Affinity Plate for Haptoglobin Phenotype Determination, Kit Comprising It, and Method of Haptoglobin Phenotype Determination by Means of Affinity Plates in Combination with Desorption Ionization Mass Spectrometry Techniques

Abstract

The invention relates to the affinity plate for haptoglobin phenotype determination, the kit comprising it, and the method of haptoglobin phenotype determination by detection of its a subunits by means of mass spectrometry desorption ionization techniques after preceding preconcentration of haptoglobin on the surface of the affinity plate with immobilized anti-haptoglobin antibody that was deposited onto this surface from gas phase after preceding electrospray ionization and heat desolvation. Then the signals of mass spectra corresponding to 1 and/or 2 subunits can be found and thus determined the haptoglobin phenotype, whereas only al subunit exists in phenotype 1-1, both the subunits a1 and 2 exist in phenotype 2-1, and only 2 subunit exists in phenotype

Claims

1. The affinity plate for the determination of haptoglobin phenotype characterized in that it consists of the substrate, the surface of the substrate being provided with anti-haptoglobin antibody in the form of a layer and its resistivity is lower than 10.sup.20 .Math.m.

2. The affinity plate according to the claim 1 characterized in that the resistivity of the substrate surface is in the range of 10.sup.8 to 10.sup.17 .Math.m, the substrate being selected from the group comprising conductive metals, alloys thereof, steel, semi conductible oxides of metals, conductive polymers, conductive forms of carbon, silicon, germanium, glass.

3. The affinity plate according to the claim 1 or claim 2 characterized in that the anti-haptoglobin antibody is selected from the group comprising polyclonal anti-haptoglobin antibody, monoclonal anti-haptoglobin antibody or single-domain anti-haptoglobin antibody, preferably goat polyclonal anti-haptoglobin antibody or rabbit polyclonal anti-haptoglobin antibody.

4. The kit for the determination of haptoglobin phenotype characterized in that it comprises the affinity plate according to any of the claim 1 to 2.

5. The method of the determination of haptoglobin phenotype characterized in that it comprises the steps of: a) depositing a biological material on the layer consisting of the anti-haptoglobin antibody, lying on the affinity plate according to any of the claims 1 to 2, then it is washed at least once with buffer, b) adding an aqueous solution of the reducing agent, and c) detection of the presence of a and subunits of haptoglobin by means of desorption ionization mass spectrometry techniques.

6. The method according to the claim 5 characterized in that the biological material is selected from the group comprising plasma, serum, hemolysate, or blood, or solutions thereof in buffer.

7. The method according to the claim 5 or claim 6 characterized in that the amount of the deposited biological material is in the range of 0.5 to 10 l.

8. The method according to the claim 5 characterized in that after the step a) the affinity plate with biological material is incubated for 5 minutes to 24 hours at the temperature of 10 to 50 C. and then it is washed with buffer.

9. The method according to the claim 5 characterized in that after the step b) the affinity plate with reducing agent is incubated for 5 minutes to 30 minutes at the temperature 20 C. to 37 C., the reducing agent being selected from the group comprising tris(2-carboxyethyl)phosphin, mercaptoethanol, or dithiothreitol.

10. The method according to the claim 5 characterized in that the concentration of the reducing agent in aqueous solution is in the range of 5 to 100 mmol/L.

11. The method according to the claim 9 or claim 10 characterized in that after the incubation of the affinity plate with the reducing agent the solution of ionization MALDI matrix is added, then it is left to dry at the temperature of 10 C. to 50 C.

12. The method according to the claim 11 characterized in that the ionization MALDI matrix is selected from the group comprising 2,5-dihydroxybenzoic acid, alpha-cyano-4-hydroxycinnamic acid, sinapic acid, 2,5-dihydroxyphenylmethylketone, and ferulic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1: The apparatus for the preparation of the affinity plate for the determination of haptoglobin phenotype with the use of desorption ionization mass spectrometry comprises T-splitter 1, syringe pump 2 operating the piston 3 of the syringe 4 with stock solution A, tube 5, microspray needle 6 on the splitter 1, carrier gas inlet 7 with the possibility of preheating, high voltage source 8 for electrospray connected to the conductive part 9 of the syringe 4, evaporation area 10, surface 12 for modification, high voltage source 11 connected to substrate surface 12. When the stock solution passes the spray, charged aerosol B is formed and transformed into dry aerosol/ion beam C by passing the tube which can be optionally heated.

[0053] FIG. 2: MALDI mass spectra of haptoglobin a chains after serum incubation with polyclonal anti-human haptoglobin antibody .sub.[MD1]that was applied onto the surface. Haptoglobin is enriched after washing and its a chains can be identified in spectra after the preceding reduction of disulphide bridges. The figure shows mass spectra of three possible combinations of phenotypes: Hp1-1; Hp2-1; Hp2-2 (in the order down from top).

[0054] FIG. 3: MALDI mass spectra of haptoglobin a chains after incubation of hemolysate with polyclonal anti-human haptoglobin antibody, applied onto the surface. Haptoglobin is enriched after washing and its a chains can be identified in spectra after the preceding reduction of disulphide bridges. The figure shows mass spectra of three possible combinations of phenotypes: Hp1-1; Hp2-1; Hp2-2 (down from top respectively).

[0055] The signals corresponding to the molecules of and haemoglobin can be observed in spectra due to the formation of haptoglobin-haemoglobin complex. Haemoglobin is not present in serum spectra in the FIG. 2, because the blood cellserythrocytes containing haemoglobin are removed during the preparation of serum.

[0056] FIG. 4: The pictures of surfaces modified with anti-haptoglobin antibody obtained by means of electron microscopy. Rightthe surface before washing; formed with the layer of antibody and the layer of salt. Leftthe surface after washing with distilled water. Black coloured spots present the applied antibody.

[0057] FIGS. 5a to 5d: The results of analysis of determination of haptoglobin phenotype for particular samples by means of immunostaining with anti-haptoglobin antibody after the separation by means of gel electrophoresis (the photograph inserted at the top part) and by means of enrichment on the affinity surface with mass spectrometry detection. It can be seen from the picture 5a that both the methods give the same results. Mass spectra shows the presence of 1 and 2 chains of haptoglobin in the form of spectral peaks, while gel electrophoresis in the form of electrophoretic zones (bands). It can be seen from the picture that the graphical presentation by means of spectrometric peaks and electrophoretic bands is the same for each of the samples 1-9, which indicates equivalency of both the techniques. The results of the analysis of determination of haptoglobin phenotype for the samples 10-36 are presented in FIGS. 5b to 5d.

[0058] FIG. 6: A) Mass spectra of haptoglobin a chains after the incubation of the serum with polyclonal anti-human haptoglobin antibody, provided by Fitzgerald Company, that was applied onto Indium Tin Oxide surface. The figure shows mass spectra of phenotype Hp2-1. B) Mass spectra of haptoglobin a chains after the incubation of the serum with polyclonal anti-human haptoglobin antibody, provided by Sigma-Aldrich Company, that was applied onto fused quartz. The figure shows mass spectra of phenotype Hp2-1. C) Mass spectra of haptoglobin a chains after the incubation of the serum with polyclonal anti-human haptoglobin antibody, provided by Fitzgerald Company, that was applied onto stainless surface. The figure shows mass spectra of phenotype Hp2-1.

PREFERRED EMBODIMENTS OF THE INVENTION

Example 1The Preparation of the Affinity Plate for the Determination of Haptoglobin Phenotype (Surface: Stainless Steel; Resistivity: 10.SUP.8 ..Math.m; Antibody Used: Anti-Haptoglobin, Fitzgerald Company; Organism: Goat)

[0059] The modified surface was prepared by electrospray deposition with dry ions landing onto the surface for 20 minutes according to the following procedure:

[0060] The values on the apparatus according to FIG. 1 were set as follows:

Flow rate of the pump 2: 1 L/min
Voltage of the source 8 brought on the conductive part 9 of the syringe 4: 1500V
Temperature of the evaporation area of the tube shape 10: 40 C.
Voltage from the source 11 on the mask 13: 1500V
Pressure on the carrier gas inlet 7: 0.25 MPa
Carrier gas: nitrogen
Temperature of the carrier gas: 40 C.
Shape of the hole on the mask: circle of the diameter of 2 mm

[0061] Syringe pump 2 was filled with the solution of anti-haptoglobin antibody of the concentration of 2 mol/L in 5 mmol/L ammonium acetate, 30 vol. % acetonitrile (Solution A). High voltage source 8 was connected to the conductive part 9 of the syringe 4 with stock solution that was connected via capillary 5 to the splitter 1. The solution of antibody (A) was introduced into the splitter by the syringe pump 2, where it was electronebulized from the spray needle due to the high voltage and the flow of pressurized carrier gas from the inlet to form the charged aerosol (B). The formed aerosol was introduced into the area 10 of the tube shape of the diameter 5 mm where the aerosol was dried and further passed through mask 13 towards the surface from stainless steel 12. After finishing the process, high voltage from both the sources 8 and 11 was turned off, the surface was removed and washed with water. 240 pmol of antibody was used for this method and the formed layer was of a circle shape of the diameter given by the mask (2 mm).

[0062] According to the abovementioned method, the surfaces were modified with anti-haptoglobin antibody. The particular values set on apparatuses are stated individually in each example. The processes differed in other parameters:

1) Antibody concentration: A) 0.01 mol/L, B) 1 mol/L, and C) 100 mol/L.
2) Antibodies were obtained from various companies, mainly Fitzgerald and Sigma-Aldrich
3) The temperature of the evaporation area during deposition of antibody was in the range of 25-45 C.
4) The surface of the modified plate was from A) stainless steel, B) ITO glass (Indium Tin Oxide), C) aluminium.

Example 2 (Anti-Haptoglobin Antibody: Fitzgerald; Immunised Organism: Goat; Starting Material: Hemolysate, Surface: Indium Tin Oxide; resistivity: 10.SUP.4 ..Math.m)

[0063] The modified surface was prepared by preforming the electrospray deposition according to Example 1, by landing dry ions of polyclonal anti-haptoglobin antibody of the concentration of 2 mol/L on ITO (Indium Tin Oxide) glass for 5 minutes and at the temperature of the evaporation area 40 C.

[0064] The values on the apparatus according to FIG. 1 were set as follows:

Flow rate of the pump: 0.54/min
Voltage of the source 8 brought on the conductive part 9 of the syringe 4: 1300V
Temperature of the evaporation area of the tube shape 10: 40 C.
Voltage from the source 11 on the mask 13: 1000V
Pressure on the carrier gas inlet 7: 0.25 MPa
Carrier gas: nitrogen
Temperature of the carrier gas: 30 C.
Shape of the hole on the mask: circle of the diameter of 2 mm

[0065] After washing the glass with water, 2 L of hemolysate were deposited on the sites with bound antibody and left to incubate in Petri dishes for 1 h at the room temperature. Then the surface was washed for 310 min with 1 PBS solution, pH 7.2, and then with distilled water for 15 min. After the surface dried, 1 L of 15 mmol/l aqueous solution of TCEP was added and the surface was incubated for 30 min in Petri dish at the room temperature. After removing the surface from the Petri dish, matrix was added to the samples with bound haptoglobin, the matrix having following composition: 7.6 mg of DHAP dissolved in 375 L, of ethanol +125 L of DHAC (diammonium hydrogen citrate) of the concentration of 18 mg/mL. This matrix solution was mixed with 0.1% trifluoroacetic acid in the ratio of 1:1. One microliter of the matrix was mixed with the sample directly on the surface and left to crystalize at the temperature of 35 C. The samples were analysed by means of MALDI mass spectrometry (FIG. 2).

Example 3 (Anti-Haptoglobin Antibody: Fitzgerald Company; Immunised Organism: Goat; Starting Material: Serum, Surface: ITO; Resistivity: 10.SUP.4 ..Math.m)

[0066] The modified surface was prepared by preforming the electrospray deposition according to Example 1, by landing the dry ions of polyclonal anti-haptoglobin antibody (source: Fitzgerald Company, organism: goat) of the concentration of 2 mol/L, on the ITO (Indium Tin Oxide) glass for 5 minutes, whereas the temperature of the evaporation area during substance deposition was 40 C.

The values on the apparatus according to FIG. 1 were set as follows:
Flow rate of the pump: 2 L/min
Voltage of the source 8 brought on the conductive part 9 of the syringe 4: 1700V
Temperature of the evaporation area of the tube shape 10: 35 C.
Voltage from the source 11 on the mask 13: 1200V
Pressure on the carrier gas inlet 7: 0.25 MPa
Carrier gas: nitrogen
Temperature of the carrier gas: 40 C.
Shape of the hole on the mask: circle of the diameter of 2 mm

[0067] After washing the glass with water, 2 L of serum were deposited on the sites with bound antibody and left to incubate in Petri dishes for 1 h at the room temperature. Then the surface was washed for 310 min with 1 PBS solution, pH 7.2, and then with distilled water for 15 min. After the surface dried, 1 L of 50 mM aqueous solution TCEP was added and the surface was incubated for 30 min in Petri dish at the room temperature. After removing the surface from the Petri dish, matrix was added to the samples with bound haptoglobin, the matrix having following composition: 7.6 mg of DHAP dissolved in 375 L of ethanol +125 L of DHAC (diammonium hydrogen citrate) of the concentration of 18 mg/mL. This matrix solution was mixed with 0.1% trifluoroacetic acid in the ratio of 1:1. One microliter of the matrix was mixed with the sample directly on the surface and left to crystalize at the room temperature. The samples were analysed by means of MALDI mass spectrometry (FIG. 6A).

Example 4 (Anti-Haptoglobin Antibody: Sigma-Aldrich; Immunised Organism: Rabbit; Starting Material: Serum, Surface: Fused Quartz; Resistivity: 10.SUP.17 ..Math.m)

[0068] The modified surface was prepared by preforming the electrospray deposition according to Example 1, by landing the dry ions of polyclonal anti-haptoglobin antibody (Sigma-Aldrich) of the concentration of 3 mol/L on the fused quartz for 5 minutes and at the temperature of the evaporation area of 40 C.

[0069] The values on the apparatus according to FIG. 1 were set as follows:

Flow rate of the pump: 1 L/min
Voltage of the source 8 brought on the conductive part 9 of the syringe 4: 1400V
Temperature of the evaporation area of the tube shape 10: 35 C.
Voltage from the source 11 on the mask 13: 1400V
Pressure on the carrier gas inlet 7: 0.25 MPa
Carrier gas: nitrogen
Temperature of the carrier gas: 35 C.
Shape of the hole on the mask: circle of the diameter of 2 mm

[0070] After washing the glass with water, 2L of serum were deposited on the sites with bound antibody and left to incubate in Petri dishes for 1 h at the room temperature. Then the surface was washed for 310 min with 1 PBS solution, pH 7.2, and then with distilled water for 15 min. After the surface dried, 1 L of 50 mmol/L TCEP aqueous solution was added and the surface was incubated for 30 min in Petri dish at the room temperature. After removing the surface from the Petri dish, matrix was added to the samples with bound haptoglobin, the matrix having following composition: 7.6 mg of DHAP dissolved in 375 L of ethanol +125 L of DHAC (diammonium hydrogen citrate) of the concentration of 18 mg/mL. This matrix solution was mixed with 0.1% trifluoroacetic acid in the ratio of 1:1. One microliter of the matrix was mixed with the sample directly on the surface and left to crystalize at the room temperature. The samples were analysed by means of MALDI mass spectrometry (FIG. 6B).

Example 5 (Anti-Haptoglobin Antibody: Fitzgerald; Immunised Organism: Goat; Starting Material: Serum, Surface: Stainless; Resistivity: 10.SUP.8 ..Math.m)

[0071] The modified surface was prepared by preforming the electrospray deposition according to Example 1, by landing the dry ions of polyclonal anti-haptoglobin antibody (Fitzgerald) of the concentration of 4 mol/L on the stainless surface for 5 minutes and at the temperature of the evaporation area of 40 C.

[0072] The values on the apparatus according to FIG. 1 were set as follows:

Flow rate of the pump: 2.5 L/min
Voltage of the source 8 brought on the conductive part 9 of the syringe 4: 1500V
Temperature of the evaporation area of the tube shape 10: 60 C.
Voltage from the source 11 on the mask 13: 1600V
Pressure on the carrier gas inlet 7: 0.25 MPa
Carrier gas: argon
Temperature of the carrier gas: 35 C.
Shape of the hole on the mask: circle of the diameter of 2 mm

[0073] After washing the stainless with water, 2 L of serum were deposited on the sites with bound antibody and left to incubate in Petri dishes for 1 h at the room temperature. Then the surface was washed for 310 min with 1 PBS solution, pH 7.2, and then with distilled water for 15 min. After the surface dried, 1 L of 50 mmol/L TCEP aqueous solution was added and the surface was incubated for 30 min in Petri dish at the room temperature. After removing the surface from the Petri dish, matrix was added to the samples with bound haptoglobin, the matrix having following composition: 7.6 mg of DHAP dissolved in 375 L of ethanol +125 L of DHAC (diammonium hydrogen citrate) of the concentration of 18 mg/mL. This matrix solution was mixed with 0.1% trifluoroacetic acid in the ratio of 1:1. One microliter of the matrix was mixed with the sample directly on the surface and left to crystalize at the room temperature. The samples were analysed by means of MALDI mass spectrometry (FIG. 6C).

Example 6 (Anti-Haptoglobin Antibody: fitzgerald; Immunised Organism: Goat; Starting Material: Serum, Surface: Porous Silicon; Resistivity: 10.SUP.2 ..Math.m; without the Presence of Matrix and without the Incubation)

[0074] The modified surface was prepared by preforming the electrospray deposition according to Example 1, by landing the dry ions of polyclonal anti-haptoglobin antibody (Fitzgerald) of the concentration of 2.5 mol/L on the porous silicon surface for 5 minutes and at the temperature of the evaporation area of 40 C.

[0075] The values on the apparatus according to FIG. 1 were set as follows:

Flow rate of the pump 1 L/min
Voltage of the source 8 brought on the conductive part 9 of the syringe 4: 1500V
Temperature of the evaporation area of the tube shape 10: 37 C.
Voltage from the source 11 on the mask 13: 300V
Pressure on the carrier gas inlet 7: 0.5 MPa
Carrier gas: helium
Temperature of the carrier gas: 20 C.
Shape of the hole on the mask: circle of the diameter of 2 mm

[0076] After washing the surface with water, 1 L of serum was deposited on the sites with bound antibody. Then the surface was washed for 310 min with 1 PBS solution, pH 7.2, and then with distilled water for 15 min. After the surface dried, 1 L of 50 mmol/L TCEP aqueous solution was added to the samples. The surface was washed with 1% aqueous solution of formic acid. The samples were analysed by means of MALDI mass spectrometry without the presence of the ionization matrix.