PREDIABETES AND DIABETES

FIELD OF THE INVENTION

The present invention relates generally to protein markers for use in detecting (pre)diahetes, and more specifically to use of LAM A.2, PLXDC2 and MLL4 for detecting, preventing and treating prediabetes and diabetes.

BACKGROUND OF THE INVENTION

Diabetes is a life-threatening metabolic disease characterized by hyperglycemia and poorly regulated carbohydrate metabolism resulting from insulin resistance and/or b-ceil dysfunction.

Despite several improvements and advances in type 2 diabetes (T2D) diagnosis and therapy over the past years, it is still an incurable disease. Accumulating evidence suggests that prevention is better than treatment because early prevention and intervention can significantly reduce the incidence of T2D and its complications. For instance, diet control, exercise and bariatric surgery prevented T2D in high-risk subjects. Prophylaxis with metformin also decreased the incidence of T2D. Therefore, .identification of subjects at high risk for T2D before its clinical onset holds the key to prevention of the disease.

Many efforts have been made to identify genetic and protein markers for T2D. Although genetic markers have high reliability, they are not satisfactory because they show up at T2D rather than prediabetes stage and have modest sensitivity and specificity. On the other hand, protein markers have high sensitivity and specificity because they reflect the progression of the disease

systematically and dynamically. Moreover, protein levels are tightly regulated by cellular stimulation. Thus, protein markers are potentially useful for diagnosis.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a diagnostic kit for detecting and identifying prediabetes and/or diabetes, comprising (i) a substrate having a top surface and a bottom surface opposite to the top surface, aid a top end and a bottom end opposite to the top end; (ii) a sample loading area; (iii) a capture antibody area, containing capture antibodies to capture prediabetes and diabetes protein markers comprising MLL4, LAMA2, and PLXDC2; (iv) a reagent area, containing a conditioning reagent; (v) a detection antibody area, containing detection antibodies to detect the captured prediabetes and diabetes protein markers comprising the MLL4, LAMA2, and PLXDC2; and (vi) optionally a positive control area; wherein the sample loading area, the detection antibody area, the reagent area, the capture antibody area, and the positive control area are located on the top surface of the substrate, allowing these areas to be in fl idi i ti n, the sample loading area being located at the top end and the capture antibody area located at the bottom end with the optionally positive control area located either after or before the capture antibody area

in one embodiment, the capture antibody area contains capture antibodies to capture one or more additional prediabetes and diabetes protein markers besides the MLL4, LAMA2, and PLXDC2.

In another embodiment the diagnostic kit may further comprise (vii) an instruction sheet showing directions of performing a method of detecting and identifying predi abates and/or diabetes according to the method invention.

The conditioning reagent may comprise a buffer solution, a detergent, a protease inhibitor, a salt, and/or a divalent cation. The reagent area may further comprise a color-forming reagent and a conditioning reagent.

In one embodiment, the capture antibodies, the conditioning reagent and the detection antibodies are coated onto the top surface of the substrate. The substrate may be a nitrocellulose membrane.

In another embodiment, the diagnostic kit device/apparatos of the invention is optimized for performance by lateral flow immunoassay.

In another aspect, the invention relates to a method for detecting and identifying prediabetes and/or diabetes, comprising (a) providing the diagnostic kit of die invention, (b) supplying a serum sample from a subject in need thereof; and (e) detecting whether the levels of prediabetes and diabetes protein markers comprising ML-L4, L AMA2, and PLXDC2 in the serum sample are increased as compared with a healthy control, wherein an increase hi the levels of the protein markers MLL4, LAMA2, and PLXDC2 is indicative of the subject in need thereof having prediabetes or diabetes.

Further in another aspect, the invention relates to a method for detecting and identifying prediabetes and/or diabetes, comprising (a) supplying a serum sample: from a subject in need thereof) and (b) detecting whether the levels of prediabetes and diabetes protein markers comprising MLL4, LAMA2, and PLXDC2 in the serum sample are increased as compared with a healthy control, wherein an increase in the levels of the protein markers MLL4, LAMA2, and PLXDC2 is indicative of the subject in need thereof having prediabetes or diabetes.

In one embodiment, the levels of the protein markers MLL4, LAMA2 and PLXDC2 exhibiting an area under an Receiver Operating Characteristic (ROC) curve of greater than 0.91 is indicative of the subject in need thereof having prediabetes or diabetes.

In another embodiment, the levels of the protein markers MLL.4, LAMA2 and PLXDC2 exhibiting an area under the ROC curve of greater than 0.95 is indicative of the subject in need thereof having prediabetes or diabetes. In another embodiment, the prediabetes and diabetes protein markers may further comprise one or more additional prediabetes and diabetes protein markers.

The detecting step may further comprise (d) providing capture antibodies specific against the MLL4, LAMA2, and PLXDC2, to form captured protein markers MLL4, LAMA2, and PLXDC2, respectively; (e) affording a conditioning reagent; and (f) supplying detection antibodies specific against the captured protein markers MLL4, L.AMA2, and PLXDC2. respectively.

The supplying step may further comprise the step of providing a diagnostic kit for detecting and identifying prediabetes and/or diabetes, in which the diagnostic kit comprises: (i) a substrate having a top surface and a bottom surface opposite to the top surface, and a top end and a bottom end opposite to the top end; (ii) a sample loading area; (lii) a capture antibody area, containing capture antibodies to capture prediabetes and diabetes protein markers comprising MLL4, LAMA2, and PLXDC2; (iv) a reagent area, containing a conditioning reagent; (v) a detection antibody area, containing detection antibodies to detect the captured prediabetes and diabetes protein markers comprising the MLL4, LAMA2, and PLXDC2; and (vi) optionally a positive control area: wherein the sample loading area, the detection antibody area, the reagent area, the capture antibody area, and the positive control area are located on the top surface of the substrate, allowing these areas to be in fluidic communication, the sample loading area being located at the top end and the capture antibody area located at the bottom end with the optionally positive control area located either after or before the capture antibody area.

In another embodiment, the detecting step may be performed by visualizing a color change.

The capture antibodies and detection antibodies may be polyclonal antibodies. In another embodiment, the capture antibodies and detection antibodies are monoclonal antibodies. Further in another embodiment, the capture antibodies are polyclonal antibodies and detection antibodies are monoclonal antibodies, or vice versa.

In another embodiment, the detection antibodies are labeled with colloidal gold, or a- color- generating enzyme, and the conditioning reagent comprises a substrate for the color-generating enzyme.

In another embodiment, the method is performed by a lateral flow immunoassay.

The invention further relates to use of a set of probes for detecting and identifying prediabetes and/or diabetes in a patient.

Further in another aspect, the invention relates to use of a set of probes with specific binding affinities to prediabetes and diabetes protein markers comprising MLL4, LAMA2, and PLXDC2 in the manufacture of the diagnostic kit for detecting prediabetes and/or diabetes of the invention. The set of probes comprises (a) a first probe having a specific binding affinity to the MLL4; (b)a second probe having a specific binding affinity to the LAMA2; and (c) a third probe having a specific binding affinity to the PLXDC2

in another aspect the invention relates to use of a set of probes with specific binding affinities to prediabetes and diabetes protein markers comprising MLL4, LAMA2, and PLXDC2 in the manufacture of a diagnostic kit for detecting pvediabetes and/or diabetes, wherein the set of probes comprises (a) a first probe having a specific binding affinity to the MLL4; (b) a second probe having a specific binding affinity to the LAMA2; and (c) a third probe having a specific binding affinity to die PLXDC2.

In another embodiment, the use of the set of the probes further comprise use of conditioning reagents, and a substrate in the manufacture of the diagnostic kit for detecting prediabetes and/or diabetes of the invention.

The set of probes may further comprise one or more additional probes with specific binding affinities to prediabetes and diabetes protein markers other than the MLL4, LAMA2, and PLXDC2.

In another embodiment the probes are antibodies comprising capture antibodies and detection antibodies.

Moreover, the invention relates to screening prediabetes and diabetes for care and/or treatment. Yet in another aspect, the invention relates to a method for care and treatment of prediabetes and/or diabetes, comprising the steps of: (I) detecting and identifying prediabetes and/or diabetes in a subject in need thereof; and (II) subjecting the subject in need thereof being identified as having die prediabetes or diabetes to a. care and/or treatment regime for the prediabetes or diabetes. The detecting and identifying step further comprises; (a) supplying a serum sample from a subject in need thereof; and (b) detecting whether the levels of prediabetes and diabetes protein markers comprising MLL4, LAMA2, and PLXDC2 in the serum sample are increased as compared with a healthy control wherein an increase in the levels of the protein markers MLL4, LAMA2, and PLXDC2 is indicati ve of the subject in need thereof having prediabetes or diabetes.

These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with die following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

Hie accompanying drawings illustrate one or more embodimen ts of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possib!e, the same reference numbers are used throughout the dra wings to refer to the same or like elemen ts of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart indi cating the experimental designs for discovery of serum proteins of mouse and human origins, followed by validation of their presence in human sera. Seram samples were collected from 3 healthy mice/ volunteers and 3 prediabetie mice/patients with 16 and 12 hrs of fasting. Serum from 3 heathy mice were pooled together and labeled with iTRAQ ^® 114. Serum from 3 prediabetic mice were labeled with iTRAQ* 115, 116 and 117, respectively. Subsequently, four of them were pooled together for tTRAQ^-based discovery. The same experimental processing was applied to human samples for iTRAQ*~hased discovery. The proteins with high confidence (average relative ratio and/? value) and novelty were selected as potential markers for immunoblotting validation.

FIG. 2 shows transformed volcano plot analysis of the selected proteins from human sera. Total proteins from human sera were processed using a combination of iTRAQ* and mass spectrometry (MS), followed by Mascot software identification. A transformed volcano plot was used to analyze

Iog2 (ratio of the level of one serum protein in prediabetic patient to its average level in healthy subjects). The serum proteins, detected in human and mouse sera, are labelled by solid dots. The ones, only detected in humans, are-labelled by hollow dots. Up-regulated and down-regulated proteins with P < 0.05 (*) were labelled by red and green colors, respectively. The proteins with P < 0 01 (**) and average expression ratio over 1 3 were selected for further analysis using

INGENUITY ^®' Pathway Analysis (IPA ^® ). Student’s /-test was used to compare the differences between heathy and prediabetic mice and humans

FIG. 3 shows pathway analysis of the selected serum proteins from human sera using IRAN The network is related to connective tissue disorders, dermatological diseases and conditions and developmental disorders. The markers which increased in prediabetie mice and patients were labelled in red color.

FIG. 4. shows results of immunoblotting analysis, diagnostic efficacy and diagnostic values of LAMA2, PL.XDC2 and MLL4 in healthy and (pre)diabetic sera of human origin. A Seram samples from healthy and (pre)diabetic subjects were collected and then lysed with lysis buffer. After centrifugation, total lysates were prepared for immunoblotting analysis with antibodies as indicated. B. Diagnosis efficacy was analyzed using ROC curve. C. Sensitivity, specificity and accuracy were evaluated for diagnostic value. D. An image of a. diagnostic kit apparatus useful for detecting (pre)diabetes using multiple protein markers. FIG, 5 shows pie charts of gene ontology for biological process (A), molecular functions (B) and cellular components (C) of the selective serum proteins with statistical significance (P < 0.05) in pre- diabetic patients compared to healthy volunteers.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

As used herein, the term“(pre)diabetes” shall generally mean both prediabetes and diabetes.

The term“a healthy control” shall generally mean a healthy subject who is neither a prediabetes nor a diabetes.

As used herein, a diagnostic kit shall mean a set or collection of (pte)-diahetes protein markers for diagnosis of prediabetes and/or diabetes, or a set or collection of (pre)-diabetes protein markers from which a diagnostic kit device/apparatus can be assembled, or shall mean a diagnostic kit device/apparatus that contain such a set or collection of (pre)-diabetes protein markers.

Ore term "a reagent area”, exchangeable with“a reagent depot”, shall generally mean a region containing“conditioning reagents”.

The term“conditioning reagents” means reagents that are required to optimize the detection or assay method.

The term“detection antibodies” shall generally mean antibodies specific against prediabetes and diabetes protein markers, which are conjugated to either an enzyme or other molecule to visualize the bi ndi ng reaction of protein mar kers to captured antibodies.

The term“capture antibodies” shall generally mean antibodies specific against prediabetes and diabetes protein markers. A capture antibody captures a protein marker by specific binding to the protein marker .

The terms“capture antibody area” and“a test reading area” are interchangeable.

The ten»“a care and/or treatment regime” shall generally mean a medicine or medical regimen to prevent or minimize the chance of the prediabetes from development into full diabetes, or to treat the prediabetes or diabetes.

The amino acid sequences of the prediabetes and diabetes protein markers are as follows: MLL4 (SEQ ID NO: 1); LAMA2 (SEQ ID NO: 2); PLXDC2 (SEQ ID NO: 3).

Abbreviations: BW, body weight: FBG, fasting blood glucose; IDF, International Diabetes Federation; lPA ^¾ , INGENUITY^ pathway analysis; ITRAQ ^® , isobarie tags for relative and absolute quantitation; MS, Mass spectrometry; T2D, type 2 diabetes; TRIG, triglyceride; an ROC curve, an Receiver Operating Characteristic curve; AUC, Area under the ROC Curve The invention relates to identificati on and use of protein markers for diagnosis and treatment of prediabetes and diabetes.

A method for identifying prediabetes protein markers is disclosed, which comprises (a) collecting serum samples from non-prediabetic (healthy) and prediabetic subjects, respectively; (b) depleting high-abundance proteins in the serum samples; (c) digesting remaining proteins with trypsin to obtain digested protein fragments in the serum samples; (d) labeling each of the digested protein fragments with a different isobaric tags to obtain isobaric tag-labeled protein fragments for relative and absolute quantitation; (e) mixing the isobaric tag-labeled protein fragments in the serum samples; (f) fractionating the isobaric tag-labeled protein fragments in the serum samples by chromatography to collect fractions; (g) identifying low-abundance proteins in the fractions with L€~ MS/MS and Mascot analyses: (h) selecting candidates for prediabetes protein markers by subjecting the identified low-abundance proteins to a volcano plot analysis; (i) grouping the selected candidates according to biological function; and (j) identifying the candidates with p < 0.01 and average relative ratio >1.3 as prediabetes protein markers. The selecting step may select candidates exhibiting a fold change of greater than 1.3 with a p value of equal to or smaller than 0.05 in the volcano plot analysis .

The abundant proteins may comprise albumin and IgG.

Three proteins Lama2, PLXDC2 and MLL4 were discovered as potential diagnostic biomarkers for prediabetes and diabetes. These protein markers Lama2, PLXDC2 and MLL.4 have clinical applications for care and treatment of prediabetes and diabetes patients, including, but not limiting to, use of antibodies specific against Lama2, FLXDC2 and MLL4 in the manufacture of a diagnostic kit, a method of detecting and identifying prediabetes and/or diabetes, a. diagnostic kit for detecting prediabetes and/or diabetes, and methods of using the diagnostic kit. The antibodies comprise capture and detection antibodies.

In the diagnostic kit device/apparatus, the levels of the capture and detection antibodies are optimized so that the test area only shows color signals when the levels of the markers are above a clinically significant threshold.

EXAMPLES

Methods

Chemicals and reagents. The eheniicals/reagents used include acetonitrile (ACN), ^' Iris 2~ carboxyethyl phosphine (TCEP), methyl methaoefhiosulfonate (MMTS), triethylamonium bicarbonat (TEAB), trifluoroacetic acid (TFA), Potassium dihydrogen phosphate (KH2P04) and potassium chloride (KC1). Mice and human serum samples collection. Healthy humans and patients diagnosed with prediabetes were recruited at the Tri-Service General hospital for blood collection. Blood samples were collected from healthy volunteers and prediabetic patients under 12 hr fasting condition. The serum samples were separated from whole ^" blood, aliqaoted to avoid repeating freeze-and-thaw cycle and then stored at -80°C. Human body weight and height were measured for body mass index (BMI).

The fasting blood glucose (FBG), hemoglobin A le (HbAlc), triglyceride (TRIG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), insulin and albumin -were assayed by automated clinical laboratory methods. C57B1/6J and C57B1/6J obese (db/db) mice were obtained from the National Laboratory Animal Center {Taipei, Taiwan) and the Jackson Laboratory' (Bar Harbor, ME, USA), respectively. At 4 and 6 weeks of age, body weights, FBG, HbAlc, TRIG, TC, HDL, LDL, insulin and albumin level were measured as previously mentioned (n - 3/each group). Blood samples were obtained from the mice which had fasted 16hr The serum were separated from whole blood by centrifugation and stored at -80°C. The mice were housed and fed standard mouse chow and water in a specific pathogen-free animal room with controlled temperature (22 ± :2'’C), humidity (55 ± 1.0%) and light/dark cycle (12hr/12hr). Ail the animals were cared and used base on the protocol of the Institutional Animal Care and Use

Committee.

Protein depletion. To augment the detection and identification of low-abundance proteins, the PROTEOFREP* immunoaffinity Albumin and IgG Depletion Kit from SIGMA-ALDRICH* was used to evaluate the efficiency of high abundance protein depletion from serum samples using the manufacture’s protocol. The protein concentration was calculated using the BCA protein assay kit from THERMO FISHER SCIENTIFIC*.

Protein digestion and ITRAQ* labeling. An equal amount of total protein (1 OOug) per depleted sample was diluted with 0.5M TEAS, reduced with 5mM TCEP at 60°C for 1. hr, alkylated using lOmM MMTS at room temperature for 10 min and then digested with Mug trypsin at 37°C for 16 hr. Subsequently, each sample from mice and humans was labeled with different ITRAQ* tag as follows: ITRAQ* -1 14 for 3 pooled healthy mice and humans, ITRAQ* -1 15, 116, 1 17 for 3 prediabetic mice and humans, respectively. The four samples from mice and humans were combined respectively, dried by speedvac, dissolved in 2CXM 5% ACN in 0.5% TEA and then desalted using Cl 8 spin column. After drying by speedvac again, each sample was dissolved in 400 mΐ of 25%

ACN/ 0.1 FA.

Strong cation exchange chromatography (SCX) fractionation. The iTRAQ* labeled samples were fractionated separately via strong cation exchange chromatography using polysulfoethyl A column (2.1 x 200mm, Sum particle size, 300 A pore size with the flow rate of0.3m!/min. The mobile phase (A) is iOmM KTEPCri in 25% ACN, pH 3.0 and (B) is IM KCL and 10 mM KH2PO4 in 25% ACN, pH 3.0. The gradient of fractionation is showed below: 0% B for 5 min, 0-20% B for 55 min, 20%-60% for 10 min, 60% for 1.0 min and 60-0% B for 20 min. Fractions were dried by speedvac.

LC-MS/MS analysis. The dried fractions were dissolved in 20OuI of 5% ACN/0.5% TFA, desalted using€18 spin column, dried by speedvac again and dissolved with 40 mΐ of 5% ACN/0.1% FA for LC-MS/MS analysis. Q EXACTIVE ^™ mass spectrometer (THERMO FISHER

SCIEN TIFIC ^4·' , Waltham, MA, USA) coupled with HCD fragmentation mode was used to generate MS and MS/MS spectra. ULTIMATE ^™ 3000 RSLC system (THERMO FISHER SCIENTIFIC ^® ) equipped with a CIS column (ACCLAIM ^™ PBPMAP ^™ RSLC, 75 uni x 150 mm, 2um, 100 A) was used for LC separation with the flow rate of 0.25ul/mhi and the mobile phase (A) 0.1% FA and (B) 95% ACN/0.1% FLA. The gradient of analysis is showed below: 1% B for 5 min, 1-25% B for 25 min, 25%~60% for 15 min, 60-80% tor 5 min, 80% B for 10 min, 80-99% for 5 min and 99% for Smin.

ITRAQ ^® data analysis. Relative protein ratio and peptide identification were processed .by Proteom Discover 1.4 for Mascot database search. All tandem mass spectra were searched for species of Mm mmculm and Homo sapiens against the International Protein Index da tabase.

Protein signaling pathways and functional analysis. Functions and signaling pathway of serum proteins with differential expression between the health and prediabetic mice and humans were analyzed by INGENUITY ^® Pathway Analysis (1PA ^® ) and PUBMED ^® .

Immtmoblotting. Serum samples were collected from C57BL/6 (B6), non-diabetic (ND) and prediabetic (PD) db/db mice and then lysed by RIP A lysis buffer. Total protein (50 tig) of each organ/tissue from control and prediabetic mice was resolved by 6% and 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membrane, immunoblotted with the antibodies against LAMA2 (1 :250Q, LifeSpan BioSciences, Seattle, WA, USA), MLL4 (1:500, Santa Cruz, Dallas, TX, USA), PLXDC2 (1 ; 1000, Novas Bio!ogicals, Littleton, Colorado, USA) and horseradish peroxidase (HRP)-conjugated goat and rabbit anti-mouse IgG as secondary antibody. The membranes were detected using FLUORCHEM™ HD2 system (BIO-TECHNE™, Minneapolis, MN, USA) after developing with enhanced chemiluminescence (ECL) substrate (EMD Millipore, Billerica, MA, USA).

ROC curve The true positive rate (sensitivity) is plotted in function of the false positive rate (100-specificity) for different cut-off points of a parameter in a ROC curve. Each point on the ROC curve represents a sensitivity/speeificiiy pair corresponding to a particular decision threshold. The area under the ROC carve (AUC) of MLL4, LAMA2 and PLXDC2 is a measure of how well a parameter can distinguish between two diagnostic groups (diseased/normaf).

Statistical analysis. The data am presented as mean ± standard error of the mean (SEM).

Student’s /-test was used to compare the difference between two groups unless indicated otherwise.

Comparisons between multiple groups were made with ANOVA. P values less than 0.05 were considered statistically significant.

Results

Comparison of the differentially expressed proteins in healthy and prediabetic sera of mouse and human origins using a quantitative protect mie approach. To characterize novel and reliable markers for (pre)diabetes, a combination of iTRAQ ^® and MS techniques was used to analyze the sera of non-prediabetic (healthy) and prediabetic db/db mice and humans (FIG. 1). Table 1 shows mouse body weight (BW) and serum biochemistry characteristics.

Table 1

The parameters of the groups are indicated as mean ± standard error. The parameters with significant change (P < 0.05) between the healthy and pre-diabetic mice are indicated with asterisk(s).

Healthy and prediabetic mice were grouped based on their fasting blood glucose (FBG). We found that BW, FBG, HbAle, triglyceride (TRIG) and fasting insulin were significantly different between both groups (Table 1 ,). Serum samples from both groups were collected and their abundant proteins were then depleted, followed by trypsin digestion individually. Three health mouse sera were pooled together to minimize individual variability and then labeled with iTRAQ ^® ! 14. Three prediabetic mouse sera were labeled with iTRAQ^ 115, 1 16 and 117, respectively. Finally, four iTRAQ ^® samples were mixed up and analyzed by LC-MS/MS (FIG. 1 ). The identity of the serum proteins from healthy and prediabetic mice was confirmed using Mascot software (FIG. 1) We identified total 442 serum proteins from mice at the peptide score of > 20, the peptide matches > 2 and the unique peptide matches > 1.

We followed the same approach to characterize the serum proteins of healthy and prediabetic subjects to compare and validate the markers between men and mice (Discovery, FIG. 1) The body mass index (BMi) and serum biochemistry' of human subjects were analyzed (Table 2) We found that age, FBG, HbAIc, fasting insulin and albumin are significantly different (P < 0 05) between healthy and prediabetic groups (Table 2). Serum samples from men were collected. After abundant proteins were depleted, the remaining proteins were digested with trypsin. The sera of 3 healthy volunteers were pooled and labeled with iTRAQ ^® 1 14. The sera of 3 prediabetic ones were labeled with iTRAQ* 1 15, 116 and 1 17, respectively. A pool of four iTRAQ * samples were analyzed by LC-MS/MS and their identity was ascertained using the Mascot analysis (FIG. 1). Total 500 proteins were identified in the sera of both groups at the peptide score > 20 the peptide matches > 2 and the unique peptide matches > 1 (Table 3). Table 2 shows characteristics of humans.

Table 2

The parameters of the groups are indicated as mean ± standard error. The parameters with significant change (P < 0.05) between the healthy and pre-diabetic subjects are indicated with asterisk/ s). Table 3 shows serum proteins with statistical significance (P < 0.05) in pre-diabetic patients compared fo healthy volunteers.

Table 3

**: CK, Cytokine: ENZ, Enzyme: 1C, ion channel; OT, Other; PD, Peptidase: TMR, transmembrane receptor; TR, transcription regulator; TP, Transporter; UM, Unmapped.

To further evaluate the potential of serum proteins as prediabetic markers, a total of 500 serum proteins of human origin were subject to volcano plot analysis based on both average relative ratio and p value (FIG. 2). Hie transformed volcano plot data indicated that among the human serum proteins, 70 proteins with fold change >1.3 and P < 0.05 could be candidate markers for prediabetes (FIG. 2 and Table 3) and need to be verified.

Gene ontology and pathway analysis of the selected serum proteins.

To gain insightful information about the biological function of the selected 70 proteins as shown in Fable 3, these proteins were analyzed by gene ontology and PUBMED ^® references searching (FiGs. 5 A-5C) Those proteins can be classified into 6 functional categories related to diabetes, diabetic complications, obesity, inflammatory immunity, coagulation and others (FIG. 5A).

Next, we narrowed down the number of candidate markers by picking up those proteins with P < 0.01 and average relative ratio > 1.3. Seven proteins, laminin subunit alpha 2 (LAMA2), serum amyloid A 2 (SAA2), plexin domain containing 2 (PLXDC2), monocyte differentiation antigen CD 14 (CD 14), CD99 antigen (CD.99), histone-lysine N-methy! transferase MLL4 (MLL4), and ciiisterin (CL!]), stood out under this stringent selection conditions. This screening strategy for the identification of potential markers tor (pre)diaheies worked well. For example, CD99 (EP1828774 Al l and CLU (US8673644 B2) were patented as diabetic markers. SAA was reported to be increased in plasma of obese and insulin resistant humans and was a marker of insulin resistance in mice.

CD 14 was reported to modulate inflammation-dri ven insulin resistance and was identified as an inflammatory marker in women with diabetes and impaired glucose tolerance. These 4 proteins showed that our data are highly reliable. Furthermore, several lines of evidences showed that the rest of 3 proteins are novel markers for diabetes MLL4 was reported to interact with the transcription factors to regulate islet b-cell function. LAMA2 mutation was shown to cause merosin-deficient congenital muscular dystrophy. PLXDC2 was known to regulate differentiation and proliferation during the development of nervous system.

To better understand the biological meaning of the changes in these proteins before and during T2D, the web-based iPA ^® and PUBMED* database searching were used to predict protein signaling pathways (FIG. 3). IPA* generated the network of a total of 35 proteins related to connecti ve tissue disorders, dermatological diseases arid conditions, and developmental disorders. The putative signaling pathways need to be ascertained with further experiments,

Confirmation of MLL4, LAMA2 and PLXDC2 for potential markers by immimoblotting. To verify the feasibility of using the 7 serum proteins MLL4, LAMA.2 JPLXDC2 ¹ CD99, CLU, SAA2 and CD 14 as prediabetic markers, MLL4, LAMA2 and PLXDC2 were selected due to their high statistical confidence and novelty; The published markers, CD99, CLU, SAA2 and CD! 4, were used to compare with novel markers for reliability. We confirmed the data with immunobtotting. The immimobiotttng data pointed out that the serum level of MLL4, LAMA2 and PLXDC2 was up- regulated in 5 prediabetic subjects (FIG. 4A). The ROC curve was used as a tool for diagnostic test evaluation. The ROC diagram was used to illustrate the diagnostic efficacy of the serum MLSL4, LAMA2 and PLXDC2. Their discrimination thresholds and the area under the curve (AUC) was used to evaluate the diagnostic value of each protein. The AUC of MLL4, LAMA2 and PLXDC2 were 0.95795, 0.9257 and 0.91445, respectively (FIG. 4B). The sensitivity, specificity and accuracy of MLL.4, SLAMA2 and PLXDC2 were all 71.42%, 71.42% and 71.42%, respectively (FIG. 4C).

FIG. 4D illustrates a diagnostic kit device/apparatus (“diagnostic kit”) 400 for detecting prediabetes and/or diabetes using multiple protein markers. The diagnostic kit 400 comprises the following: (i) a substrate 402 having a top surface 404 and a bottom surface 406 opposite to the top surface 404, and a top end 408 and a bottom end 410 opposite to the top end 408; (iij a sample loading area 420; (iti) a capture antibody area (a test reading area) 426, containing capture antibodies to capture prediabetes and diabetes protein markers comprising MLL4 428, LAMA2430, and PLXDC2 432; (iv) a reagent area 424, being coated with a conditioning reagent: (v) a detection antibody area 422, containing detection antibodies to visualize the captured prediabetes and diabetes protein markers comprising the MLL.4 428, LAMA2 430, and PLXDC2 432 in the captured antibody area (test reading area) 426; and (vi) optionally a positive control area 434, wherein the sample loading area 420, the detection antibody area 422, the reagent area 424, the capture antibody area 426, and the positive control area 434 are located on the top surface 404 of the substrate 402, allowing these areas to be in fluidic communication, the sample loading area 420 being located at the top end 408 and the capture antibody area 426 located at the bottom end 410 with the optionally positive control area 434 located either after or before the capture antibody area 426

Other prediabetes and diabetes protein markers may also be included together with the MLL4 428, LAMA2430, and PLXDC2 432 in the diagnostic kit device/apparatus 400 of the invention Linder this situation, the capture antibodies further comprise antibodies specific against other prediabetes and diabetes protein markers. The sample loading area 420, the detection antibody area 422, the reagent area 424, and the capture antibody area 426 may be sequentially located on the top surface 404 of the substrate 402 with the optionally positive control area 434 being located either after or before the capture antibody area 426. Alternatively, the sample loading area 420, the reagent area 424, the detection antibody area 422, and the capture antibody area 426 are sequentially located with the optionally positive control area 434 being located either after or before the capture -antibody area 426.

The capture antibody area 426 contains antibodies (primary antibodies) against the analytes (protein markers), which are immobilized to the area 426 The detection antibody area 422 contains antibodies (secondary antibodies) against the analytes (protein markers) which are conjugated to either an enzyme or other molecule to- visualize the binding reaction in the capture antibody area (or test reading area) 426 Examples of the enzyme may be horseradish peroxidase (HRP) or alkaline phosphatase (AP). The reagent area may contain color-forming suhstrate(s) and buffer(s) when enzyme-based assay detection system is used, or only buffers when non-enzyme -based assay detection system is used. The positive control area serves to show that the diagnostic kit functions properly. It may contain an immobilized non-eonjugated enzyme, or an immobilized antibody against one of more the detection antibodies. All the antibodies and reagents are either adsorbed, coated or immobilized onto the substrate.

The diagnostic kit device/apparatus of the invention was designed to perform lateral flow immunoassay such as disclosed by US Patent No. 8,399,261 and Serebrennikova et al. (2018) (“Hierarchical Nanogold Labels to Improve the Sensitivity of Lateral Flow Immunoassay” Nano- Micro Lett. 10:24), both of which are incorporated herein in their entireties by reference.

In summary, we used a combination of ITRAQ ^¾ and MS techniques to identity proteins in human and mouse sera and quantify their amounts INGENUITY* pathway analysis (iPA ^:S ) was used to predict the likely interaction network and pathways of the selected proteins. The level of three serum proteins was further confirmed using immunoblotting analysis and the receiver operating characteristic (ROC) curve analysis. The data suggest that a combination of iTRAQ* and MS techniques is able to identify serum proteins as potential markers for (pre)diabetes. MLL4, LAM A2 and PLXDC2 could be suitable diagnostic markers for (pre)diabetes. Among these proteins, MLL4 is the most potential marker for diagnosis.

The embodiments and examples were chosen and described to explain the principles of the invention and their practical application so as to enable others skilled In the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.