DIAGNOSTIC TEST

Abstract

The method according to the invention enables the confirmation of infections/carriage caused by Streptococcus agalactiae bacterial species. The method employs a specific reaction of selected epitopes that are part of immunoreactive proteins of clinical isolates of Streptococcus agalactiae with antibodies present in the serum.

Claims

1. A protein comprising an amino acid sequence selected from Seq. No. 1-2 and epitopes contained in it.

2. Epitope specific for infectious Streptococcus agalactiae having an amino acid sequence selected from Seq. No. 3-15 and its derivatives in which at least one of the amino acids was removed or replaced with another amino acid, preferably selected from Ala or Gly, or its biotinylated form.

3. The epitope according to claim 2, characterized in the fact that it has an amino acid sequence which is a derivative of the sequence selected from Seq. No. 3-15, in which at least one of the amino acids was removed or replaced with another amino acid, preferably selected from Ala or Gly, or its biotinylated form; at the same time, the said epitope has increased immunoreactivity.

4. The epitope according to claim 2, characterized in the fact that it has an amino acid sequence selected from Seq. No. 16-27.

5. A method to detect a patient's infection with a Streptococcus agalactiae strain, characterized in the fact that, in the sample taken from the patient, the presence is checked of the protein determined in claim 1 or antibodies specific to this protein, wherein the presence of that protein or such antibodies indicates infection of the patient with a Streptococcus agalactiae strain.

6. The method according to claim 5, characterized in the fact that the test is carried out using well-known immunochemical methods, in particular Western Blotting or ELISA.

7. The method according to claim 5, characterized in the fact that human serum is used as the sample studied, especially one diluted 100-10000 times.

8. The method according to claim 5, characterized in the fact that the presence of a protein, epitope or antibodies signals the patient's carriage of the Streptococcus agalactiae strain.

Description

[0019] Example realizations of the invention have been presented in Figures, in which:

[0020] FIG. 1 presents amino acid sequences of immunoreactive proteins of S. agalactiae.

[0021] FIG. 2 presents an example picture from Western blot analysis using three strains: S55 (1736/08), S57 (13793/08), D129. The following sera from umbilical cord blood were used to check immunogenicity: A) 3/KP (inactivated), B) 1/KP, C) 6/KP. Immunoreactive proteins were NRID5 and NRID6 with weights of about 55 kDa.

[0022] FIG. 3 presents specificity of polypeptide core epitopes of S. agalactiae with a mix of GBS+ sera and a mix of GBS sera.

[0023] FIG. 4 presents an example of reactivity of peptides modified by alanine substitution for a derivative of the core epitope MVTTGIIDPVKV (Ep9) of the NRID6 protein.

[0024] FIG. 5 presents an example of specificity of the derivative of the epitope GQVLAKPGSINPHTKF (Ep. 4) of the NRID4 protein of the sequence KPGSINPHTKF assessed in the ELISA test against selected sera: 6/KPserum from a patient with GBS carriage, GBS 2aserum from a patient with GBS infection, 13/KPserum from a non-GBS patient (negative control); GASserum from a patient with S. pyogenes infection (negative control). FIG. 5A presents the results obtained for IgG antibodies, FIG. 5B presents results for IgM antibodies.

[0025] FIG. 6 presents an example ELISA result comparing protein reactivity elongation factor Tu (EF-Tu, NRID4 protein) and the derivative of one of its epitopes (Ep. 4) of the sequence KPGSINPHTKF. Legend: GBS+serum from an S. agalactiae carrier patient, GBSserum from a non-GBS patient (negative control), EF-Tuelongation factor Tu.

[0026] Moreover, the method according to the invention was presented more closely on the examples described below.

EXAMPLE 1

Determination of Immunoreactivity of Epitopes Using PEPSCAN [J. Mark Carter 1996]

[0027] A library of several dozen epitopes was obtained as a result of chemical synthesis using polyethylene pins (NCP Block of 96 gearsMimotopes, cat. no.: MIA10750001) with the application of Fmoc amino acid derivatives according to the procedure: [0028] 1. DEPROTECTIONpins we incubated in piperidine solution (PIP) 20% dissolved in dimethylformamide (DMF) for 1 h. [0029] 2. RINSINGpins were rinsed once with DMF for 2 min, then four times with methanol (MeOH) for 2 min and dried. [0030] 3. COUPLING (amino acid acylation)pins were activated with DMF for 5 min and then coupled with 60 mM amino acid derivative dissolved in DMF with 65 mM 1-hydroxy-7-azabenzotriazole (HOAt) and 60 mM diisopropylcarbodiimide (DIC) and 50 M bromophenol blue used as an indicator of the end of the acylation reaction. Aminoacylation was carried out at room temperature (22 C.) throughout the night or for 4 h in a sealed container, to prevent evaporation of the solution. [0031] 4. RINSING [0032] Variant A: if synthesis was continued, pins were rinsed once with MeOH for 5 min, air-dried for 5 min, and then incubated for 5 min in DMF. [0033] Variant B: at the end of the synthesis, pins were rinsed once with MeOH for 5 min, air-dried for 5 min, then incubated in DMF for 5 min. After its completion, pins were rinsed twice with MeOH for 2 min and air-dried for 30 min [0034] 5. ELONGATIONconsisted in cyclical repetition of stages 1-4. [0035] 6. N-ACETYLATION (optional)acetylation of -amino groups of the synthesized peptides was carried out in wells of a polyethylene plate using acetylation cocktail (3% acetic anhydride, 0.5% N,N-diisopropylethylamine (DIEA) dissolved in DMF) for 90 min. [0036] 7. RINSINGpins were rinsed once with MeOH for 10 min and air-dried for 15 min. [0037] 8. SIDE GROUP DEBLOCKING/REMOVALblocking side groups were removed from amino acids through 3-4 h of incubation in a bath containing deblocking cocktail (2.5% anisole, 2.5% 1,2-dithioethane in trifluoroacetic acid (TFA)) [0038] 9. RINSINGpins were rinsed once with MeOH for 10 min and then incubated in acetic acid solution (0.5% acetic acid, 50% MeOH diluted in water) for 60 min. Afterwards, pins were washed twice with MeOH for 2 min and dried throughout the night over desiccant resin. [0039] 10. DISRUPTIONpins were placed in a sonicator filled with disruption buffer (1% sodium dodecyl sulfate, 0.1% 2-mercaptoethanol, 0.1 M sodium phosphate; pH 7.2) heated to about 60 C. and sonicated for 10 min (7 kW/25 kHz). Afterwards, pins were removed from the buffer and washed with water heated to 60 C.

[0040] Following disruption, pins were stored under anhydrous conditions (e.g., in the presence of a water-absorbing substance or in a desiccator under vacuum conditions) or were employed directly for ELISA.

EXAMPLE 2

ELISA Test [Andersson et al., 1989]

[0041] 1. Synthetic epitopes on pins were submerged in Tris/HCl blocking buffer with 0.05% Tween 20 (TBS-T) containing 1% BSA and incubated for 1 h at room temperature. [0042] 2. Pins were washed three times with TBS-T buffer for 5 min. [0043] 3. After rinsing, pins were incubated in human serum solution diluted 1:1000 for 2 h at room temperature. [0044] 4. Pins were washed three times with TBS-T buffer for 5 min [0045] 5. Pins were submerged in a solution containing goat anti-human antibodies coupled to alkaline phosphatase diluted 1:10 000 and incubated for 1 h. [0046] 6. Pins were washed three times with TBS-T buffer for 5 min [0047] 7. After washing, pins were submerged in a solution with a substrate for alkaline phosphatase and, for 30 min, a color reaction was induced. [0048] 8. Pins were removed and absorbance was read at 2=405 nm.

EXAMPLE 3

[0049] Synthesis on Wang Resin by Fmoc Applied to Obtain Core Epitopes and their Modified Derivatives [Bachem, 2016] [0050] 1. Resin was activated in 20% piperidine solution (PIP) diluted in dimethylformamide (DMF) for 15 min [0051] 2. Resin was rinsed six times in 2 ml of DMF. [0052] 3. Additional amino acids were attached to the resin by adding the appropriate number of them, determined according to the formula:

M.sub.compound=n(active spaces)*2.5 eq*M.sub.compound [0053] and 43.7 l 1-hydroxy-7-azabenzotriazole (HOAt) and 43.9 l diisopropylcarbodiimide (DIC). Incubation was carried out from 6 to 36 h depending on the nature of the amino acid attached. [0054] 4. Resin was rinsed six times in 2 ml of DMF. [0055] 5. Stages 2-4 were repeated cyclically until a peptide of the sequence desired was obtained. [0056] 6. The peptide was removed from resin using 95% trifluoroacetic acid solution (TFA) dissolved in water. [0057] 7. The peptide was precipitated with ether and centrifuged, then dissolved in water and lyophilized.

EXAMPLE 4

ELISA Test for Biotinylated Peptides

[0058] 1. The well of a 96-well plate was coated with a streptavidin solution diluted in carbonate buffer at a concentration of 1 g/ml at 4 C. overnight. [0059] 2. The wells were rinsed three times (with saline buffered with phosphate with 0.05% Tween 20 (PBS-T). [0060] 3. Biotinylated peptides diluted in carbonate buffer at a concentration of 1 g/ml were added and incubated for 20 min at room temperature. [0061] 4. The wells were rinsed three times with PBS-T. [0062] 5. The wells were blocked with 1% BSA solution diluted with PBS-T for 1 h at room temperature. [0063] 6. Incubation was carried out with human serum diluted 1:100 for 2 h at room temperature. [0064] 7. Unbound antibodies were rinsed three times with PBS-T. [0065] 8. Incubation was carried out with a solution of goat anti-human IgM and/or IgG antibodies conjugated with horseradish peroxidase or alkaline phosphatase 1:10,000 for 1 h at room temperature. [0066] 9. Unbound antibodies were rinsed five times with PBS-T. [0067] 10. After rinsing, the substrate for horseradish peroxidase or alkaline phosphatase was added to each well and incubated for 30 min in the dark. [0068] 11. After the incubation was complete, the reaction was stopped by adding a sulfuric acid solution. [0069] 12. The absorbance was measured at a wavelength of 450 nm.

EXAMPLE 5

ELISA Test for Recombinant Protein

[0070] 1. The well of a 96-well plate was coated with protein dissolved in saline and incubated at 4 C. overnight. [0071] 2. The wells were rinsed three times with PBS-T (saline buffered with phosphate with 0.05% Tween 20. [0072] 3. Biotinylated peptides diluted in carbonate buffer at a concentration of 1 g/ml were added and incubated for 20 min at room temperature. [0073] 4. The wells were rinsed three times with PBS-T. [0074] 5. The wells were blocked with 1% BSA solution diluted with PBS-T for 1 h at room temperature. [0075] 6. Incubation was carried out with human serum diluted 1:100 for 2 h at room temperature. [0076] 7. Unbound antibodies were rinsed three times with PBS-T. [0077] 8. Incubation was carried out with a solution of goat anti-human IgM and/or IgG antibodies conjugated with horseradish peroxidase or alkaline phosphatase 1:10,000 for 1 h at room temperature. [0078] 9. Unbound antibodies were rinsed five times with PBS-T. [0079] 10. After rinsing, the substrate for horseradish peroxidase or alkaline phosphatase was added to each well and incubated for 30 min in the dark. [0080] 11. After the incubation was complete, the reaction was stopped by adding a sulfuric acid solution. [0081] 12. The absorbance was measured at a wavelength of 450 nm.

Results and Conclusions

[0082] As a result of the experiments conducted, 13 core epitopes were identified (Table 1), which were recognized in a highly specific way by human antibodies present in umbilical cord blood serum of GBS-infected patients and/or carriers (GBS+). The reaction was not observed with serum of GBS-negative people (GBS) (FIG. 3).

[0083] Modifications consisting in substituting individual amino acids with, among others, alanine or glycine as well as biotinylation of peptides caused an increase in immunoreactivity ranging from 7 to 80%, which is a non-obvious result (FIG. 4). It was also demonstrated that using two and/or three epitope derivatives in combination test increases the specificity in the reaction with umbilical cord antibodies up to about 40% in comparison to using a single epitope.

[0084] Biotinylated epitopes were characterized by high specificity in recognizing infection (high reactivity in the presence of IgM antibodies and average reactivity against IgG antibodies) and carrier state (high reactivity in the presence of IgG antibodies and low reactivity against IgM antibodies). The reaction was not observed with the serum of GBS-negative people (GBS) or GAS-positive serum, which is a non-obvious result (FIG. 5).

[0085] Furthermore, biotinylated epitopes of the immunogenic EF-Tu protein have been demonstrated to more strongly and more specifically recognize anti-GBS antibodies than the whole protein (FIG. 6).

REFERENCES

[0086] Andersson G., Ekre H. P., Alm G., Perlmann P. Monoclonal antibody two-site ELISA for human IFN-gamma. Adaptation for determinations in human serum or plasma. J Immunol Methods. 1989; 125:89-96. [0087] Solid phase synthesis Bachem (002363) published by Global Marketing, Bachem Group, February 2016. [0088] Carter J. M. Epitope Mapping of a Protein Using the Geysen (PEPSCAN) Procedure. The Protein Protocols Handbook; 1996, Part V, 581-593.

TABLE-US-00001 TABLE1 Polypeptidecoreepitopesof Streptococcusagalactiae. Corepeptide Protein Epitope aminoacid identification symbol sequence no. Ep1 RAAADYLEVPLYSYLG NRID2 Ep2 DRAMIALDGTPNKG NRID2 Ep3 LTAAITTVLARRLP NRID4 Ep4 GQVLAKPGSINPHTKF NRID4 Ep5 VVKVGIGPGSICTTR NRID5 Ep6 QGRKFKTYRG NR1D5 Ep7 KAFGSPLITN NRID6 Ep8 AGGVAVIKVGAA NRID6 Ep9 MVTTGLIDPVKV NRID6 Ep10 KLQERLAKLA NRID6 Ep11 AATETELKEMKLR NRID6 Ep12 KVTRSALQNA NRID6 Ep13 LQNAASVASLILTTE NRID6

TABLE-US-00002 TABLE 2 Characteristics of clinical strains of bacteria from the species S. agalactiae. Alp Strain Clinical Patient/ family Resistance ermB mefA/E No name material diagnosis Serotype genes phenotype gene gene 1 1736/08 urine newborn V alp2 cMLS.sub.B ermB UTI* 2 D129 urine woman III rib UTI* 3 D437 urine woman Ib epsilon UTI* 4 D280 urine man UTI* Ia epsilon 5 D481 urine woman V rib UTI* 6 G413 urine woman V alp2 UTI* 7 G408 urine woman Ib epsilon UTI* 8 G437 urine woman III rib UTI* 9 G361 urine man UTI* IV epsilon 10 286378 urine man UTI* II rib cMLS.sub.B ermB 11 300666 urine man UTI* V alp2 iMLS.sub.B 12 305139 urine newborn II bca UTI* 13 306723 urine newborn III alp2 UTI* 14 13793/08 urine newborn V alp2 cMLS.sub.B ermB UTI* 15 13723/07 urine newborn III rib cMLS.sub.B ermB UTI* 16 2992/08 urine newborn V rib UTI* 17 5303/08 urine newborn Ia epsilon UTI* 18 PP4 vaginal woman n/a alp2/3 swab carriage 19 PP6 vaginal woman n/a alp2/3 swab carriage 20 PP7 vaginal woman n/a bd swab carriage 21 PP8 vaginal woman n/a alp2/3 swab carriage 22 PP9 vaginal woman n/a rib swab carriage 23 NPP1 vaginal woman n/a n/a swab carriage 24 GAS1 pharyngeal child n/a n/a swab tonsillitis 25 GBS1 pharyngeal child tonsil n/a alp2/3 swab infection 26 GBS2 wound woman n/a n/a swab necrotizing fasciitis 27 2337/08 mouth newborn Ia epsilon swab colonization 28 CM47 blood newborn II rib ckMLS.sub.B ermB EOD** 29 13793/08 blood newborn V alp2 cMLS.sub.B ermB EOD** 30 2992/08 urine newborn V rib UTI* *UTIurinary tract infection (S. agalactiae >10.sup.5 CFU/ml); **EODearly onset disease; n/anot available; none; cMLS.sub.Bconstitutive resistance to macrolides, lincosamides, streptogramin B; iMLS.sub.Binducible resistance to macrolides, lincosamides, streptogramin B.

TABLE-US-00003 TABLE 3 List of GBS-positive and GBS-negative sera employed to evaluate the specificity of immunoreactive proteins of S. agalactiae and their epitopes in immunochemical tests. Venous blood Umbilical cord blood GBS-positive sera: SK1, SK2, PP4, PP6, PP7, PP8, PPG, NPP1, 1/KP, 2/KP, 3/KP, 4/KP, 5/KP, 6/KP, 8/KP, GBS1, GBS2a, GBS2b, PP9, SB3b, SB4b, PP7, 10/KP, 14/KP, 15/KP, 16/KP 28/3, 14/3, 10/3, 3/3, 42/3 GBS-negative sera: SK8, SB7, SB8, SB9, SB1a, SB1b, SB3a, 5/3, 12/KP, 13/KP, 24/KP, 28/KP, 29/KP 13/3, 22/3, 34/3

TABLE-US-00004 TABLE4 Percentincreaseinimmunoreactivityof derivativesoftheepitopeMVTTGHDPVK againstGBS-positiveserum,obtained asaresultofalaninesubstitution ofsubsequentaminoacids. %increasein reactivity Average compared absorb- tothe ance starting (A405nm sequence Peptide GBS+) (MVTTGIIDPVK) MVT1-GIIDPVA 1.38510001 anincreaseof45% MVTTGIIDPAK 1.01940002 anincreaseof7% MVTTGIIDAVK 1.32013333 anincreaseof38% MVTTGIIAPVK 1.15009999 anincreaseof21% MVTTGIADPVK 0.89886667 adecreaseof6% MVTTGAIDPVK 1.28863335 anincreaseof35% MVTTAIIDPVK 1.50660002 anincreaseof58% MVTAGIIDPVK 1.24636666 anincreaseof31% MVATGIIDPVK 1.2343667 anincreaseof30% MATTGIIDPVK 1.69836664 anincreaseof78% AVTTGIIDPVK 1.34063331 anincreaseof40% MVTTGIIDPVK 0.95466667