Protein microarray for characterizing the specificity of the monoclonal immunoglobulins of MGUS or myeloma patients

Abstract

The present invention concerns materials and methods for characterizing monoclonal immunoglobulin specificity of a Monoclonal Gammopathy of Undetermined Significance (MGUS) or Myeloma patients using a protein microarray comprising (a) a substrate, (b) antigens immobilized on the substrate, said antigens being selected from a defined group consisting of infectious agent antigens and/or self-antigens. In particular said protein microarray may be used to improve diagnosis, for the prognosis of myeloma or MGUS, for preventing transformation of MGUS toward myeloma, for adapting treatment of MGUS and myeloma or for monitoring the response to therapy of MGUS and myeloma patients.

Claims

1. A method for determining whether a Gammopathy of Undetermined Significance (MGUS) or myeloma is specific for an infectious agent, wherein said method comprises a protein microarray assay comprising: a) incubating a purified monoclonal immunoglobulin sample of the MGUS or myeloma patient with a protein microarray comprising (a) a substrate and (b) antigens immobilized on the substrate, said antigens comprising infectious agent antigens which comprise at least one virus-specific antigen and at least one bacteria-specific antigen, and b) detecting if said monoclonal immunoglobulin is bound to said antigens.

2. The method according to claim 1, wherein said infectious agent antigens further comprise at least one parasite-specific antigen.

3. The method according to claim 2, wherein said infectious agent antigen is: a virus specific antigen specific for an infectious agent selected from the group consisting of Hepatitis C virus (HCV), Epstein-Barr Virus (EBV), Hepatitis B virus (HBV), Human immunodeficiency virus (HIV), cytomegalovirus (CMV), varicella-zoster virus, HHV-1, HHV-2, HHV-6, HHV-8, coxsackie virus B4, influenza A and B viruses and Rubella virus and Measles virus, and/or a bacteria specific antigen specific for an infectious agent selected from the group consisting of Helicobacter pylori, Staphylococcus aureus, Streptococcus A, Chlamydia trachomatis, Mycoplasma pneumoniae, Haemophilus influenza, Borrelia burgdorferi; Bartonella Hensalae, Porphyromonas gingivalis and Prevotellaceae, and/or a parasite specific antigen specific for Toxoplasma gondii and Candida albicans.

4. The method according to claim 3, wherein said parasite specific antigen is a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a variant or fragment thereof.

5. The method according to claim 1, wherein said virus-specific antigen is specific for an infectious agent selected from the group consisting of Hepatitis C virus (HCV) and Epstein-Barr Virus (EBV), and/or said bacteria-specific antigen is specific of Helicobacter pylori bacterium.

6. The method according to claim 1, wherein said virus specific antigen is a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO 11, SEQ ID NO: 12, SEQ ID NO 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 SEQ ID NO: 18; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 SEQ ID NO: 51; SEQ ID NO:52 SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56; SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO: 60, or a variant or fragment thereof.

7. The method according to claim 6, wherein said virus specific antigen is a polypeptide comprising the amino acids 1-300, 301-582, 583-1063, 301-534, 583-1028 or 1050-1063 of sequence SEQ ID NO 51 or the amino acids 1-1301 or 1302-2116 of sequence SEQ ID NO: 52 or a variant or fragment thereof.

8. The method according to claim 1, wherein said bacteria specific antigen is a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66 and a variant or fragment thereof.

9. The method according to claim 1, wherein said infectious agent antigen is at least one HCV specific antigen comprising an HCV lysate and/or at least one polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, the amino acids 1192-1459 of SEQ ID NO: 11, SEQ ID NO 12, SEQ ID NO: 13, the amino acids 1691-1710 of SEQ ID NO: 13, the amino acids 1712-1733 of SEQ ID NO: 13, the amino acids 1921-1940 of SEQ ID NO: 13, a variant or fragment thereof, at least one EBV specific antigen comprising an EBV lysate and/or at least one polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 16, amino acid sequence 1-162 of SEQ ID NO: 15, a variant or a fragment thereof, and/or at least one H. pylori specific antigen comprising an H. pylori lysate and/or at least one polypeptide selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, a variant or a fragment thereof.

Description

BRIEF DESCRIPTION OF THE DRAWING(S)

(1) FIG. 1 is a diagram illustrating the EBV/CMV/T. gondii/HCV protein microarray according to the invention. Each antigen or lysates was spotted in four replicates. Tag controls (His, GST), and negative controls (BSA, PBS) are used to validate the specificity of the IgG detection. Concentrations of antigens: EBNA (8 M), VCA C1 (8 M), VCA C2 (32 M); Core, NS-3 and NS-4 (16 M); Mix 5Ag for CMV (16 M); p24 (12 M). Lysate were printed at C1 (200 g/m L) and C2 (400 g/mL).

(2) FIG. 2 illustrates the fluorescence minus background noise (FLU (F-B)) collected from single antigen arrays: EBV-EBNA (FIG. 2A), CMV-glycoprotein (FIG. 2B), HCV-core (FIG. 2C). For each germ, serum dilutions of a patient found positive for the germ by ELISA were tested on a single antigen array. The median fluorescence intensity minus background noise (FLU (F-B)) was, collected at different concentrations of antigens (32-258 g/mL (1-8 M) for glycoprotein B of CMV, and 92-736 g/mL (2-16 M) for EBV-EBNA and 22.5-220 g/mL (2-16 M) for HCV-core) for different quantities of hybridized IgG (12.5-800 g/mL). Positive and negative results obtained with patient sera tested for EBV (n=42), CMV (n=44) and HCV (n=60) are shown.

(3) FIGS. 3 to 5 illustrates the fluorescence minus background noise (FLU (F-B)) collected from the multiplexed infectious protein (MIP) microarray. The MIP microarray used carried several antigens from EBV (FIG. 3A), CMV (FIG. 3B), T. gondii (FIG. 4) and HCV (FIGS. 5A and 5B). Results obtained for each germ are shown separately in FIGS. 3 to 5. The data show results obtained for 10 patients: patients were selected with serum found negative for EBV (n=4), CMV (n=5) and T. gondii (n=5), and patients with serum found positive for EBV (n=6), CMV (n=5) and T. gondii. (n=5). The threshold of significant fluorescence was determined for each germ using the negative control. Graphs represent the median fluorescence intensity minus background noise (FLU (F-B)) collected from the arrayed antigens: EBV (FIG. 3A), CMV (FIG. 3B), T. gondii (FIG. 4) and HCV (FIGS. 5A and 5B). PC: Positive control, NC: Negative Control. Then the MIP microarray was tested for monoclonal immunoglobulin (5th and last figure). For these studies we selected two patients with serum found negative for HCV, and three patients with serum found positive for HCV. Both sera and purified monoclonal immunoglobulin were analyzed on the same slide. The threshold of significant fluorescence was determined for each germ using the negative control. Graphs represent the median fluorescence intensity minus background noise (FLU (F-B)) obtained for core NS-3 and NS-4 antigens from HCV. S: serum, mc: monoclonal immunoglobulin.

(4) FIG. 6 illustrates serum protein electrophoresis: (A): serum without spike in the gamma-globulins zone (B): serum with a monoclonal spike in the gamma-globulins zone.

(5) FIG. 7 is a diagram illustrating the 8-germs MIP microarray according to the invention. Each antigen or lysates was spotted in three replicates. Tag controls (His, GST), and negative controls (PBS) are used to validate the specificity of the mc Ig detection. Concentrations of antigens: H. pylori Ag (500 g/mL), H. pylori lysate (500 g/mL), HSV-1 gD (12 M), HSV-1 lysate (130 g/mL), HSV-1 gG (12 M), HSV-2 lysate (150 g/mL), HSV-2 gG (16 M), HSV-2 gD (14 M), VZV gE (14 M), VZV ORF 26 (16 M), EBNA (8 M), VCA (10 M), T. gondii lysate (200 g/mL), T. gondii p24 (12 M), CMV lysate (400 g/mL), mix 5Ag for CMV (16 M), HCV Core (16 M), HCV NS-3 (16 M).

(6) FIG. 8 illustrates the median fluorescence intensity minus background noise [FLU (F-B)] collected from the arrayed antigens: (A): The results shown were obtained with sera from 4 patients found positive for EBV (D36, D41 D42 and D45) and 2 patients with serum found negative for EBV (D23 and D24). Purified mc Ig from patients D36, D41 and D42 specifically recognize EBNA. Negative control (C), positive control (C+), Sera (S) and purified mc Ig (Mc) were analysed on the same slide. The threshold of significant fluorescence was determined using the negative control.

(7) FIG. 9 illustrates the median fluorescence intensity minus background noise [FLU (F-B)] collected from the arrayed antigens: The results shown were obtained with serum from a patient (D24) found positive for H. pylori. Sera and purified mc Ig were analysed on the same slide. The threshold of significant fluorescence was determined using the negative control (C).

EXAMPLES

Example 1

A/ Patients and Methods

(8) Serum Samples

(9) The study was performed on a panel of 70 human sera obtained from a heterogeneous group constituted of a majority of hospitalized patients and a few outpatients, including 34 women and 36 men, of age ranging from 7 to 73 (49.311.9) years and for whom one or several serological analyses had been prescribed. With consent, samples of venous blood were collected without anticoagulant. After coagulation, blood samples were centrifuged at 3500 rpm for 15 minutes at 4 C. and sera were collected and stored at 20 C. until analysis. The sera were provided by laboratories of the University Hospital of Nantes (Virology, Bacteriology and Parasitology laboratories).

(10) Determination of Serological Status

(11) Serological status for one or several pathogens including EBV, CMV, HCV and T. gondii was determined using classical ELISAs, as described below.

(12) HCV:

(13) HCV serological status was determined by chemiluminescent ELISA immunoassay on an Architect Abbott analyzer using the Abbott anti-HCV kit (ref. 6C37). This assay detects antibodies directed against structural and non-structural proteins of HCV using the following antigens: HCr 43, composed of the products of two non-contiguous coding regions of the HCV genome, amino-acids (aa) 1192 to 1457 from the NS-3 sequence and aa 1-150 from the HCV core sequence; C-100-3, a chimeric fusion protein with a part of human superoxiydase dismutase (h-SOD) and aa 1569-1931 of the NS-3 and NS-4 sequences.

(14) CMV:

(15) CMV serological status was determined by quantitative sandwich chemiluminescent immunoassay on a Diasorin Liaison analyzer using the LiaisonCMV IgG kit (ref. 310740). This assay detects antibodies directed against human CMV by using magnetic particles coated with inactivated human CMV (type AD169). The second antibody is a monoclonal mouse antibody directed against anti-human IgG, conjugated with isoluminol.

(16) EBV:

(17) EBV serological status was determined by quantitative sandwich chemiluminescent immunoassay on a Diasorin Liaison analyzer using the LiaisonEBNA IgG kit (ref. 310520) and Liasion VCA IgG kit (ref. 310510) on a Diasorin Liaison analyzer. These assays detect antibodies directed against EBV nuclear antigen (EBNA) by using magnetic particles coated with synthetic peptide EBNA-1, or against the viral capsid antigen (VCA) by using magnetic particles coated with synthetic VCA peptide p18. The second antibody is a monoclonal mouse antibody against anti-human IgG conjugated with isoluminol.

(18) T. gondii:

(19) Determination of serological status against T. gondii was performed using quantitative ELISA on AxSYM System from Abbott. This assay detects IgG directed against the whole tachyzote T. gondii using coated microparticles; the main antigen is represented by T. gondii membrane protein p30. The secondary antibody is an anti-human IgG alkaline phosphatase conjugate; revelation is done by addition of 4-methylumbelliferyl phosphate.

(20) Determination of IgG Concentration

(21) The IgG concentration of each serum sample was determined with an immuno-nephelemetric assay performed on a Beckman Immage Analyser. Then for each serum, IgG concentrations were adjusted from 12.5 to 800 g/mL in PBS with 1% bovine serum albumin (BSA) and 0.1% Tween 20 (T-PBS) for further use on protein microarray (80 L per incubation pad). Purification of mc Ig G from MGUS and myeloma patients was performed separating monoclonal Igs from polyclonal Igs and beta globulins by electric charge, using electrophoresis on agarose gels (kit Paragon SPE-II; Beckman Coulter, Villepinte, France). A portion of the agarose gel is stained using Coomassie Brilliant Blue and bands corresponding to monoclonal (apparent MW, pI) or polyclonal Igs (MW, pI) are then cut on the unstained portion of the agarose gel and proteins are eluted from gels into PBS. Purity may be verified using immunofixation (SAS-MX; Helena Biosciences, Gateshead, United Kingdom) or/and isoelectrofocusing and immunoblotting. IgG concentrations in eluates of purified monoclonal immunoglobulin were determined using the same immuno-nephelemetric assay as for serum. Tween 20 0.1% (TPBS) prior to hybridization onto the microarray.

(22) Design of the Multiplexed Protein Microarray

(23) Selected Antigens and Lysates

(24) Antigens were supplied by Abcam (Cambridge, United Kingdom), Advanced Biotechnologies Inc. (Columbia, Md., USA) and Virogen (Watertown, Mass., USA). Lysates were supplied by Advanced Biotechnologies Inc. (Columbia, Md., USA).

(25) For EBV the three Ag used were: Viral Capsid Antigen (VCA) p23 (sequence SEQ ID NO: 15; ref. ab43145, Abcam), p23 region 1-162aa (ref. 00211-V, Virogen) and Epstein-Barr Nuclear Antigen (EBNA) recombinant protein EBNA-1 of sequence SEQ ID NO: 16 (ref. 10-523-001, Advanced Biotechnologies).

(26) For CMV, a mixture of five antigens was used: region 297-510 of Cytomegalovirus pp65 IE having the sequence SEQ ID NO: 60 (ref. ab54103, Abcam); immunodominant region of CMV pp28 (UL99; SEQ ID NO: 2) (ref. ab43038, Abcam); immunodominant region of CMV pp52 (UL44; sequence SEQ ID NO: 3) (ref. ab43044, Abcam); immunodominant region of glycoprotein B (SEQ ID NO: 4) (ref. ab43040, Abcam); and immunodominant region of CMV pp38 (UL80a; SEQ ID NO: 5) (ref. ab73042, Abcam) as well as a purified viral lysate (ref. 10-144-000, Advanced Biotechnologies).

(27) For T. gondii, one antigen was used e.i. p24 (GRA1) protein of sequence SEQ ID NO: 6 (ref. ab43137, Abcam) and a purified trachyzoites lysate (ref. 10-279-001, Advanced Biotechnologies).

(28) For HCV, three antigens were used: core protein composed of 119 aa (1-119) having the sequence SEQ ID NO: 8 (ref. ab49015, Abcam); NS-3 protein recombinant fragment subtype 1c (1192-1459 aa) (ref. ab91395, Abcam, SEQ ID NO 11) and NS-4 recombinant mosaic protein containing the HCV NS-4 immunodominant regions 1691-1710 aa, 1712-1733 aa, 1921-1940 aa from 1, 2, 3, 5 genotypes (SEQ ID NO: 13; ref. ab49027, Abcam). Some antigens contain histidine-tag or glutathione S-transferase (GST) fusion proteins.

(29) Before being printed, the adequate concentration range of each antigen and lysate was determined. For this purpose, antigens were diluted in PBS from 1 to 16 M, and lysates were diluted from 10 to 400 g/mL. Lysates were ultra-sonicated prior to dilution to avoid aggregates.

(30) Preparation of the MIP Microarray

(31) Antigen (10 L, 1-16 M) or lysate (10 L, 10-400 g/mL) solutions were pipetted in 384-well microtiter plates (PDC 90 Porvair Sciences Ltd., Shepperton, United Kingdom). Then, samples were transferred onto FAST slides 16 pad of nitrocellulose (Whatman, Maidstone, United Kingdom) using the sciFLEXARRAYER S3 Piezo Electric Dispenser (Scienion, BerlinGermany). In all cases 6 drops were printed; each drop is estimated to contain 500 L. Antigens, tag, fusion proteins and negative controls were also spotted.

(32) As shown in FIG. 1, the arrays consisted of 88 matrices that included: (i) seven Ag: 3 for EBV, 3 for HCV, 1 for T. gondii; (ii) two lysates (CMV, T. gondii) in two concentrations; (iii) mix of CMV lysates and five Ag; (iv) two tag controls (GST, histidine); (v) two negative controls (PBS, BSA). Spotting was performed inside a chamber at 25 C. and 60% humidity. FIG. 1 shows the design of the microarray, and the concentrations of antigens, lysates and CMV mix.

(33) Processing of Microarray Slides

(34) Printed slides were saturated for 1 hour at room temperature with TPBS and 5% BSA in order to prevent non specific antibody binding. After washing with TPBS, slides were incubated with 80 l of diluted serum or purified monoclonal immunoglobulin (12.5 to 800 g/mL), for two hours at room temperature. After a second washing, slides were incubated with a labelled secondary antibody (0.1 to 4 g/mL, Dylight 680 Labelled Goat anti-human IgG (H+L), from Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md., USA) while shaking in the dark, then washed with TPBS. The testing of each serum and monoclonal immunoglobulin was repeated at least three times. Fluorescence signal, detected with the Odyssey infrared imaging system scanner at 21 m resolution (LI-COR Biosciences, NE, USA), was used to determine the serological status of each sample.

(35) Data Analysis

(36) Specific fluorescence was quantified using the GenePix Pro 4 Microarray Acquisition & Analysis Software (Molecular Devices, Sunnyvale, Calif., USA). For each sample, the median fluorescence intensity (FLU) was determined after subtraction of background slide fluorescence. The intensity of each spot was analysed and the ratio of fluorescence minus background was calculated. Results for each sample (patients and controls) were represented on histograms using GraphPad 5.0 software (San Diego, Calif., USA). FLU values represent the mean of four replicates from one experiment. Experiments were repeated three times on different arrays for each patient FLU values obtained for each sample were compared to positive and negative controls for each germ. For each antigen, a positivity threshold was determined, that corresponds to a level of fluorescence above all negative controls. Patients for whom the mean FLU value was higher than the positivity threshold were considered positive for the antigen tested. When several antigens were used for the same pathogen, if one or more positive results were obtained for a serum, the patient was considered as positive.

B/ Results

(37) Determination of Sera and Antigen Concentrations

(38) In order to optimize the microarray assay, we performed different experimental conditions allowing the best ratio of antigen-antibody. For each germ, a range of concentration from 1 to 16 M of each antigen or 10-400 g/mL lysate was first spotted onto the MIP protein microarray. Then, hybridization with different concentrations of serum IgG (from 12.5 to 800 g/mL), as well as different concentrations of labelled secondary antibody (from 0.1 to 4 g/mL, dilutions 1/250 to 1/10000) were tested in different pads of the same protein array. Detection of antibodies against EBV was performed using two major antigens: Epstein-Barr Nuclear Antigen (EBNA) and Viral Capsid Antigen (VCA). Examples of results obtained are shown in FIG. 2. The concentrations used ranged from 92 to 736 g/mL for EBNA (FIG. 2A), from 32 to 258 g/mL for glycoprotein B of CMV (FIG. 2B) and from 22.5 to 220 g/mL for the core protein of HCV (FIG. 2C). For EBNA, CMV and HCV, the intensity of the fluorescent signal was too low when antigen concentration was 1 M, and when IgG serum concentrations were less than 50 g/mL.

(39) For CMV, the inventors tested different antigens including glycoprotein B, and also virus lysates. FIG. 2B shows the fluorescence signals obtained with a CMV-positive serum used at different concentrations of IgG.

(40) For HCV, the inventors tested three different antigens (core protein, NS-3 and NS-4). FIG. 2C shows the florescence obtained for HCV core protein with a positive serum. For the 4 germs, the intensity of the fluorescence signal increased with the concentration of antigen and IgG. The data obtained allowed us to define that the most suitable quantity of IgG to be used per pad was 100 to 400 g/mL (8 to 32 g/80 L). Table 1 presents the optimal concentrations of antigens and lysates to be used for spotting and subsequent hybridization, in order to compare serological status determined by Elisa and by MIP. The final IgG concentration for hybridization was 400 g/mL (32 g/80 L per pad), and the final secondary antibody concentration for detection was 0.2 g/mL (dilution 1/5000).

(41) TABLE-US-00001 TABLE 1 Optimal concentration of antigens and lysates for the Epstein-Barr virus, Cytomegalovirus, T. gondii, Hepatitis C Virus used in MIP microarray assay. Infectious agent Type Protein Concentrations EBV Antigen EBNA 8 M VCA 32 M CMV Lysate 400 g/mL Antigen Glycoprotein B 8 M Mix 5 Ag pp65, pp28, 16 M pp52, pp38 glycoprotein B Lysate + 200 g/mL mix 5 Ag 16 M T. gondii Lysate 400 g/mL Antigen P24 12 M HCV Antigen Core 16 M NS-3 16 M NS-4 16 M

(42) TABLE-US-00002 TABLE 2 Results obtained with 70 sera analyzed by classical ELISAs (E) used routinely in hospital diagnostic laboratories, and by the MIP microarray immunoassay (MA) CMV T. gondii EBV Lysate + 5 Lysate + HCV EBNA VCA Ag mix Ag Core ID Age Sex IgG (g/L) E MA E MA E MA E MA E MA 1BOA 25 F 6.7 + NA NA 2DEE 25 M 24.2 + + + (*) NA NA 3DEC 67 F 3.9 + NA NA 4BIT 36 M 11.7 + + + NA NA NA NA 5RYE 34 M 8.9 NA NA 7ANM 27 F 12.2 NA NA NA NA NA NA 9OUC 60 F 15.5 + + + + + + NA NA + 11CHI 11 M 13.1 NA NA NA NA NA NA NA NA 12LET 28 M 12.2 NA NA NA NA NA NA NA NA ++++ + 13LEG 61 M 17.4 NA NA NA NA NA NA NA NA + + 14DEM 57 F 12 NA NA NA NA NA NA NA NA + + 15XXA 22 M 11 + + NA + NA NA 16MAH 20 F 13.9 + + + + +++ + + + 17GRN 41 M 10.9 NA NA NA NA +++ + NA NA 18ZOL 47 M 12 + + + + + + NA NA 19GUC 30 M 8.6 + + + + + + 20LED 28 M 8.4 + + + + NA NA NA NA 21GRP 45 F 12.1 + + NA NA NA NA 22BES NA NA 8.8 + + + + + + NA NA NA NA 23ROF 48 M 3.5 + + + + + NA NA 25GBX 35 F 18.8 NA NA NA NA NA NA NA NA + + 26DIA 43 F 28.8 NA NA NA NA NA NA NA NA + + 27 PAJ NA H 33.1 NA NA NA NA NA NA NA NA + + 28VAP 46 M 10.8 NA NA NA NA NA NA NA NA + + 29GIP 42 M 9.1 NA NA NA NA NA NA NA NA + + 30ROA 63 M 16.1 NA NA NA NA NA NA NA NA + + 31DIH 43 F 23.8 + + + + + + NA NA + + 32BRL 49 M 10.2 + + + + + + NA NA 33BEV 50 F 7.7 + + + + + + NA NA 34SAO 71 M 16.7 NA NA NA NA NA NA NA NA 35POL 20 F 4.7 NA NA NA NA NA NA 36SHM 37 F 8.9 NA NA NA NA NA NA ++++ + 37 HEC 59 M 5.4 + + + + + 38CHH 24 M 10.4 + + + + + + + + 39DUT 34 M 4.4 + + + + 40MUB 32 M 7.7 NA NA NA NA + (**) + 41SEE 24 F 4.9 NA NA NA NA NA NA 42KAM 51 M 7.9 + + + + + + NA NA 43LEA 36 F 6.9 NA NA NA NA NA NA NA NA 44MAC 40 M 11.1 + + + + + (***) + NA NA 45HUA 17 F 10.9 + + NA NA 46REE 45 M 14.5 + + + + + + NA NA 47DEC 7 F 9.1 NA NA 48DOD 34 F 11.5 NA NA NA NA NA NA + + 49TEO 33 M 22.1 + + + + + + 50BAM 32 F 10.4 NA NA NA NA NA NA + + NA NA 51MIM 53 F 10.5 + + + + + + + + 52HAK 26 M 12.8 + + + + + + 53BEK 35 F 6.1 + + + + + + + + 54CHG 73 M 8.3 + + + + + + 55SAS 34 F 7.9 NA NA NA NA + + 56SAO 22 F 18.9 NA NA NA NA NA NA NA 58ZOK 27 M 11.1 NA NA NA NA NA NA NA NA NA 59LEB 47 M 8.1 + + + + + + NA NA 60COY 61 F 6.4 + + + + NA NA 61CLJ 59 M 7.6 + + + NA NA 62PAC 43 M 12.8 + + + + + + 63MAJ 37 M 13.3 + + + + + + + 64ABR 60 M 8.41 + + + + 65DIA 53 F 17.5 + + + + + + + + 66YAH 36 F 15.1 NA NA NA NA NA NA + + 67SOD 24 F 10.6 NA NA NA NA NA NA + + 68CHC 32 F 10.1 NA NA NA NA NA NA + + NA NA 69MEN 21 M 14.9 + + + + + + + + 70BIA 31 F 13.2 NA NA NA NA NA NA + + 71TER 57 M 22.5 + + + 72TAH 29 F 12.0 + + + + + + + + NA NA 73PAD 66 M 6.8 + + + + + + 74LEC 49 F 14.3 + + + + + + NA NA 75THF 44 F 14.1 + + + + + + + + E: ELISA; MA: Array; NA: Not available, : involve patients with results near the detection threshold), (*): past infection; (**): low; (***) recent infection.

(43) Analysis of Serum Reactivity Against Arrayed Antigens

(44) The inventors assessed the antibody reactivity of the 70 human sera against EBV, CMV, T. gondii, and HCV (a summary of the serological characteristics of patients is presented in Table 2). The data shown are representative of three experiments, independently performed.

(45) Each serum was tested three times for each germ. FIGS. 3 to 5 present results obtained for EBV, CMV, T. gondii and HCV for a representative selection of ten patients. Fluorescence scanning of microarray slides incubated with representative sera of the different groups of patients showed that the MIP microarray assay allows the detection of antibodies specifically directed against microbial antigens in accordance with ELISA results (Tables 3 and 4).

(46) TABLE-US-00003 TABLE 3 Discordant results between ELISA and MIP microarray immunoassay. CMV T. gondii EBV Lysate + Lysate + IgG EBNA VCA mix 5 Ag Ag Pt ID Age Sex (g/L) E MA E MA E MA E MA 1BOA 25 F 6.7 + ND ND 2DEE 25 M 24.2 + + + ND ND 3DEC 67 F 3.9 + ND ND 4BIT 36 M 11.7 + + + ND ND ND ND 5RYE 34 M 8.9 ND ND 15XXA 22 M 11 + + ND ND ND ND 37HEC 59 M 5.4 + + + + + 39DUT 34 M 4.4 + + + + 55SAS 34 F 7.9 ND ND ND ND + + 61CLJ 59 M 7.6 + + + ND ND 63MAJ 37 M 13.3 + + + + + + + 71TER 57 M 22.5 + + + Age, sex, Ig G concentration and results of EBV, CMV and T. gondii ELISA (E) and MIP microarray (MA) for each patient presenting a discordant result. Pt: patient; : results near the detection threshold; ND: not done.

(47) For EBV, the presence of IgG against the two antigens EBNA and/or VCA antigens was determined by the two techniques on sera obtained from 42 patients. For EBNA the same 32 patients were found positive, and the same 3 patients were found negative, by ELISAs and by MIP microarray. Five patients were classified uncertain by ELISA (considered borderline with the detection limit). Among these 5 patients, 3 were found negative and two were found positive by MIP microarray. For two patients the detection of IgG against EBNA was positive by ELISA and negative by MIP microarray. One presented with a high concentration of IgG directed against the varicella-zoster virus (VZV), suggesting that a false positivity due to EBV/VZV cross reactivity was possible. For the other patient presented a high level of IgG (22.5 g/L) and this patient was found negative for VCA of EBV by ELISA and MIP microarray. For VCA, 3/35 patients showed discordant results; 2 were found positive using ELISA and negative using MIP microarray. The patient with a high concentration of antibodies directed against VZV was also discordant for EBNA. The other patient presented with a high concentration of IgG (24 g/L); for this patient the detection of antibodies against EBNA was uncertain by ELISA and positive using MIP microarray. For one patient, the inventors found a positive detection of IgG against VCA using the multiplexed protein microarray and negative results using ELISA. For this patient, the detection of IgG directed against EBNA was positive by both ELISA and MIP microarray, suggesting a greater sensitivity for VCA for the MIP microarray (Table 3).

(48) For CMV, 3/44 patients were found discordant using the two techniques: these patients had positive results by ELISA and negative results by MIP microarray (see table 3). For 1 patient, a similar discordance was found for T. gondii (positive ELISA and negative MIP microarray). Considering the two other patients with positive ELISA and negative MIP microarray tests, one presented a high serum IgG concentration (23 g/L), and the other a low concentration of IgG in serum (3.9 g/L) (table 3).

(49) For T. gondii, results were discordant for 3/33 patients. Two patients who were found positive by ELISA were found negative by MIP microarray. For these 2 patients, the IgG concentration was lower than 8 g/L. One of the patients was also negative for CMV by MIP microarray but positive by ELISA.

(50) One patient was found positive by MIP microarray and negative by ELISA. For the patient with a very low level of IgG directed against T. gondii when tested by ELISA, serum should be used less diluted to obtain a positive result by MIP microarray. Nonlinear relationship was evident and suggested the presence of a hook effect. Under specific conditions, a high analyte concentration can simulate false negative signals (Table 4).

(51) For HCV, 60 patients were compared using the two techniques. There was no discordance between ELISA and MIP microarray results. The 48 sera found negative by ELISA were also negative using the MIP microarray technique, and the 12 sera found positive by ELISA were also positive using the MIP microarray assay. Hence the MIP microarray had excellent sensitivity (100%) and specificity (100%) for the detection of anti-HCV IgG.

(52) The two methods, MIP microarray and ELISA assays, were compared using the Chi-squared test on discordant results (+/ and /+). No statistical difference was found: values were 0, 0.57, 1.73 and 0.57 for EBNA, VCA, CMV and T. gondii, respectively (Table 4).

(53) TABLE-US-00004 TABLE 4 Agreement rate between results obtained assessing serum IgG reactivity by ELISA and by MIP microarray assay. Agreement A B C D E F (%) Elisa + + MIP + + + EBV EBNA + VCA 35 1 2 1 2 0 95 EBNA 31 3 3 2 2 0 94 VCA 33 4 0 1 2 1 92.5 CMV 24 17 0 0 3 0 93 T. gondii 22 8 0 0 2 1 91 HCV 12 48 0 0 0 0 100

(54) Results are given as numbers of concordant results: A, positive ELISA and positive MIP array; B, negative ELISA and negative MIP array; and discordant results: E, positive ELISA and negative MIP array; F, negative ELISA and positive MIP array. C and D were available only for EBV (EBNA and VCA), for which ELISA gave a result near the detection threshold () and the MIP array gave either a positive (C) or a negative (D) result. The % agreement for each germ was calculated on concordant results; the Chi-squared test performed on discordant patients was not significant for EBV, CMV, and T. gondii.

(55) Analysis of Mc Ig Specificity with the MIP Microarray Assay

(56) The inventors tested the suitability of the EBV/CMV/T. gondii/HCV microarray assay for analysis of the specificity of purified mc Igs. Sera and purified mc Igs of 3 HCV-positive patients presenting with either myeloma (2 patients) or MGUS (1 patient) were tested. For both serum and purified mc Ig, the MIP microarray showed specific detection of a single HCV antigen, either the core protein (2 patients) or NS-4 (1 patient) (FIG. 5).

C/ Discussion

(57) The inventors showed that the MIP microarray assay can be used to detect the presence of IgG directed against various infectious epitopes in human sera. The MIP microarray allows the generation of high quality fluorescence signals suitable for the determination of serological status of patients for EBV, CMV, T. gondii and HCV. Agreement between results obtained by ELISAs and the MIP microarray assay was 95% for EBV, 93% for CMV, 91% for T. gondii, and 100% for HCV. For EBV, CMV and T. gondii, discordance was most often due to a positive ELISA and negative MIP microarray assay. However, among the 5 sera found uncertain for EBNA by ELISA, the MIP microarray found 3 positive. Hence the MIP assay uses lesser volumes and is as sensitive as ELISAs and more sensitive than ELISA for anti-EBNA antibody detection. Regarding EBV, the detection of two types of antibodies minimized the risk of false negativity of false positivity: combining EBNA and VCA results, there was only one false negative serum by MIP microarray. Comparison of the MIP microarray and the ELISA assays on discordant results showed no statistical difference.

(58) Altogether these results highlight the importance of IgG concentration: in all cases in cases with discordant results, the IgG concentration was outside of normal values, most often high. For the ELISA technique, plasma samples are used at fixed volume and dilution, without taking the IgG level into account. In contrast, for the MIP microarray assay, a dilution is performed for each sample so as to obtain 400 g/mL IgG, deposited on a pad. For instance one patient with a positive ELISA assay but negative MIP microarray result for T. gondii was tested again on the MIP microarray using a higher dilution of sample (200 g/mL g of IgG instead of 400 g/mL), this time with positive result. This is consistent with a hook effect for sera with high levels of specific antibodies. Thus, in order to avoid false negative results, it is advisable to test plasma samples at two Ig concentrations: 200 and 400 g/mL.

(59) It is worth noting that the EBV/CMV/T. gondii/HCV MIP microarray was found suitable to study the anti-infectious specificity of mc Ig. This is of importance since mc Ig can be specific for infectious antigens, as demonstrated for HCV-infected MGUS or myeloma patients whose mc Ig are directed against HCV proteins. The EBV/CMV/T. gondii/HCV MIP microarray detected the HCV specificity of mc Ig from 3/3 HCV-infected patients. For the purpose of analyzing mc Ig specificity, the MIP microarray presents the advantage of allowing simultaneous analysis against several germs with very small amount and volume of purified mc Ig.

(60) As an ever-increasing variety of microarray formats becomes available (patterned microarrays, three-dimensional pads, flat surface spot microarrays), these versatile tools will be more and more used in high-throughput functional genomics and proteomics. Those results demonstrate that this test format has important advantages, and that reliable and reproducible analytical and clinical data can be obtained with microarrays. The novel EBV/CMV/T. gondii/HCV microarray assay is a suitable alternative assay for simultaneous serodiagnostics of infectious diseases in small volume samples in a clinical context. In the future, the design of the MIP microarray can be completed by the addition of more antigens and lysates of new viruses, bacteria or parasites.

(61) This approach presents a wide range of potential applications for epidemiologic research as well as for the diagnosis of infectious diseases.

(62) In conclusion, a major novel aspect of the MIP microarray assay resides in the combination of epitopes from a selection of infectious agents known to cause chronic infection, to be used for the diagnostic work-up of patients with a variety of diseases linked to chronic infection such as allergies, inflammatory diseases, auto-immune diseases or chronic monoclonal gammapathies. The MIP microarray assay with EBV, CMV, T. gondii and HCV antigens will allow testing patients with a single assay rather than a series of ELISAs. This is particularly interesting for biological samples typically available only in small volumes, such as purified mc Igs or cerebrospinal fluid.

(63) The microarray test format should become in the near future a tool of choice for rapid diagnosis of infectious diseases and pathologic conditions linked to infection, as well as for the characterization of the infectious specificity of mc Ig.

Example 2

A/ Methods

(64) Patients, Samples, and Mc Ig Purification

(65) After informed consent, serum was obtained from patients diagnosed with MGUS or myeloma in different centers: Nantes, Dijon, Paris. Serum samples were aliquoted and kept at 20 C. until analysis. The presence and type of mc Ig was verified using serum electrophoresis onto agarose gel and immunofixation A homogeneous spike-like peak in a focal region of the gamma-globulin zone indicates a monoclonal gammopathy (FIG. 6). The mc Ig was purified using modification of serum electrophoresis ie elution from agarose gel electrophoresis of the zone corresponding of the mc Ig in the gamma globulin migration, then elution from into PBS and then the purity of purified mc Ig is verified using isoelectofocalisation and immunoblotting as previously described (Bigot-Corbel E. et al., Blood 2008; 112: 4357-4358; Fron D. Et al. Analytical Biochem 2013; 433: 202-209).

(66) Selected Antigens and Lysates

(67) Antigens (Ag) were supplied by Abcam (Cambridge, United Kingdom), Advanced Biotechnologies Inc. (Columbia, Md., USA), Virogen (Watertown, Mass., USA), EastCoast Bio (North Berwick, USA). Lysates were supplied by Advanced Biotechnologies Inc. (Columbia, Md., USA), EastCoast Bio (North Berwick, USA).

(68) For EBV the three antigens have been used: Viral Capside Antigen (VCA) p23 (ref. ab43145, Abcam), p23 region 1-162aa (ref. 00211-V, Virogen) and Epstein-Barr Nuclear Antigen (EBNA) recombinant protein EBNA-1 (ref. 10-523-001, Advanced Biotechnologies).

(69) For CMV, a mixture of five antigens has been used: region 297-510 of CMV pp65 IE (ref. ab54103, Abcam); CMV pp28 (UL99) immunodominant region (ref. ab43038, Abcam); CMV pp52 (UL44) immunodominant region (ref. ab43044, Abcam); glycoprotein B immunodominant region (ref. ab43040, Abcam); and CMV pp38 (UL80a) immunodominant region (ref. ab73042, Abcam) as well as a purified viral lysate (ref. 10-144-000, Advanced Biotechnologies).

(70) For T. gondii, one antigen have been used, p24 (GRA1) protein (ref. ab43137, Abcam) and a purified tachyzoite lysate (ref. 10-279-001, Advanced Biotechnologies).

(71) For HCV, three antigens have been used: core protein composed of 119 aa (1-119) (ref. ab49015, Abcam); NS-3 protein recombinant fragment subtype 1c (1192-1459 aa) (ref. ab91395, Abcam) and NS-4 recombinant mosaic protein containing the HCV NS-4 immunodominant regions aa1691-1710, aa1712-1733, aa1921-1940 from genotypes 1, 2, 3, 5 (ref. ab49027, Abcam).

(72) For Helicobacter pylori (H. pylori), one antigen extract (ref. FC 509, EastCoast Bio) and one bacterial lysate (ref. FC504, East Cost Bio) have been used.

(73) For Herpes Simplex Virus 1 (HSV1), two antigens were used: HSV1 gD immunodominant regions (ref.ab43045, Abcam) and HSV-1 gG immunodominant regions (ref.ab43048, Abcam) as well as purified viral lysate (ref.10-145-000, Advanced Biotechnologies).

(74) For Herpes Simplex Virus 2 (HSV2), two antigens have been used: HSV-2 gD immunodominant regions with 33aa (266-39) (ref.ab48971, Abcam) and aminoacids 525-578 of HSV2 gG Envelope Protein (ref.ab67703, Abcam) as well as purified viral lysate (ref.10-146-000, Advanced Biotechnologies).

(75) For Varicella Zoster Virus (VZV), two antigens have been used: one contains immunodominant regions of protein gE and the other immunodominant regions of ORF26. Some antigens contain histidine-tag or glutathione S-transferase (GST) fusion proteins. Before being printed, the adequate concentration range of each antigen and lysate was determined. For this purpose, antigens were diluted in PBS from 1 to 16 M, and lysates were diluted from 10 to 500 g/mL. Lysates were ultra-sonicated prior to dilution to avoid aggregates.

(76) Preparation of the 8-Germs MIP Microarray

(77) Ag (10 L, 1-16 M) or lysate (10 L, 10-400 g/mL) solutions were pipetted in 384-well microtiter plates (Porvair Sciences Ltd., Shepperton, United Kingdom). Then, samples were transferred onto FAST slides 16 pad of nitrocellulose (Whatman, Maidstone, United Kingdom) using the sciFLEX ARRAYER S3 Piezo Electric Dispenser (Scienion, BerlinGermany). In all cases 6 drops were printed; each drop is estimated to contain 500 L. Ag, tag, fusion proteins and negative controls were also spotted. The arrays consisted of 88 matrices that included: (i) thirteen Ags: 2 for EBV, 3 for HCV, 1 for T. gondii, 1 for H. pylori, 2 for HSV1, 2 for HSV2, 2 for VZV; (ii) five lysates (CMV, T. gondii, H. pylori, HSV1, HSV2); (iii) mix of five Ag; (iv) two tag controls (GST, histidine); (v) one negative control (PBS) (FIG. 7). Spotting was performed inside a chamber at 25 C. and 60% humidity.

(78) Processing of Microarray Slides

(79) Printed slides were saturated for 1 hour at room temperature with T-PBS and 5-10% BSA in order to prevent non-specific Ab binding. After washing with TPBS, slides were incubated with 80 L of diluted serum (100 to 800 g/mL) or purified mc Ig (12.5 to 200 g/mL), for two hours at room temperature. After a second washing, slides were incubated with a labelled secondary Ab (0.1 to 4 g/mL) of Dylight 680-labelled goat anti-human IgG (H+L) (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md., USA) or Dylight 680 goat anti-human IgA from antibodies-online or monoclonal antibody to lambda light chains Alexa Fluor 700-conjugated (Exbio, Czech Republic) or goat anti-human kappa chains Alexa Fluor 700-conjugated (Invitrogen, Belgium) while shaking in the dark, then washed with T-PBS. Fluorescence signal, detected with the Odyssey infrared imaging system scanner at 21 m resolution (LI-COR Biosciences, NE, USA) was used to determine the serological status of each sample.

(80) Data Analysis

(81) Specific fluorescence was quantified using the GenePix Pro 4 Microarray Acquisition & Analysis Software (Molecular Devices, Sunnyvale, Calif., USA). For each sample, the median fluorescence intensity (FLU) was determined after subtraction of background slide fluorescence. Results for each sample (patients and controls) were represented on histograms using GraphPad 5.0 software (San Diego, Calif., USA). FLU values represent the mean of four replicates from one experiment. Experiments were repeated three times on different arrays for each patient. FLU values obtained for each sample were compared to positive and negative commercial controls for each germ. For each antigen, the positivity threshold was the mean plus 2 standard deviations of fluorescence values obtained for the negative control for each germ. Patients or whom the mean FLU value was higher than the positivity threshold were considered as positive for the antigen tested. When several antigens were used for the same pathogen, if one or more positive results were obtained for a serum, the patient was considered as positive.

(82) Western Blot Analysis

(83) The MP Diagnostics (MPD) HELICO BLOT 2.1 Western Blot kit assay consists of a Western Blot made from bacterial lysate of H. pylori strain ATCC 49503 and a recombinant antigen called CIM. The test strip contains H. pylori antigens with molecular weights of 116 kDa (CagA), 89 kDa (VacA), 37 kDa, 35 kDa, 30 kDa (Urease A), and 19.5 kDa as separate lines. The CIM had been originally identified by screening of immunogenic proteins of H. pylori and was synthesized by recombinant technology. The test was done and interpreted according to instructions of the manufacturer. Patient sera were diluted at 1/60. Mc Ig were concentrated at 0.025-0.05 g/L. Diluted and non-diluted sera and purified mc Igs were incubated with the strips according to instructions of the manufacturer. The manufacturer's recommended criteria for determining H. pylori positivity by Helico Blot 2.1 were as follows:

(84) (1) 116 kDa (CagA) positive, where CagA has to be present with at least one of the following bands89 kDa (VacA), 37 kDa, 35 kDa, 30 kDa (UreA), or 19.5 kDa, or with CIM,

(85) (2) presence of any one band at 89 kDa, 37 kDa, or 35 kDa, with or without CIM,

(86) (3) presence of both 30 kDa and 19.5 kDa band with or without CIM.

(87) These criteria were used to validate the positivity of serum but not for mc Ig because we look for specificity for only one antigen.

B/ Results

(88) Analysis of Mc Ig Specificity with the 8-Germ MIP Microarray Assay

(89) It has been previously shown that mc Ig of patients who are infected with HCV typically recognize HCV core or NS4. Sera and purified mc Ig from 90 patients with myeloma or MGUS were analysed using the novel, 8-germ MIP microarray. For 59 patients with EBV-positive serum, mc Ig specifically recognized EBNA (Epstein Barr nuclear Ag) in 19% cases (FIG. 8), and for 25 patients with H. Pylori-positive serum, mc Ig was found specific for one H. Pylori antigen (CagA protein) in 8% of cases (FIG. 9).

(90) Specificity of mc Ig for H. pylori CagA protein was confirm using western blot analysis.

(91) In contrast, for 45 patients found positive for CMV, no CMV specificity was found for the mc Ig. For 38 patients found positive for HHV-1 or HHV-2, no HHV-1/2 specificity was found for the mc Ig. For 41 patients found positive for VZV, no VZV specificity was found for the mc Ig, and for 16 patients found positive for T. gondii, no T. gondii specificity was found for the mc Ig.

(92) Altogether, for 23 of the 101 (22.8%) patients studied, the mc Ig was found specific for one Ag of these 3 germs: HCV, EBV, H. pylori. Using the 8 germ MIP array immunoassay, in the cohort studied, no patient was found to have a mc Ig directed at Ag from CMV, HHV-1, HHV-2, VZV, or T. gondii. In this series, 20.6% of MGUS and 24.2% of myeloma patients (non significant difference, p=0.810) present with a mc Ig that is directed against HCV, EBV or H. pylori.

C/ Discussion

(93) This study is the first systematic analysis of the specificity of mc Ig of MGUS and myeloma patients. The MIP array immunoassay designed for this study is uniquely suited to successfully analyse the specificity of mc Ig: prior to the protein array technology, screening mc Ig for a panel of Ag using classical assays such as ELISAs required large quantities of purified mc Ig, usually not available. The inventors found that antigens from three germs known to cause other B-cell malignancies (EBV, HCV, H. pylori) can be the targets of mc Ig of more than 20% of MGUS and myeloma patients. The exact proportion of patients with mc Ig specific for these three germs will have to be determined in large cohorts of MGUS and myeloma patients. In contrast, in the same cohort the inventors found no evidence that Ag from CMV, HHV-1, HHV-2 or VZV (all common germs) or T. gondii (less frequent) are targeted by mc Ig. Nevertheless, these observations will have to be confirmed in large cohorts of patients; these studies are on-going in the laboratory of the inventors.

(94) These findings regarding HCV, EBV and H. pylori demonstrate that chronic antigen stimulation is a key step in the pathogenesis of MGUS and myeloma for subsets of patients, estimated in this first study to represent more than 20% of MGUS and myeloma patients. The inventors provide a new tool to stratify patients according to the antigen-specificity of mc Ig. This novel assay, based on the protein array technology, allows testing the specificity of minute amounts of purified mc Ig for up to 36 different Ag in a single assay. This technology is considerably more efficient for studies of mc Ig specificity than phage display, epitope reconstruction or epitope mediated antigen prediction (E-MAP), which all proved disappointing. Two reasons explain the paucity of results using these techniques: first, the technical complexity makes it difficult to use in clinical practice; second, these techniques are predictive only and it is necessary to confirm the specificity of mc Ig with other assays. The new array will allow the stratification of patients according to particular antigen-specificity of their mc Ig.

(95) Indeed the Ag-specificity of mc Ig may be associated with specific patient characteristics, risk of disease progression or/and response to treatment, in view of personalized medicine in myeloma and MGUS. Indeed, differences and distinct disease evolution and response to treatment are observed among myeloma patients. Chronic stimulation by various categories of Ag can contribute to the diversity of clinical, biological and cytogenetic presentation, as well as the heterogeneity of response. New protocols taking antigen specificity of mc Ig into account could lead to curative treatment in MGUS and improved response to treatment in myeloma: treatment including antibiotics or antiviral drugs could cure subsets of MGUS and facilitate response to treatment in myeloma.