METHOD FOR LABELING INTRACELLULAR AND EXTRACELLULAR TARGETS OF LEUKOCYTES

Abstract

The present invention relates to methods for labeling intracellular and extracellular targets of leukocytes, as well as to kits for performing said methods.

Claims

1. A kit for performing a method for labeling a marker of leukocytes, the kit comprising: a first solution comprising: a cross-linking agent that is formaldehyde, wherein a concentration of the cross-linking agent in the first solution is 0.5% to 2% (v/v); a second solution comprising: (i) the first solution, (ii) a solution comprising a permeabilizing agent and a neutralizing agent comprising a mixture of sodium chloride and (NaCl) and ammonium chloride (NH.sub.4Cl) and optionally glycine, tris(hydroxymethyl)aminomethane (Tris), N-Lauroyl sarcosine, ethanolamine, or a mixture thereof, in a concentration of 1 to 100 mM in the solution, wherein the neutralizing agent neutralizes a cross-linking activity of the cross-linking agent and a pH of the solution is in a range of 6.0 and 6.6; and (iii) a binding agent that binds a marker of a leukocyte, wherein the binding agent comprises a detectable agent, wherein the permeabilizing agent of (ii) is added in an amount sufficient to permeabilize leukocytes, and so as to lyse red blood cells to effect lysis of substantially all red blood cells contained in a cellular composition comprising leukocytes and red blood cells, while a majority of leukocytes contained in the cellular composition are not lysed.

2. The kit according to claim 1, wherein the binding agent binds an intracellular target of the leukocytes.

3. The kit according to claim 1, wherein the binding agent is in a binding agent cocktail that includes extracellular and intracellular binding agents.

4. The kit according to claim 1, wherein the concentration of the neutralizing agent in the solution is 5 to 20 mM.

5. The method according to claim 4, wherein the volume of the first solution is 1 to 10 times a volume of the second solution.

6. The kit according to claim 5 wherein a volume of the first solution is 4 to 8 times greater than the volume of the second solution.

7. The method according to claim 5, wherein the volume of the first solution is six times greater than the volume of said second solution.

8. The kit of claim 1, wherein the binding agent is in a binding agent cocktail that includes extracellular and intracellular binding agents.

9. The kit according to claim 1, wherein the permeabilizing agent contains a C.sub.12-type alkane residue.

10. The kit according to claim 1, wherein the kit contains one vial of the first solution, four vials of the second solution, and one vial of a third solution, wherein the third solution comprises a fixative and a detergent.

11. The kit according to claim 1, wherein said permeabilizing agent is N-Lauroyl sarcosine.

12. The kit according to claim 1, wherein the binding agent comprises a first antibody binding agent which specifically binds to an extracellular target of leukocytes, and a second antibody binding agent which specifically binds to an intracellular target of leukocytes, wherein said first antibody binding agent comprises a first detectable agent and said second antibody binding agent comprises a second detectable agent.

13. A kit for performing a method for labeling a marker of leukocytes, the kit comprising: (a) a first solution, comprising a compound, selected from the group consisting of formaldehyde, paraformaldehyde, and glutaraldehyde; (b) a second solution, comprising (i) a quaternary ammonium salt or an amine containing compound, and (ii) a detergent; and (c) a third solution, comprising (i) a compound, selected from the group consisting of formaldehyde, paraformaldehyde, and glutaraldehyde, and (ii) a detergent.

14. The kit according to claim 13, wherein a concentration of the compound, selected from the group consisting of formaldehyde, paraformaldehyde, and glutaraldehyde is in a range of from about 1 to 100 mM of the first solution.

15. The kit according to claim 14, wherein the concentration of the compound, selected from the group consisting of formaldehyde, paraformaldehyde, and glutaraldehyde is in a range of from about 5 to 20 mM of the first solution.

16. The kit according to claim 13, wherein the kit contains one vial of the first solution, four vials of the second solution, and one vial of a third solution, wherein the third solution comprises a fixative and a detergent.

17. The kit according to claim 13, wherein a pH of the second solution is in a rang of 6.0 to 6.6.

18. The kit according to claim 13, wherein the first solution is added in an amount sufficient to permeabilize leukocytes, and so as to lyse red blood cells to effect lysis of substantially all red blood cells contained in a cellular composition comprising leukocytes and red blood cells, while a majority of leukocytes contained in the cellular composition are not lysed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1:

[0041] Workload comparison showing the amount of time and work needed to perform methods known in the art (“Intraprep” and “VersaLyse/Fix”), and the method of the present invention (“INCA”). Each bar represents 5 minutes of time; light gray bars show incubation time, dark gray bars show work time.

[0042] FIG. 2:

[0043] Staining of normal human whole with a PE-labeled anti-ZAP-70 antibody by a method known in the art (“Intraprep”) and the method of the present invention, either with or without an additional washing step (“INCA wash” and “INCA no wash”). The dot plots show the intracellular staining with the antibody (FL2) versus sideward scatter.

[0044] FIG. 3:

[0045] Staining of normal human whole blood with a FITC-labeled anti-CD4 antibody and an Alexa647-labeled anti-FoxP3 antibody by a method known in the art (“Intraprep”) and the method of the present invention, either with or without an additional washing step (“INCA wash” and “INCA no wash”). The dot plots show surface staining with the anti-CD4 antibody (FL1) versus intracellular staining with the anti-FoxP3 antibody (FL4).

[0046] The present invention will now be further illustrated in the following examples without being limited thereto.

EXAMPLES

Example 1

[0047] Staining of Normal Human Whole Blood with a PE-Labeled Anti-ZAP-70 Antibody

[0048] Normal human whole blood was stained with a PE-labeled anti-ZAP-70 antibody by a method known in the art (“Intraprep”) and the method of the present invention, either with or without an additional washing step (“INCA wash” and “INCA no wash”) and subsequently analyzed by flow cytometry. FIG. 2 shows the respective stainings with the antibody (FL2) versus sideward scatter. The methods according to the present invention stained equal amounts of cells compared to the prior art method and showed a clearer separation from the unstained cells with an RMFI (relative mean fluorescence intensity, which is a measure for the signal-to-noise ration) of 30 (INCA wash) and 15 (INCA no wash).

Example 2

[0049] Staining of Normal Human Whole Blood with a FITC-Labeled Anti-CD4 Antibody and an Alexa647-Labeled Anti-FoxP3 Antibody

[0050] Normal human whole blood was stained with a FITC-labeled anti-CD4 antibody and an Alexa647-labeled anti-FoxP3 antibody by a method known in the art (“Intraprep”) and the method of the present invention, either with or without an additional washing step (“INCA wash” and “INCA no wash”) and subsequently analyzed by flow cytometry. FIG. 3 shows the respective stainings with the anti-CD4 antibody (FL1) versus the intracellular stainings with the anti-FoxP3 antibody (FL4). The method known in the art stained only a small population of 0.36% of the cells, whereas the method of the present invention containing an additional washing step stained a substantial population of 4.3%.

Example 3

[0051] Staining of Normal Human Whole Blood with Various Intracellular Antibodies

[0052] Normal human whole blood was stained with various intracellular antibodies by a method known in the art (“Intraprep”) and the method of the present invention, either with or without an additional washing step (“INCA wash” and “INCA no wash”) as described above. Results of the respective stainings are shown in Table 1 below.

[0053] Results show that the method of the present invention provides at least comparable and in many cases superior stainings as a prior art method.

TABLE-US-00001 TABLE 1 Quality of stainings Antibody Intraprep INCA wash INCA no wash CD125-PE ++ ++ + APO2.7-PE + ++ ++ DAP-12-PE + ++ + FoxP3 - Alexa647 − ++ − Tia-l-PE ++ ++ ++ ZAP-70-PE + ++ + MPO-FITC ++ + + Lactoferrin-PE ++ + + CD79a-PE + ++ ++ IFNg-PE ++ ++ + TNFa-PE ++ ++ + CyclinA2-FITC − ++ ++ TdT-FITC − ++ ++ ++: best conditions +: not optimal but staining works −: no adequate staining

Example 4

[0054] Staining of Human Whole Blood without Washing Steps

[0055] In the following human whole blood samples were stained with Krome Orange—labeled anti-CD45 antibody, FITC-labeled anti-CD5 antibody, APC-labeled anti-CD19 antibody or a PE-labeled anti-ZAP-70 antibody. While the anti-CD19 antibody is directed against an extracellular epitope, the anti-ZAP-70 antibody is directed against intracellular epitope.

[0056] The following solutions were prepared and used within this example: [0057] Solution 1:Formaldehyde 5.5% wt/vol [0058] Na2HPO4 3 mM [0059] NaH2PO4 3 mM [0060] NaCL 47 mM [0061] pH 6.8 [0062] Solution 2:NaCl 140 mM [0063] NH4C1 10 mM MES 20 mM [0064] NaLS 0.15% [0065] BSA 0.5% [0066] Proclin 300 0.05% [0067] pH 6.3 [0068] Solution 3:Na2HPO4 11 mM [0069] NaH2PO4 29 mM [0070] NaCL 260 mM [0071] NaLS 0.05% [0072] Pluronic F68 0.1% [0073] Formaldehyde 0.5% wt/vol [0074] pH 7.2

[0075] In order to show whether intra- and extracellular epitopes can be stained using the present invention, a human whole blood sample (WBS) was processed in the following manner.

[0076] First, 50 μl of WBS were combined with 5 μl of solution 1 and incubated for 15 minutes. Then 300 μl of solution 2 were added, together with 10 μl of the labeled antibodies indicated. After 40 minutes of incuvation 3000 μl of solution 3 were added, and the mixture was vortexed.

[0077] The analysis of the above mixture was carried out using a flow cytometer device. It was shown that both intra- and extracellular epitopes were stained successfully. No difference could be seen when comparing the method outlined above having a washing step with a method as outlined above containing no washing step. Both method showed a sufficient staining.

Example 4a

Variations of Example 4

[0078] In order to find out whether the method of the present invention is a robust method which can be successfully used for many conditions, several variations of the solutions 1, 2, and 3 disclosed in Example 4 were tested.

[0079] In each case, 50 μl of WBS were combined with 5 μl of solution 1 (R1) and incubated for 15 minutes. Then 300 μl of solution 2 (R2) were added, together with 10 μl of each of the labeled antibodies indicated. After 30 minutes of incubation 3000 μl of solution 3 (R3) were added, and the mixture was vortexed.

[0080] As shown in Table 2 herein below, a setup was chosen where either the amount of solution 1 was varied or the concentrations of the ingredients of solution 2 was varied and/or the concentration of the ingredients of solution 3 was varied. In the various settings the concentration of several reagents was changed by + or −20% from the reference sample. The reference example is experiment No. 1 in Tables 2 and 3.

[0081] In the following table solution 1 is called R1, solution 2 is called R2, and solution 3 is called R3.

TABLE-US-00002 TABLE 2 Variations of solutions 1, 2, and 3 FIX PERM (R2) WASH (R3) (R1) MES NaCl NH4CL BSA NALS Proclin FA LS F68 No. μL (mM) (mM) (mM) (%) (%) (%) pH (%) (%) (%) 1 5 10 140 10 0.5 0.15 0.05 6.3 0.5 0.05 0.1 2 6 12 168 12 0.6 0.18 0.06 6.5 0.6 0.06 0.12 3 6 12 112 8 0.4 0.18 0.04 6.1 0.6 0.04 0.12 4 6 12 168 8 0.4 0.12 0.06 6.1 0.4 0.06 0.08 5 4 12 168 12 0.4 0.12 0.04 6.5 0.4 0.04 0.12

[0082] The above experimental settings were tested regarding their deviation of the signal to noise ratio in the detection of intra- and extracellular epitopes when compared to the reference experiment No. 1.

[0083] The following table 3 provides signal/noise values with regard to 3 intracellular targets and 2 extracellular targets. The conjugates used were Anti-MPO-FITC, anti-CD79a-PE, anti-CD3-ECD (IOTest3 cocktail PN IM3464U), and Anti-CD14-PC7 (PN A22331).

TABLE-US-00003 TABLE 3 Signal to Noise ratios of the settings of Table 2 Sample S/N MPO S/N MPO S/N S/N S/N no. Mono Granu CD79a CD3 CD14 1 18.6 32.2 32.9 54.3 354 2 14.1 21.4 27.3 52.5 348 3 15.6 26.3 25.2 54.4 347 4 30.4 31.7 34.9 51.5 325 5 25.8 34.8 37.9 55.7 339

[0084] As can be seen all experimental variations outlined in table 2 were working equally well regarding the signal to noise ratio. Accordingly, the present invention provides a robust system and method for detecting intra- and extracellular epitopes which works well over a great variety of buffer compositions including various concentrations of the buffer ingredients.

Example 5

Influence of an Additional Washing Step

[0085] In order to find out whether an additional washing step has any negative influence on the results obtained above, the conditions of Example 4 were varied insofar as a further washing step was added.

[0086] In particular, solutions 1, 2, and 3 as outlined in Example 4, above, were prepared and the following method for staining a human whole blood sample (WBS) with labeled antibodies was carried out.

[0087] First, 50 μl of WBS were combined with 5 μl of solution 1 and incubated for 15 minutes. Then 300 μl of solution 2 were added, together with 10 μl of each of the labeled antibodies indicated. After 40 minutes of incubation 3000 μl of solution 3 were added, and the mixture was vortexed. Finally, in addition to the above, the cell suspension was centrifuged (500 g, 5 min.), the supernatant was discarded, and the cell pellet is resuspended in 500 μl of the solution 3.

[0088] The results obtained using the above method did not differ from the results obtained in Example 4. Thus, an additional washing step does not have any essential influence on the result achieved. This again shows that the system and method according to the present invention is robust over various parameters.

Example 6

Influence of pH Variations

[0089] In order to show the influence of differences in the pH value the solutions used on the outcome of the system and method according to the present invention, an experimental setup was chosen, wherein the pH of solution 2 was varied.

[0090] Regarding the experimental setup, the method for staining a human whole blood sample as outlined in Example 4 was carried out using the variations of solutions 1, 2, and 3 as outline for reference experiment No. 1 given in Table 2 of Example 4a. In particular, it was determined as to whether the signal to noise ratio is drastically influenced by a variation of the pH of solution 2. The staining of the human whole blood sample was done by using a PE-labeled anti-ZAP-70 antibody.

[0091] As can be taken from Table 4, below, the signal to noise ratio did not differ significantly when the pH of solution 2 was varied from pH 6.00 to pH 6.91.

TABLE-US-00004 TABLE 4 Variation of the pH of solution 2 pH LYSE 6.00 6.12 6.26 6.35 6.46 6.60 6.74 6.91 RMFI 34.3 33.4 35.9 35.9 33.6 30.9 28.0 26.9