CN107022028B

CN107022028B - Specific anti-CitH 3 monoclonal antibody and application of enzyme-linked immunosorbent assay kit thereof in sepsis diagnosis

Info

Publication number: CN107022028B
Application number: CN201710096048.0A
Authority: CN
Inventors: 哈桑·阿拉姆; 李永清; 崇巍
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-02-22
Filing date: 2017-02-22
Publication date: 2020-07-28
Anticipated expiration: 2037-02-22
Also published as: CN107022028A

Abstract

The invention discloses a specific anti-CitH 3 monoclonal antibody and an E L ISA kit containing the antibody and used for quantitatively detecting CitH 3.

Description

Specific anti-CitH 3 monoclonal antibody and application of enzyme-linked immunosorbent assay kit thereof in sepsis diagnosis

Technical Field

The invention relates to the field of sepsis diagnosis, and particularly provides a guanidine-aminated histone H3 (CitH 3) specific antibody, a method for detecting CitH3 by using the antibody enzyme-linked immunosorbent assay for diagnosing sepsis and a corresponding kit.

Background

The early signs of sepsis are often unspecific and difficult to detect, and prodromal symptoms are easily confused with non-infectious diseases.A valid blood culture is the gold standard for diagnosing sepsis, however, a positive result in blood culture takes more than 48h to obtain, and blood culture is often negative due to low bacterial density in blood at an early stage of infection.thus, those alternative markers with high sensitivity, specificity and predictability can contribute to early detection of sepsis, monitoring of the progression of sepsis, and its therapeutic effect.A number of currently discovered biological markers, including procalcitonin, interleukin 1 β (I L-1 β), interleukin 6 (I L-6), tumor necrosis factor α (TNF- α), etc., where many biological markers have been applied due to the short half-life of the ring, are ideally applied to diagnosis of sepsis, and other non-infectious trauma-specific markers are also difficult to differentiate between early stage death.

The inventors of the present invention found that guanidine-aminated histone H3 (citrullated histone H3, CitH 3) is an ideal diagnostic marker for sepsis and septic shock. Pan et al found that CitH3 protein in peripheral blood of mice in septic shock model appeared and persisted in early stage of injury (0.5 h), and could be used for early diagnosis of sepsis, and for assessing prognosis and severity of illness and reflecting treatment effect of sepsis patients, see Pan et al, 12th academic clinical Congress (2017).

The existing method for detecting the CitH3 is western blotting (western blotting), but the method can only qualitatively detect the CitH3, namely, the method can only detect whether the CitH3 exists in a sample, but cannot detect the actual concentration of the CitH3, and is complex and time-consuming to operate, and the defects seriously limit the practicability of the CitH3 as a sepsis diagnostic marker, compared with the existing commercial monoclonal antibody, the monoclonal antibody provided by the invention has higher affinity to the CitH3, compared with the western immunoblotting, an enzyme-linked immunosorbent assay (E L ISA) performed by using the monoclonal antibody provided by the invention can more sensitively and accurately detect the specific concentration of the CitH3 in the sample, and when the concentration of the CitH3 in blood of a patient exceeds a threshold value, the sepsis diagnosis can be prompted, and the kit can be applied to the detection of a human sample, such as a whole blood sample (such as ascites, cerebrospinal fluid, urine, saliva or blood serum, so that the clinical application of the CitH3 in the human sample is expanded.

Disclosure of Invention

In order to overcome the problem that the prior art lacks a CitH 3E L ISA detection method and a corresponding detection antibody:

in one aspect, the invention provides an antibody specific for CitH3, wherein the antibody is made using a peptide having the amino acid sequence of SEQ ID NO. 1.

Further, the antibody comprises the light chain CDR1 of the amino acid sequence of SEQ ID NO. 2, the light chain CDR2 of the amino acid sequence of SEQ ID NO. 3, the light chain CDR3 of the amino acid sequence of SEQ ID NO. 4, the heavy chain CDR1 of the amino acid sequence of SEQ ID NO. 6, the heavy chain CDR2 of the amino acid sequence of SEQ ID NO. 7, and the heavy chain CDR3 of the amino acid sequence of SEQ ID NO. 8.

Further, the antibody comprises the light chain variable region of the amino acid sequence of SEQ ID NO. 5 and/or the heavy chain variable region of the amino acid sequence of SEQ ID NO. 9.

Further, the antibody amino acid sequence has greater than 90% sequence identity to SEQ ID NO 18.

Further, the amino acid sequence of the antibody is SEQ ID NO. 18.

Further, the antibody is a monoclonal antibody.

In another aspect, the invention provides the use of an anti-CitH 3 specific monoclonal antibody in the preparation of a kit for detecting CitH3 in a biological sample.

Further, the biological sample is selected from the group consisting of a serological sample, cerebrospinal fluid, urine, saliva, and ascites fluid.

Further, the biological sample is from a human, non-human primate, rodent, dog.

Further, the detection process includes:

exposing a biological sample of the subject to an antibody of any one of the preceding claims; quantifying the amount of CitH3 present in said sample; comparing the amount of CitH3 present in the sample to known standards.

Further, the detection process includes:

exposing a biological sample of the subject to an antibody of any one of the preceding claims; quantifying the amount of CitH3 bound by said antibody present in said sample; comparing the amount of CitH3 present in the sample to: a. a known standard, or b. a comparison of biological samples obtained from the subject at an earlier time point; and determining whether the subject has a level of CitH3 that is indicative of a diagnostic, severity, therapeutic, and prognostic effect of sepsis or septic shock.

Further, the known standard is the level of CitH3 derived from subjects identified as not having sepsis or the level of CitH3 derived from subjects identified as having sepsis or septic shock.

In another aspect, the invention provides an immunoassay kit of the E L ISA for the specific detection of a guanidinium histone H3 (CitH 3) protein, the kit comprising a first antibody as described in any preceding claim, and at least one second antibody that binds to CitH 3.

Further, the kit further comprises peptides for use as standards and for calibration purposes.

Further, the peptide used as a standard and for calibration purposes has the amino acid sequence of SEQ ID NO 1.

Further, the kit is used for determining CitH3 in a body fluid sample.

Further, the body fluid sample is selected from the group consisting of a serological sample, cerebrospinal fluid, urine, saliva, and ascites fluid.

Further, one of the first or second antibodies in the kit is immobilized on a surface.

Further, the kit also includes a container for containing the antibody when not in use, and instructions for use.

In another aspect, the invention provides a polynucleotide encoding an antibody specific for CitH3 comprising light chain CDR1 of the amino acid sequence of SEQ ID NO. 2, light chain CDR2 of the amino acid sequence of SEQ ID NO. 3, light chain CDR3 of the amino acid sequence of SEQ ID NO. 4, heavy chain CDR1 of the amino acid sequence of SEQ ID NO. 6, heavy chain CDR2 of the amino acid sequence of SEQ ID NO. 7, heavy chain CDR3 of the amino acid sequence of SEQ ID NO. 8

Further, the polynucleotide comprises the nucleotide sequences of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.

Further, the polynucleotide comprises the nucleotide sequences of SEQ ID NO 13 and SEQ ID NO 17.

The antibodies provided by the invention specifically bind to fragments of humoral CitH3, as demonstrated by comparison with control antibodies, which are prepared from a peptide as the antigen. The peptide is selected from amino acid residues 1 to 30 from the amino terminal of human-derived Histone (Histone) H3, and has an amino acid sequence described in SEQ ID NO: 19.

The second antibody in the kit of the invention can be selected from a commercial anti-CitH 3 polyclonal antibody (ab 5103, Abcam, Cambridge, MA, USA) or other CitH3 antibodies, and the specific binding substance of the second antibody can be selected from a commercial peroxidase-labeled goat anti-rabbit immunoglobulin (111-.

Drawings

FIG. 1 shows a schematic of possible sources of serum guanylated histone H3 (CitH 3) and histone H3 (H3).

FIG. 2 shows the levels of CitH3 protein in peripheral blood of mice, a septic shock model, over different time periods (E L ISA method).

Figure 3 shows the levels of serum I L-1 β at different time points in a mouse model of septic shock and after treatment.

Figure 4 shows the levels of serum PCT at different time points in a mouse model of septic shock and after treatment.

FIG. 5 shows histograms of serum TNF- α levels in mice in the low dose (SD) lipopolysaccharide (L PS) group and the high dose (L D) L PS group.

FIG. 6 shows densitometric quantitative determination of the level of CitH3 protein expression in the low dose (SD) L PS group and the high dose (L D) L PS mouse model.

Figure 7 shows the levels of CitH3 in plasma of healthy humans, septic patients and trauma patients.

FIG. 8 shows the results of detection of CitH3 by Western blot and E L ISA.

Figure 9 shows the levels of serum I L-6 at different time points in a mouse model of septic shock and after treatment.

Examples

Example 1

Clinical studies have shown that circulating CitH3 increases in patients with sepsis due to multiple infections.

Healthy subjects

And (3) admission standard: the age is more than or equal to 18 years old; (ii) is capable of answering; plain body is healthy; body weight is more than or equal to 110 pounds (49.9 kg)

Exclusion criteria: age <18 years old; patients with chronic inflammatory diseases; acute illness; application of immunosuppressive drugs; AIDS; patients with malignant diseases; transfusions of blood or blood products over the past 24 hours; weight <110 pounds (49.9 kg), last 3 weeks with history of blood donations

Trauma patient

And (3) admission standard: severely traumatized patients; lesion severity score (ISS) > 16; the age is more than or equal to 18 years old; the weight is more than or equal to 110 pounds (49.9 kg);

exclusion criteria: age <18 years old; patients with chronic inflammatory diseases; application of immunosuppressive drugs; AIDS; patients with malignant diseases; body weight <110 pounds (49.9 kg); there was severe blood loss, say >100ml, with a history of blood transfusions in the past three weeks.

Patients with sepsis

And (3) admission standard: patients with sepsis were clinically diagnosed according to the guidelines of the united states department of chestnuts and the guidelines of the critical care society.

Exclusion criteria: age <18 years old; patients with chronic inflammatory diseases; acute illness; application of immunosuppressive drugs; AIDS; patients with malignant diseases; transfusions of blood or blood products over the past 24 hours; body weight <110 pounds (49.9 kg); there was severe blood loss, say >100ml, with a history of blood transfusions in the past three weeks.

A total of 5 healthy volunteer samples were collected, 5 from 5 trauma patients and 6 from 6 sepsis patients.

Preparation of human guanylated histone 3 (cityliden H3, CitH 3) enzyme-linked immunosorbent system three-step enzyme-linked immunosorbent assay Systems were prepared with anti-CitH 3 monoclonal antibody (primary antibody), commercial anti-CitH 3 polyclonal antibody (secondary antibody), and commercial peroxidase-labeled goat anti-rabbit immunoglobulin (second specific binding substance) described herein, the primary antibody was diluted to 2ug/ml with sodium carbonate/sodium bicarbonate (pH 9.6) (Sigma Aldrich, St. L ouis, MO, USA) and then 100ul of the resulting solution was added to each well of an immunoplate (R & D Systems inc., Minneapolis, MN, USA) and overnight at 4 ℃.

Human serum preparation serum was separated by conventional centrifugation or the like and stored at minus 80 ℃ when CitH3 in blood circulation was measured qualitatively or quantitatively using the kit, DNase (Sigma Aldrich, St. L ouis, MO, USA) at a concentration of 150 units/ml was added to mouse and human serum while supplementing 1mM calcium ions, and reacted in a 37 ℃ water bath for one hour.

Enzyme-linked immunosorbent assay the enzyme-linked immunosorbent assay used protein-free blocking solution as diluent, mouse and human serum samples of normal individuals and sepsis were prepared by diluting serum 20-fold, respectively, after completion of the reaction, the samples were washed four times with PBS containing 0.05% tween 20 and 100ul second antibody diluted to 0.3ug/ml with blocking solution was added to each well, after 2 hours of reaction at room temperature, the plates were washed 4 times in the same manner as above, and 100ul second specific binding substance diluted to 0.02ug/ml with blocking solution was added to each well and reacted at room temperature for 2 hours, after completion of the reaction, the plates were washed 4 times in the same manner as above and tetramethylbenzidine (TMB, Thermo Scientific, Rockford, I L) was added to each well, after 20 minutes of reaction at room temperature, and then measured using 0.5M sulfuric acid solution (R D & l., MN & lty > molecular absorbance, USA, measured using a spectrophotometer, measuring absorbance measurement system, map, USA using molecular absorbances, spectral analysis, map 52.

Establishment of a standard curve: the system of the invention was prepared by the method described above and a standard curve was established by the system using a standard preparation of artificially synthesized human-derived CitH3, and CitH3 was measured quantitatively. Standard curves were established using the methods shown above using blocking solutions to make 0, 0.32,0.63, 1.25, 2.55, 10 and 20ng/ml gradient dilutions of the standard formulation.

Furthermore, in enzyme-linked immunosorbent assays, the assay is carried out by using peroxidase-labeled antibodies as an alternative method. This alternative method is performed by forming a sandwich complex of antibody-antigen-enzyme-labeled antibody.

The level of CitH3 in the normal healthy human circulation is low and not easily detectable. Thus, blood samples from mouse endotoxic shock models and healthy subjects can serve as positive and negative controls, respectively.

As shown in FIG. 7, CitH3 was not detectable in normal human blood, and interestingly CitH3 was also not detectable in blood from patients with hemorrhagic shock. Sepsis can cause significant increases in blood CitH3 compared to normal subjects and patients with blood loss, suggesting increased levels of CitH3 in patients with sepsis. Furthermore, the above response may be specific for sepsis and thus not seen in patients with hemorrhagic shock.

Example 2

Comparison of enzyme-linked immunosorbent assay and Western blot assay for mouse serum CitH3

Experiments were divided into two groups, experiment one, using the specific CitH3 enzyme-linked immunosorbent assay (E L ISA) described in this statement, i.e.96-well plates coated overnight with CitH3 monoclonal antibody, incubated with mouse serum for 2 hours and then with CitH3 polyclonal antibody and HRP-associated anti-rabbit antibodies, synthetic CitH3 was used to prepare a standard curve (0-20 ng/ml), experiment two, using peptidylarginine deiminase 4 (peptidylarginine deiminase-4, PAD 4) inhibitor YW3-56 as an intervention, C57 4/6J mice were randomly divided into three groups (n = 7/group), intraperitoneally injected with (1) Dimethyl sulfoxide (Dimethyl foxide, DMSO), (2) L PS (35 mg/kg/3) 24W + 56W 56, Western 635W 5 + 56, DMSO 24-5, DMSO + 26 g, and Western blot 26, using a variance analysis of plasma survival rates after (24 mg/kg, 26, 8 kg, 26).

The method comprises the following specific implementation steps:

(1) mouse guanidinated histone 3 (citrullinated histone H3, CitH 3) enzyme-linked immunosorbent system preparation three-step enzyme-linked immunosorbent assay system was prepared using the anti-CitH 3 monoclonal antibody (primary antibody), commercial anti-CitH 3 polyclonal antibody (secondary antibody), and commercial peroxidase-labeled goat anti-rabbit immunoglobulin (second specific binding substance) described herein, the primary antibody was diluted to 2ug/ml with sodium carbonate/bicarbonate (pH 9.6) (Sigma Aldrich, St. L ouis, MO, USA) and 100ul of the resulting solution was added to each well of an immunoplate (R & D Systems inc., Minneapolis MN, USA) and overnight at 4 ℃.

(2) Mouse serum preparation blood was obtained from mice by a conventional blood collection method such as heart, peripheral vein, etc., serum was separated by a conventional centrifugation method, etc., and stored at minus 80 ℃ when CitH3 in blood circulation was measured qualitatively or quantitatively using the kit, deoxyribonuclease (Sigma Aldrich, St. L ouis, MO, USA) at a concentration of 150 units/ml was added to mouse and human serum while supplementing 1mM calcium ions, and reacted in a 37 ℃ water bath for one hour.

(3) The E L ISA method determines CitH3 the E L ISA method uses a protein-free blocking solution as a diluent, and prepares mouse and human serum samples of normal individuals and sepsis by diluting 20 times the serum, respectively, adding 100ul of diluted sample per well and reacting at room temperature for 2 hours after the reaction is completed, the sample is washed four times with PBS containing 0.05% Tween 20 and 100ul of a second antibody diluted to 0.3ug/ml with blocking solution is added to each well after the reaction is completed at room temperature, the plate is washed 4 times in the same manner as above after 2 hours of reaction, and 100ul of a second specific binding substance diluted to 0.02ug/ml with blocking solution is added to each well and reacted at room temperature for 2 hours after the reaction is completed, the plate is washed 4 times in the same manner as above and tetramethylbenzidine (TMB, Thermo Scientific, Rockford, I L) is added to each well after 20 minutes of reaction at room temperature and the absorbance of the protein is measured using a Biotechnology photo spectrometer (Calif., Calif. the results are given in the present invention, using a protein-Min & S.S.S.S.S.S.S.A.A.S. is able to be measured using a spectrophotometric, using a spectrophotometric (Calif. No. 2. As indicated, Calif. 1, Calif. Min.S.S.S.S.S.S.S.S.S.S.S.S..

(4) Western blot assay CitH3 equal amounts of plasma were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 15% polyacrylamide gel, proteins were subsequently transferred to nitrocellulose membranes (Bio-Rad L analytes, Hercules, Calif.), blocked with 0.05% PBS-Tween (PBST) and 5% milk (Bio-Rad L analytes, Hercules, Calif.), incubated overnight at 4 ℃ in primary antibody, and then incubated with horseradish peroxidase-coupled secondary antibody (PBST containing 5% milk at 1: 3000) for 1: 2h at room temperature to bind to primary antibody binding was added to Wem stemilk L staining Reagent Plus (Perkin Elmer L, AS., Boston, Mass.) and detected by Chemiluminescence detection under standard Imaging procedures, and quantified by scanning on Biosample strips (Biochemical scanning systems, Inc.) L).

The result is shown in FIG. 8, Western blot and E L ISA can both detect the increase of CitH3, but only E L ISA can accurately quantify the concentration of blood CitH3, which proves that compared with Western blot, the specificity CitH 3E L ISA established in the laboratory, which is a CitH3 quantitative detection method, is more reliable and accurate.

Example 3

CitH3 is an ideal early diagnosis and treatment marker for septic shock

C57B L/6J mice were randomly divided into three groups (n =7/10 groups) and were each injected intraperitoneally with (1) Dimethyl sulfoxide (DMSO), (2) L PS (35 mg/kg) + DMSO, (3) L PS + YW3-56 (5 mg/kg, dissolved in DMSO, injected after L PS), and blood samples of 0h, 0.5h, 3h, 12h and 24h were collected and plasma CitH3 levels were measured using enzyme-linked immunosorbentary assay (E L ISA) as described in this statement, while interleukin (I L) -1 β, I L-6 and Procalcitonin (PCT) levels were measured using E L ISA.

Mouse CitH3 elisa system preparation three-step elisa experimental Systems were prepared using anti-CitH 3 monoclonal antibody (primary antibody), commercial anti-CitH 3 polyclonal antibody (secondary antibody), and commercial peroxidase-labeled goat anti-rabbit immunoglobulin (second specific binding substance) described herein the primary antibody was diluted to 2ug/ml with sodium carbonate/bicarbonate (pH 9.6) (Sigma Aldrich, St. L ouis, MO, USA) and 100ul of the resulting solution was added to each well of an immune plate (R & D Systems inc., Minneapolis, MN, USA) and overnight at 4 ℃.

Mouse serum preparation blood was obtained from mice by a conventional blood collection method such as heart, peripheral vein, etc., serum was separated by a conventional centrifugation method, etc., and stored at minus 80 ℃ when CitH3 in blood circulation was measured qualitatively or quantitatively using the kit, deoxyribonuclease (Sigma Aldrich, St. L ouis, MO, USA) at a concentration of 150 units/ml was added to mouse and human serum while supplementing 1mM calcium ions, and reacted in a 37 ℃ water bath for one hour.

Enzyme-linked immunosorbent assay the enzyme-linked immunosorbent assay used a blocking solution without protein as a diluent, mouse and human serum samples of normal individuals and sepsis were prepared by diluting serum 20-fold, respectively, after completion of the reaction, the samples were washed four times with PBS containing 0.05% tween 20 and 100ul of a second antibody diluted to 0.3ug/ml with a blocking solution was added to each well and reacted at room temperature for 2 hours, the plates were washed 4 times in the same manner as above after the reaction at room temperature, and 100ul of a second specific binding substance diluted to 0.02ug/ml with a blocking solution was added to each well and reacted at room temperature for 2 hours, after completion of the reaction, the plates were washed 4 times in the same manner as above and tetramethylbenzidine (TMB, Thermo Scientific, Rockford, I L) was added to each well after reaction for 20 minutes at room temperature, and then the absorbance was measured using a 0.5M sulfuric acid solution (R & ltx., USA) and the absorbance was measured using a spectrophotometer for each well as indicated by using a absorbance map, USA, measuring absorbance of soluble protein, USA, inc 2 nm.

The results are shown in FIGS. 2-4,9, that CitH3 levels in L PS group were elevated at an early stage of septic shock (0.5 h) compared to the control group, and were significantly different, that CitH3 in blood was significantly reduced when YW3-56 treatment was given, that septic shock and its therapeutic effects could be suggested at an early stage, and that the above-mentioned indications were maintained for a longer period of time (24 h) compared to CitH3, although it could reflect the therapeutic effects of YW3-56 on septic shock mice, I L-1 β could not be detected at an early stage of onset of disease, I L-6 could not show the progress of treatment continuously at every time point, PCT as a clinically common infection marker, did not significantly change within 24h of septic shock, thereafter began to significantly increase, and could further guide treatment.

SEQUENCE LISTING

<110> Han mulberry Alamem Li Yongqing Wei

<120> application of specific anti-CitH 3 monoclonal antibody and enzyme-linked immunosorbent assay kit thereof in sepsis diagnosis

<160>19

<170>PatentIn version 3.5

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Claims

1. An anti-CitH 3 specific monoclonal antibody, wherein the antibody is prepared with a peptide of the amino acid sequence of SEQ ID NO. 1;

the antibody comprises the light chain CDR1 of the amino acid sequence of SEQ ID NO. 2, the light chain CDR2 of the amino acid sequence of SEQ ID NO. 3, the light chain CDR3 of the amino acid sequence of SEQ ID NO. 4, the heavy chain CDR1 of the amino acid sequence of SEQ ID NO. 6, the heavy chain CDR2 of the amino acid sequence of SEQ ID NO. 7, and the heavy chain CDR3 of the amino acid sequence of SEQ ID NO. 8.

2. The antibody of claim 1, comprising the light chain variable region of the amino acid sequence of SEQ ID NO. 5 and/or the heavy chain variable region of the amino acid sequence of SEQ ID NO. 9.

3. The antibody of claim 2, having an amino acid sequence with greater than 90% sequence identity to SEQ ID NO. 18.

4. The antibody of claim 3 having the amino acid sequence of SEQ ID NO 18.

5. Use of an antibody according to any one of claims 1 to 4 in the preparation of a kit for detecting CitH3 in a biological sample.

6. The use of claim 5, wherein the biological sample is selected from the group consisting of a serological sample, cerebrospinal fluid, urine, saliva, and ascites fluid.

7. The use of claim 5, wherein the biological sample is from a human, a non-human primate, a rodent, a dog.

8. The use of any of claims 5-7, wherein the detection process comprises:

exposing a biological sample to the antibody of any one of claims 1-4; quantifying the amount of CitH3 present in said sample; comparing the amount of CitH3 present in the sample to known standards.

9. The application of claim 8, wherein the detection process comprises:

exposing a biological sample to the antibody of any one of claims 1-4; quantifying the amount of CitH3 bound by said antibody present in said sample; comparing the amount of CitH3 present in the sample to: a. a known standard, or b. a comparison of biological samples obtained at earlier time points; and determining whether the level of CitH3 in the organism to which the biological sample belongs is indicative of a diagnostic, severity, therapeutic and prognostic effect of sepsis or septic shock.

10. The use of claim 9, wherein a. known criteria is the level of CitH3 derived from an organism identified as not having sepsis or the level of CitH3 derived from an organism identified as having sepsis or septic shock.

11. An enzyme-linked immunosorbent assay kit for specifically detecting a guanidinium histone H3 (CitH 3) protein, the kit comprising: a first antibody; and at least one second antibody that binds to CitH3, wherein the first antibody is the antibody of any one of claims 1-4.

12. The kit according to claim 11, further comprising peptides for use as standards and for calibration purposes.

13. The kit according to claim 12, wherein the sequence of the peptide used as a standard and for calibration purposes is the amino acid sequence of SEQ ID NO. 1.

14. A kit according to claim 11 wherein the kit is for the determination of CitH3 in a body fluid sample.

15. The kit according to claim 14, wherein the body fluid sample is selected from the group consisting of a serological sample, cerebrospinal fluid, urine, saliva or ascites fluid.

16. A kit according to any one of claims 11 to 15, wherein one of the first or second antibodies is immobilized on a surface.

17. The kit of claim 16, further comprising a container for containing the antibody when not in use, and instructions for use.

18. A polynucleotide encoding an antibody specific for CitH3 comprising light chain CDR1 of the amino acid sequence of SEQ ID NO. 2, light chain CDR2 of the amino acid sequence of SEQ ID NO. 3, light chain CDR3 of the amino acid sequence of SEQ ID NO. 4, heavy chain CDR1 of the amino acid sequence of SEQ ID NO. 6, heavy chain CDR2 of the amino acid sequence of SEQ ID NO. 7, heavy chain CDR3 of the amino acid sequence of SEQ ID NO. 8.

19. The polynucleotide of claim 18, comprising the nucleotide sequences of SEQ ID NO 10, 11, 12, 14, 15 and 16.

20. The polynucleotide of claim 19, comprising the nucleotide sequences of SEQ ID NOs 13 and 17.