dal-tons and carrier 2 has a molecular weight of over 120,000 daltons. Assume you have an anti-H1 antibody and an anti-H-2 antibody and a molecular-weight marker that is 100,000 daltons. Use Western blotting to determine the contents of each bottle and show the Western blots you would expect from 1, 2, 3, and 4. Your answer should also tell which antibody or combination of antibodies was used to obtain each blot.
4. The concentration of a small amount (250 nanograms/ml) of hapten can be determined by which of the following assays: (a) ELISA (chromogenic), (b) Ouchterlony method, (c) RIA, (d) fluorescence microscopy, (e) flow cytometry, (f) immunoprecipitation, (g) immunoelectron microscopy, (h) ELISPOT assay, (i) chemiluminescent ELISA.
5. You have a myeloma protein, X, whose isotype is unknown and several other myeloma proteins of all known isotypes (e.g., IgG, IgM, IgA, and IgE).
Bovine serum a. How could you produce isotype-specific antibodies that could be used to determine the isotype of myeloma protein, X?
b. How could you use this anti-isotype antibody to measure the level of myeloma protein X in normal serum?
6. For each antigen or antibody listed below, indicate an appropriate assay method and the necessary test reagents. Keep in mind the sensitivity of the assay and the expected concentration of each protein.
a. IgG in serum b. Insulin in serum c. IgE in serum d. Complement component C3 on glomerular basement membrane e. Anti-A antibodies to blood-group antigen A in serum f. Horsemeat contamination of hamburger g. Syphilis spirochete in a smear from a chancre
7. Which of the following does not participate in the formation of antigen-antibody complexes?
a. Hydrophobic bonds b. Covalent bonds c. Electrostatic interactions d. Hydrogen bonds e. Van der Waals forces
8. Explain the difference between antibody affinity and antibody avidity. Which of these properties of an antibody better reflects its ability to contribute to the humoral immune response to invading bacteria?
9. You want to develop a sensitive immunoassay for a hormone that occurs in the blood at concentrations near 10~7 M. You are offered a choice of three different antisera whose affinities for the hormone have been determined by equilibrium dialysis. The results are shown in the Scatchard plots.
r a. What is the value of K0 for each antiserum?
b. What is the valence of each of the antibodies?
c. Which of the antisera might be a monoclonal antibody?
d. Which of the antisera would you use for your assay? Why?
10. In preparing a demonstration for her immunology class, an instructor purified IgG antibodies to sheep red blood cells (SRBCs) and digested some of the antibodies into Fab, Fc, and F(ab.)2 fragments. She placed each preparation in a separate tube, labeled the tubes with a water-soluble marker, and left them in an ice bucket. When the instructor returned for her class period, she discovered that the labels had smeared and were unreadable. Determined to salvage the demonstration, she relabeled the tubes 1,2,3, and 4 and proceeded. Based on the test results described below, indicate which preparation was contained in each tube and explain how you identified the contents.
a. The preparation in tube 1 agglutinated SRBCs but did not lyse them in the presence of complement.
b. The preparation in tube 2 did not agglutinate SRBCs or lyse them in the presence of complement. However, when this preparation was added to SRBCs before the addition of whole anti-SRBC, it prevented agglutination of the cells by the whole anti-SRBC antiserum.
c. The preparation in tube 3 agglutinated SRBCs and also lysed the cells in the presence of complement.
d. The preparation in tube 4 did not agglutinate or lyse SRBCs and did not inhibit agglutination of SRBCs by whole anti-SRBC antiserum.
11. You are given two solutions, one containing protein X and the other containing antibody to protein X. When you add 1 ml of anti-X to 1 ml of protein X, a precipitate forms. But when you dilute the antibody solution 100-fold and then mix 1 ml of the diluted anti-X with 1 ml of protein X, no precipitate forms.
a. Explain why no precipitate formed with the diluted antibody.
b. Which species (protein X or anti-X) would likely be present in the supernatant of the antibody-antigen mixture in each case?
12. Consider equation 1 and derive the form of the Scatchard equation that appears in equation 2.
Where: S = antibody binding sites; [S] = molar concentration of antibody binding sites; L = ligand (monovalent antigen); [L] = molar concentration of ligand; SL = site-ligand complex; [SL] = molar concentration of site ligand complex; B is substituted for [SL] and F for [L]. Hint: It will be helpful to begin by writing the law of mass action for the reaction shown in equation 1.
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