Zenon® Mouse IgG Labeling

Introduction

Zenon® labeling technologyprovides a rapid, versatile and reliable method for producing fluorescently-labeled antibody using a variety of fluorochromes, even with small amounts of unpurified starting material such as hybridoma culture supernatant.

Antibodies labeled using Zenon® technology are suitable for use in all applications where a directly labeled antibody can be used.

Each of the Zenon® mouse IgG labeling kits is designed for use with a particular mouse monoclonal antibody isotype: IgG1, IgG2a, or IgG2b. Zenon® labeling technology uses a labeled Fab fragment directed against the Fc portion of an intact IgG primary antibody in order to form a labeling complex (Figure 1). Formation of the Fab-antibody complex occurs within 5 minutes, and complexes formed using this technology display fluorescence intensity similar to or brighter than that of directly labeled primary antibodies. Multiple Zenon® antibody complexes may be prepared individually and used in a multicolor stain after using the Zenon® blocking reagent.

The following protocols were developed for the staining of peripheral blood mononuclear cells and can be readily adapted to other cell types or whole blood. Note that an Fc blocking step is not required when using antibodies labeled with Zenon® technology by themselves, as the labeling complex hinders Fc receptor access to the antibody.

 
Zenon Technology Overview

Figure 1. How Zenon® Labeling Technology works. Noncovalent labeling with Zenon® antibody labeling vs. covalent labeling with the Microscale, Monoclonal, and Protein Labeling Kits.

Materials

Zenon® labeling reagent
Zenon® blocking reagent
Unlabeled primary mouse antibody
Sample: Cell suspension at ~1 x 10 6 cells/mL or whole blood
Staining buffer: phosphate-buffered saline with 1% BSA (PBS/BSA), or equivalent
 
Sample preparation reagents:

  • Cal-Lyse™ lysing solution (Invitrogen) or equivalent erythrocyte lysis reagent
  • FIX & PERM® reagents (Invitrogen) or equivalent cell fixation and permeabilization reagents
  • 4% formaldehyde in phosphate-buffered saline (PBS) or equivalent cell fixation reagent

Flow cytometry tubes, 12 x 75 mm
Flow cytometer

1. Zenon® Labeling Complex Formation

  1. Verify that the isotype of the mouse antibody matches the isotype specificity of the Zenon® Mouse IgG Labeling Kit. The Zenon® Mouse IgG labeling reagents in the kits are isotype specific and are not recommended for labeling antibodies that are not of the corresponding mouse IgG isotype.

  2. Add 1 µg antibody to small tube.
     
    If the concentration of mouse IgG is not specified, contact the supplier and obtain an approximate IgG content. If a concentration cannot be obtained from the supplier, Zenon® labeling reagent will need to be titered to obtain the optimum labeling conditions.
     
    If only a limited amount of antibody is available, quantities of less than 1 µg may be labeled. The volume of Zenon    ® labeling reagent should be adjusted according to degree of scale.

  3. Add 5 µl of the Zenon® mouse IgG labeling reagent (Component A) to the antibody and mix. This ratio allows for a 3-fold molar excess of Fab over antibody (molar ratio = 3). See Results and Discussion for information on how to adjust staining brightness with the molar ratio.

  4. Incubate the mixture for 5 minutes at room temperature protected from light.
     
    If long-term storage of labeling complexes is desired, stop the labeling procedure here. At this stage, the labeling mixture can be stored at 4ºC for several weeks with the addition of 2 mM sodium azide as a preservative. When ready to use, continue the complex formation with step 1.5.

  5. Add 5 µl of the Zenon® blocking reagent (Component B) to the reaction mixture and mix. The blocking reagent contains nonspecific mouse immunoglobulin.
     
    The blocking step is necessary if the Zenon® complex will be used with other labeled antibodies or with mouse target cells. It is important to always use equal volumes of Zenon® labeling reagents and Zenon blocking reagent.

  6. Incubate the solution for 5 minutes at room temperature. The complex is now ready for use. If blocking reagent has been used, the complex should be used within 30 minutes.

2. Surface Marker Using 1 Zenon® Labeling Complex

  1. Collect whole blood or prepare a cell suspension in a suitable staining buffer such as PBS/BSA.

  2. Add 100 µl cell suspension or 100 µl whole blood to a 12 x 75 mm flow cytometry tube.

  3. Prepare Zenon® complexes with primary antibody according to the Zenon® Labeling Complex Formation Protocol.

  4. Add the entire Zenon® labeling mixture to the cell suspension.

  5. Note:    If using whole blood samples, the lysis of red blood cells may be performed either before or after cell staining.

  6. Incubate the sample for 30 minutes at room temperature, protected from light.

  7. Wash the cells once: fill the tube with staining buffer, centrifuge the sample to pellet the cells, and decant the supernatant.

  8. Resuspend the cells in staining buffer.

  9. -- Optional Fixation: The sample may be fixed in 4% formaldehyde in PBS solution for 15 minutes at room temperature, protected from light.

  10. -- Wash the cells once by filling the flow tube with staining buffer, centrifuging the sample to pellet the cells, and decant the supernatant.

  11. -- Resuspend the cells in staining buffer.

  12. Analyze the sample using appropriate instrument parameters.

3. Surface Markers Using Multiple Zenon® Labeling Complexes

  1. Collect whole blood or prepare a cell suspension in a suitable staining buffer such as PBS/BSA.

  2. Add 100 µl cell suspension or 100 µl whole blood to a 12 x 75 mm flow cytometry tube.

  3. Prepare Zenon® complexes with primary antibody according to the Zenon® Labeling Complex Formation Protocol. Prepare each antibody-Zenon® complex individually.

  4. Combine the individually-prepared Zenon® complexes and add to the cell suspension at the same time.

    5. If using whole blood samples, the lysis of red blood cells may be performed either before or after cell staining.

  5. Incubate the sample for 30 minutes at room temperature, protected from light.

  6. Wash the cells once: fill the tube with staining buffer, centrifuge the sample to pellet the cells, and decant the supernatant.

  7. Resuspend the cells in staining buffer.

  8. --- Optional Fixation: The sample may be fixed in 4% formaldehyde in PBS solution for 15 minutes at room temperature, protected from light.

  9. --- Wash the cells once by filling the flow tube with staining buffer, centrifuging the sample to pellet the cells, and decant the supernatant.

  10. --- Resuspend the cells in staining buffer.

  11. Analyze the sample using appropriate instrument parameters

4. Surface Markers Using a Zenon® Labeling Complex & Direct Conjugate

  1. Collect whole blood or prepare a cell suspension in a suitable staining buffer such as PBS/BSA.
  2. Add 100 µl cell suspension or 100 µl whole blood to a 12 x 75 mm flow cytometry tube.
  3. Prepare Zenon® complex with primary antibody according to the Zenon® Labeling Complex Formation Protocol.
  4. Add the entire Zenon® labeling mixture and add any direct conjugates (appropriately titered) to the cell suspension at the same time.
    If using whole blood samples, the lysis of red blood cells may be performed either before or after cell staining.

  5. Incubate the sample for 30 minutes at room temperature, protected from light.
  6. Wash the cells once: fill the tube with staining buffer, centrifuge the sample to pellet the cells, and decant the supernatant.
  7. Resuspend the cells in staining buffer.
  8. ---  Optional Fixation: The sample may be fixed in 4% formaldehyde in PBS solution for 15 minutes at room temperature, protected from light.
  9. ---  Wash the cells once by filling the flow tube with staining buffer, centrifuging the sample to pellet the cells, and decant the supernatant.
  10. --- Resuspend the cells in staining buffer.
  11. Analyze the sample using appropriate instrument parameters.

5. Surface Markers Using a Zenon® Labeling Complex & Indirect Staining

  1. Collect whole blood or prepare a cell suspension in a suitable staining buffer such as PBS/BSA.

  2. If Fc blocking is desired, add 67 µl of 3 mg/ml normal mouse or Rabbit IgG per 1 ml of cell suspension (200ug/ml IgG final concentration.) and incubate 10 minutes at room temperature.

  3. Add 100 µl cell suspension or 100 µl whole blood to a 12 x 75 mm flow cytometry tube.

  4. Add the appropriate amount of unconjugated (or biotin-conjugated) mouse anti-human monoclonal antibody (appropriately titered) to the cell suspension.

  5. Incubate 15 minutes at room temperature.

  6. Wash the cells once: fill the flow tube with staining buffer, centrifuge the sample to pellet the cells, and then decant the supernatant.

  7. Add appropriate amount of goat anti-mouse (or streptavidin-conjugated) fluorochrome (appropriately titered) to the cell suspension.

  8. Prepare Zenon® complex with primary antibody according to the Zenon® Labeling Complex Formation Protocol.

  9. Add the entire Zenon®  labeling mixture to the cell suspension.

  10. Incubate the sample for 30 minutes at room temperature, protected from light.

  11. Wash the cells once: fill the tube with staining buffer, centrifuge the sample to pellet the cells, and then decant the supernatant.

  12. Resuspend the cells in staining buffer.

  13. --- Optional Fixation: The sample may be fixed in 4% formaldehyde in PBS solution for 15 minutes at room temperature, protect from light.

  14. --- Wash the cells once by filling the flow tube with staining buffer, centrifuging the sample to pellet the cells, and decant the supernatant.

  15. --- Resuspend the cells in staining buffer.

  16. Analyze the sample using appropriate instrument parameters.

6. Intracellular Marker with a Zenon® Labeling Complex

  1. Collect whole blood or prepare a cell suspension in a suitable staining buffer such as PBS/BSA.

  2. Add 100µl cell suspension or 100 µl whole blood to a 12 x 75 mm flow cytometry tube.

  3. Fix the cell sample in by adding 100µl FIX & PERM® Reagent A, incubate for 15 minutes at room temperature.  Any aldehyde-based fixation method may be used.

  4. Wash the cells once: fill the flow tube with staining buffer, centrifuge the sample to pellet the cells, and then decant the supernatant.

  5. Permeabilize the cell sample by adding 100 µl FIX & PERM® Reagent B.  Other permeabilization reagents may be used, such as 0.1% Triton-X-100 in PBS.

  6. Prepare Zenon® complex with primary antibody according to Protocol 1.

  7. Add the entire Zenon® labeling mixture to the cells suspended in permeabilization reagent.

  8. Incubate the sample for 30 minutes at room temperature, protected from light.

  9. Fill the tube with staining buffer and let rest for 5 minutes at room temperature, protected from light.

  10. Centrifuge the sample to pellet the cells, and decant the supernatant.

  11. Resuspend the cells in staining buffer.

  12. Analyze the sample using appropriate instrument parameters 

7. Combined Surface & Intracellular Staining

  1. Collect whole blood or prepare a cell suspension in a suitable staining buffer such as PBS/BSA.

  2. Add 100 µl cell suspension or 100 µl whole blood to a 12 x 75 mm flow cytometry tube.

  3. Prepare Zenon® complex with primary antibody for surface staining, according to the Zenon® Labeling Complex Formation Protocol.

  4. Add the entire Zenon®  labeling mixture for surface staining (or directly-conjugate antibody) to the cell suspension.

  5. Incubate the sample for 30 minutes at room temperature, protected from light.

  6. Fix the cell sample in by adding 100 µl Fix & Perm®  Reagent A, incubate for 15 minutes at room temperature.   Any aldehyde-based fixation method may be used.

  7. Wash the cells once: fill the flow tube with staining buffer, centrifuge the sample to pellet the cells, and then decant the supernatant.

  8. Permeabilize the cell sample by adding 100 µl Fix & Perm® Reagent B.  Other permeabilization reagents may be used, such as 0.1% Triton-X-100 in PBS.

  9. Prepare Zenon® complex with primary antibody for intracellular staining, according to the Zenon® Labeling Complex Formation Protocol.

  10. Add the entire Zenon® labeling mixture for intracellular staining to the cell suspension in permeabilization reagent.

  11. Incubate the sample for 30 minutes at room temperature, protected from light.

  12. Fill the flow tube with staining buffer and let rest for 5 minutes at room temperature, protected from light.

  13. Centrifuge the sample to pellet the cells, and decant the supernatant.

  14. Resuspend the cells in staining buffer.

  15.  Analyze the sample using appropriate instrument parameters

Results

Staining is Dependent on Molar Ratio

The intensity of staining achieved with Zenon reagent-complexed antibody is dependent on the molar ratio of Fab to antibody. In general, a 3-fold molar excess should be used with abundant targets. For less abundant targets, the molar ratio can be increased by adding more Zenon® labeling reagent with 1 µg antibody. Figure 2 shows the effect of increasing molar ratio on the staining intensity of anti-CD4 on human lymphocytes, and compares this to a direct conjugate of the same clone and fluorochrome. Increasing molar ratio has little effect on background labeling of negative cell populations.


Figure 2. Staining of CD4 (clone S6.5) on human lymphocytes, comparison of a direct conjugate with Zenon complexes made at molar ratios of 3 and 6, each using the same clone and allophycocyanin (APC). Increasing molar ratio has little effect on background labeling of negative cell populations, while the fluorescence intensity of the positive population increases. The overlay plot compares lymphocytes stained with directly-conjugated CD4-APC clone S6.5 (blue line), and with Zenon® APC Mouse IgG2a labeling reagent with mouse anti-human CD4 clone S6.5 at molar ratio 3 (red line) and molar ratio 6 (green line).   Y axis: Number of events; X axis: APC fluorescence.


Using Zenon® Labeling Technology  on Non-Purified Antibodies

In cases where the antibody is not a purified protein, as with ascites fluid or hybridoma culture supernatant, Zenon® labeling reagent can still be used to make Fab:Ig complexes, and the ratio will have to be estimated. In this case, the Zenon® labeling reagent will label nonspecific IgGs of the given isotype as well as the specific antibody, but the non-specific IgG complexes should not stain appreciably and should be washed away during the staining procedure.


Brightness of Zenon® Labeling Reagent Fluorescence

Zenon® complexes can be as bright as or brighter than direct conjugates, depending on the molar ratio used, and unfixed samples can retain their fluorescence patterns for several hours. In Figure 3, peripheral blood lymphocytes were labeled with Zenon® reagent-complexed antibodies against CD4 and CD8 using Protocol 3. Unfixed samples were read over 4 hours and showed neither intensity change nor evidence of the Zenon® labeling reagent used for one marker crossing over to label the other marker.

Stability two color stains

Figure 3.  Stability of two color stains. Peripheral blood mononuclear cells were stained with anti-CD4 + Zenon® R-phycoerythrin (R-PE) mouse IgG 1 labeling reagent and anti-CD8 + Zenon® Alexa Fluor® 488 mouse IgG 1 labeling reagent, both used at molar ratio 3. Samples analyzed immediately, at 2 hours and 4 hours: no change in staining pattern was observed.


Versatility of Zenon® Labeling Technology
 
Zenon® labeling technology allows different fluorophore combinations to be explored. Figure 4 shows both combinations of Alexa Fluor® 488 dye- and R-phycoerythrin-labeled CD56 and CD16 stain on lymphocytes.  It is also simple to combine staining of surface and intracellular markers. Figure 5 shows staining of samples with Zenon® labeling reagent complexes of surface CD2 (T and NK cells), surface CD19 (B cells), and ZAP-70, an intracellular marker normally expressed in T and NK cells. ZAP-70 expression in B cell chronic lymphocytic leukemia correlates with disease progression.

Peripheral blood lymphocytes
Figure 4. Peripheral blood lymphocytes were stained with CD56 and CD16 antibodies combined with Zenon® labeling reagents. The left panel shows CD56 + Zenon® R-PE mouse IgG1 labeling reagent combined with CD16 + Zenon® Alexa Fluor® 488 mouse IgG1 labeling reagent; the right panel shows the reverse fluorophore combination.

Intracellular ZAP-70 staining

Figure 5. Intracellular ZAP-70 staining combined with surface CD2 and CD19 staining of human lymphocytes using Zenon labeling reagents. Samples stained with anti-CD19 + Zenon® R-PE mouse IgG1 labeling reagent (molar ratio 3), anti-CD2 + Zenon® Alexa Fluor® 680-R-PE mouse IgG1 labeling reagent (molar ratio 3) and anti-ZAP-70 + Zenon® Alexa Fluor® 488 mouse IgG1 labeling reagent (molar ratio 3). A lymphocyte gate was used. A) Normal patient sample showing a normal ZAP-70 staining pattern copositive with CD2 lymphocytes B) Normal patient sample showing a normal ZAP-70 staining pattern mutually exclusive with CD19 lymphocytes C & D) Two different patients with B-cell chronic lymphocytic leukemia showing different ZAP-70 expressions in dual parameter testing with CD2, gated on lymphocytes. Data collected in collaboration with M. Suter, Oregon Medical Laboratories, Eugene, OR.

Troubleshooting

  • Zenon® complexes can be brighter than direct conjugates, and brightness can be controlled with the Fab:Ig molar ratio.

  • The blocking step is only necessary if the Zenon® complex will be used with other labeled antibodies or with mouse target cells.

  • Zenon® labeling complexes can be prepared in a larger volume by scaling the labeling mixture in the Zenon® Labeling Complex Formation Protocol The volume of Zenon ®complex to be used for each sample must then be calculated based on the degree of scaling.

  • Zenon® labeling complexes can be prepared using quantities of less than 1 µg by scaling the labeling mixture in the Zenon® Labeling Complex Formation Protocol. The volume of Zenon® complex to be used for each sample must then be calculated based on the degree of scaling.

  •  If long-term storage of labeling complexes is desired, stop the labeling procedure in the Zenon® Labeling Complex Formation Protocol at step 1.4 (before addition of Zenon® blocking reagent). At this stage, the labeling mixture can be stored at 4ºC for several weeks with the addition of 2 mM sodium azide as a preservative. When ready to use, the blocking agent can be added if necessary

References

  1. JA Bradford, G Buller, M Suter, M Ignatius, JM Beechem. Fluorescence intensity multiplexing: Simultaneous seven marker, two-color immunophenotyping using flow cytometry. Cytometry, 61A:142-152 (2004)

  2. JK Brown, AD Pemberton, SH Wright, HRP Miller. Primary antibody-Fab fragment complexes: A flexible alternative to traditional direct and indirect immunolabeling techniques. J Histochem Cytochem, 52(9): 1219-1230 (2004)

  3. A Bojarska-Junak, J Rolinski, J Kawiak. Modification of immunocytochemical ZAP-70 assay for potential clinical application in B-cell chronic lymphocytic leukemia. Folia Histochem Cytobiol, 43(1): 19-23(2005)

  4. P Pozarowski, DH Halicka, Z Darzynkiewicz. NF-kB inhibitor esequiterpene parthenoide induces concurrently atypical apoptosis and cell necrosis: Difficulties in identification of dead cells in such cultures. Cytometry, 54A:118-124 (2003)

  5. Shapiro, H. Practical Flow Cytometry. Fourth Edition, John Wiley and Sons, 2003, p. 348-9.
MP 25000