Method for assessing risk of human cytomegalovirus active infection in body and related kit

Abstract

The invention belongs to the fields of medicine and immunology, particularly, the field of immunological diagnosis. In particular, the invention discloses a method for assessing whether a subject is at risk of developing human cytomegalovirus (HCMV) active infection and a kit therefore. The method comprises the steps of: (1) determining the level of an antibody against a HCMV protein in a body fluid sample from the subject; and (2) comparing the level with a predetermined reference value, wherein if the level is below the predetermined reference value, the subject is determined to be at risk of developing HCMV active infection. In addition, the invention also discloses a method for screening a candidate drug which is capable of improving the ability of a subject to resist human cytomegalovirus (HCMV) active infection, and a kit therefore.

Claims

1. A method for assessing whether a subject is at risk of developing human cytomegalovirus (HCMV) active infection, comprising the following steps of: (1) determining the level of an antibody against a HCMV protein in a body fluid sample from the subject, wherein the antibody is an IgG antibody; (2) comparing the level with a predetermined reference value; wherein, the HCMV protein is pp150 or pp28; and if the level is below the reference value, the subject is determined to be at risk of developing HCMV active infection compared with those who have the antibody level above said reference value; and (3) treating the subject determined to be at risk of developing HCMV with an appropriate therapeutic regimen for said subject, so as to reduce the risk of developing HCMV active infection in the subject.

2. The method of claim 1, wherein the method is characterized by one or more of the following items: (a) the subject is human; (b) the body fluid sample is selected from blood, serum, plasma, urine and saliva; (c) the active infection is a primary infection by HCMV in a subject that has not been infected by HCMV, or, a re-infection by HCMV or activation of latent HCMV in a subject that has been infected by HCMV; (d) the level of the antibody against the HCMV protein in the body fluid sample is determined by immunologic assay; (e) the level refers to an antibody titer, and the reference value refers to a predetermined antibody titer; or the level is an antibody absolute quantity and the reference value refers to a predetermined antibody absolute quantity; and (f) the method further comprises: before the step (1), providing a body fluid sample from the subject.

3. The method according to claim 1, wherein in the step (1), the level refers to an antibody titer, and the antibody titer of the antibody against pp150 and/or pp28 in the body fluid sample is determined by ELISA; and the reference value is an antibody titer in a range of 40-320.

4. The method of claim 3, wherein the reference value is 40, and if the antibody titer of the antibody against pp150 is below or equal to 40, the subject is determined to have a relative risk of 11.2 for developing HCMV active infection, with 95% CI of 8.7-14.6; and/or, the subject is determined to have a probability of 55.37% for developing HCMV active infection; or, the reference value is 80, and if the antibody titer of the antibody against pp150 is below or equal to 80, the subject is determined to have a relative risk of 10.6 for developing HCMV active infection, with 95% CI of 7.7-14.6, and/or, the subject is determined to have a probability of 36.98% for developing HCMV active infection; or, the reference value is 160, and if the antibody titer of the antibody against pp150 is below or equal to 160, the subject is determined to have a relative risk of 14.8 for developing HCMV active infection, with 95% CI of 9.0-24.6; and/or, the subject is determined to have a probability of 23.66% for developing HCMV active infection.

5. The method of claim 3, wherein pp150 and/or an antigenic fragment thereof is used to determine the antibody titer of the antibody against pp150 in the body fluid sample by ELISA; and/or, pp28 and/or an antigenic fragment thereof is used to determine the antibody titer of the antibody against pp28 in the body fluid sample by ELISA.

6. The method of claim 5, wherein pp150 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has an amino acid sequence set forth in SEQ ID NO: 2; and/or pp28 has an amino acid sequence set forth in SEQ ID NO: 3.

7. The method according to claim 1, wherein, in the step (1), the level is an antibody absolute quantity, and the absolute quantity of the antibody against pp150 and/or pp28 in the body fluid sample is determined; and the reference value is an antibody absolute quantity in a range of 0.8-6.4 IU/ml.

8. The method of claim 7, wherein the reference value is 0.8 IU/ml, and if the absolute quantity of the antibody against pp150 is below or equal to 0.8 IU/ml, the subject is determined to have a relative risk of 11.6 for developing HCMV active infection, with 95% CI of 7.8-17.2; and/or, the subject is determined to have a probability of 60.0% for developing HCMV active infection; or, the reference value is 1.6 IU/ml, and if the absolute quantity of the antibody against pp150 is below or equal to 1.6 IU/ml, the subject is determined to have a relative risk of 15.2 for developing HCMV active infection, with 95% CI of 9.5-24.3; and/or, the subject is determined to have a probability of 50.0% for developing HCMV active infection; or, the reference value is 3.2 IU/ml, and if the absolute quantity of the antibody against pp150 is below or equal to 3.2 IU/ml, the subject is determined to have a relative risk of 19.0 for developing HCMV active infection, with 95% CI of 9.6-37.7, and/or, the subject is determined to have a probability of 31.3% for developing HCMV active infection.

9. A kit for assessing whether a subject is at risk of developing human cytomegalovirus (HCMV) active infection comprising, a reagent capable of determining the level of an antibody against a HCMV protein, and instructions of using the reagent to determine the level of an antibody against a HCMV protein in a body fluid sample from the subject so as to assess whether the subject is at risk of developing human cytomegalovirus (HCMV) active infection, wherein the HCMV protein is pp150 or pp28 and wherein the antibody is an IgG antibody; and a non-natural antibody against HCMV.

10. The kit of claim 9, wherein the reagent is capable of determining the level of an antibody against a HCMV protein by immunologic assay.

11. The kit of claim 10, wherein the reagent is selected from: the HCMV protein or an antigenic fragment thereof, a fusion protein comprising the HCMV protein or an antigenic fragment thereof, and any combination thereof.

12. The kit of claim 10, wherein the HCMV protein is pp150, and the reagent is pp150 and/or an antigenic fragment thereof; or, the HCMV protein is pp28, and the reagent is pp28 and/or an antigenic fragment thereof; or, the HCMV protein is pp150 and pp28, and the reagent comprises: pp150 and/or an antigenic fragment thereof as a first component, and pp28 and/or an antigenic fragment thereof as a second component.

13. The kit of claim 12, wherein pp150 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has an amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has an amino acid sequence set forth in SEQ ID NO: 3.

14. The kit of claim 9, wherein the kit further comprises: (i) a device for collecting or storing the body fluid sample from the subject; and/or (ii) an additional reagent necessary for the assay.

15. The kit of claim 14, wherein the additional reagent necessary for the assay is selected from a buffer, a diluent, a blocking solution, a labelled anti-antibody, a standard sample and any combination thereof.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B show that the ELISA assay platform (based on pp150 & pp150-2) established in Example 2 can be used to determine the level of an antibody against a CMV protein (an antibody against pp150), and further to determine whether a serum sample is positive or negative. FIG. 1A shows the correlation analysis of the detection results of 288 serum samples by the ELISA assay using pp150 and pp150-2 as coating antigens (abscissa) and the ELISA assay using purified virus as coating antigen (ordinate). The results show that the results determined by the two ELISA assays are highly identical, the coincidence rate of them is 99.3%, and the correlation of response intensity is significant (correlation coefficient r=0.85). FIG. 1B shows the comparison of the detection results of 36 serum samples by the ELISA assay using pp150 and pp150-2 as coating antigens and the ELISA assay using a commercially available CMV-IgG reagent (Diasorin-IgG) as coating antigen. The results show that the reactivity of the pp150 & pp150-2-based ELISA assay according to the invention is significantly stronger than the reactivity of Diasorin-based ELISA assay, and the reactivity of the latter to positive serums is substantively at a relatively low level. The results in FIGS. 1A and 1B show that the ELISA assay platform established in Example 2 can be used to determine the level of an antibody against a CMV protein (an antibody against pp150) in a serum sample accurately, reliably and effectively.

(2) FIG. 2 shows the correlation between HCMV infection dose and Elispot detection value, in the IE1 protein-based Elispot assay platform established in Example 3. The results show that there is a significant linear relationship (R.sup.2=0.9988) between HCMV infection dose and Elispot detection value in the Elispot assay platform. This indicates that the Elispot assay platform established in Example 3 can be used to determine the level of an antibody against a CMV protein (an antibody against IE1 protein) in a serum sample accurately, reliably and effectively.

(3) FIG. 3 shows the comparison of the detection results (Elispot NT50) of 61 serum samples by the IE1 protein-based Elispot assay platform established in Example 3 and the detection results by a commercially available HCMV IgG antibody assay kit (Diasorin IgG reagent), wherein, 31 serum samples, which were detected to be positive by the kit, were also detected to be positive in the detection results by the Elispot assay platform; 30 serum samples, which were detected to be negative by the kit, were also detected to be negative in the detection results by the Elispot assay platform; their results are completely consistent with each other. This indicates the accuracy and reliability of the Elispot assay platform established in Example 3.

(4) FIG. 4 shows the correlation between the detection results (Elispot NT50) of 61 serum samples by the IE1 protein-based Elispot assay platform established in Example 3 and the detection results by a commercially available HCMV IRD neutralization assay platform (IRD NT50). The results show that there is a significant correlation (R.sup.2=0.8960) between the detection results (Elispot NT50) by the Elispot assay platform and the detection results by the IRD neutralization assay platform (IRD NT50). This indicates the accuracy and reliability of the Elispot assay platform established in Example 3.

(5) FIG. 5 shows the detection results of 61 serum samples with known background by 6 ELISA assay platforms (which are based on pp150 & pp150-2, HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99), and UL48a, respectively) established in Example 2, wherein pp150 refers to the ELISA assay platform based on a mixture of pp150 and pp150-2. The results show that the ELISA assay platforms established in Example 2 (in particular, the pp150 & pp150-2-based ELISA assay platform and the pp28-based ELISA assay platform) can determine the level of an antibody against a CMV protein in a serum sample accurately, reliably and effectively, and determine whether a serum sample is negative or positive.

(6) FIG. 6A-C shows the ROC curve analysis by the pp150 & pp150-2-based ELISA assay platform (called pp150 assay platform for short) under different criteria for a virus event in Example 6, wherein abscissa represents (100%the detection specificity % of the assay platform), and the ordinate represents the detection sensitivity % of the assay platform; and,

(7) FIG. 6A shows the analysis of sensitivity and specificity of the pp150 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the occurrence of a virus event (HCMV active infection) is defined by the circumstance where the detection results of the other assay platforms are double positive or more.

(8) FIG. 6B shows the analysis of sensitivity and specificity of the pp150 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the occurrence of a virus event (HCMV active infection) is defined by the circumstance where the detection results of the other assay platforms are triple positive or more.

(9) FIG. 6C shows the analysis of sensitivity and specificity of the pp150 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the occurrence of a virus event (HCMV active infection) is defined by the circumstance where the detection results of the other assay platforms are quadruple positive or more.

(10) The results in FIG. 6A-C show that under all the three circumstances, the pp150 assay platform has an accuracy of above 80% for predicting the occurrence of a virus event (HCMV active infection), and therefore can be used to assess the risk of developing human cytomegalovirus (HCMV) active infection in a subject accurately, reliably and effectively.

(11) FIG. 7A-C shows the ROC curve analysis of the pp28-based ELISA assay platform (called pp28 assay platform for short) under different criteria for a virus event in Example 6, wherein abscissa represents (100%the detection specificity % of the assay platform), and the ordinate represents the detection sensitivity % of the assay platform; and,

(12) FIG. 7A shows the analysis of sensitivity and specificity of the pp28 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the occurrence of a virus event (HCMV active infection) is defined by the circumstance where the detection results of the other assay platforms are double positive or more.

(13) FIG. 7B shows the analysis of sensitivity and specificity of the pp28 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the occurrence of a virus event (HCMV active infection) is defined by the circumstance where the detection results of the other assay platforms are triple positive or more.

(14) FIG. 7C shows the analysis of sensitivity and specificity of the pp28 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the occurrence of a virus event (HCMV active infection) is defined by the circumstance where the detection results of the other assay platforms are quadruple positive or more.

(15) The results in FIG. 7A-C show that under all the three circumstances, the pp28 assay platform has an accuracy of above 78% for predicting the occurrence of a virus event (HCMV active infection), and therefore can be used to assess the risk of developing human cytomegalovirus (HCMV) active infection in a subject accurately, reliably and effectively.

(16) FIG. 8 shows the change in the antibody titer of an antibody against pp150 in a certain natural population as described in Example 7 over 12 months (one year), wherein, abscissa represents a baseline antibody level (antibody titer), ordinate represents the probability that the antibody level increases or decreases by 4 folds one year later. The results show that individuals with a higher baseline antibody level, have a lower probability that the level of an antibody against pp150 increases by 4 folds or more (i.e., developing HCMV active infection) one year later; while, individuals with a lower baseline antibody level of an individual, have a higher probability that the level of an antibody against pp150 increases by 4 folds or more (i.e., developing HCMV active infection) one year later. The risk of developing HCMV active infection in an individual is negatively correlated with the baseline antibody level.

(17) FIG. 9 shows the correlation between the baseline level of an antibody against pp150 and the risk of developing HCMV active infection in a certain natural population as described in Example 7, wherein, abscissa represents the baseline level of an antibody against pp150 (antibody titer), and ordinate represents the risk of developing HCMV active infection (ratio). The results show that individuals with a lower baseline antibody level, have a higher risk of developing HCMV active infection (i.e., the level of an antibody against pp150 increases by 4 folds or more) later. The risk of HCMV active infection is negatively correlated with the baseline antibody level. When the baseline antibody level (antibody titer) is lower than 10, the risk of developing HCMV active infection is up to 82.6%; and when the baseline antibody level (antibody titer) is no more than 160, the risk of developing HCMV active infection is up to 23.66%.

(18) FIG. 10 shows an antibody content-antibody reactivity standard curve plotted using a standard sample (Paul-Ehrlich-Instltut, Referenz-CMV-IgG, Juli 1996, 110 IU/ml) comprising an antibody in a known amount, wherein, abscissa represents an antibody content (expressed as IU/ml); and ordinate represents antibody reactivity (expressed by the OD value obtained by ELISA). The results show that there is a significant linear relationship between the antibody content and antibody reactivity (R.sup.2=0.9984), and the linearity range is more than an order of magnitude. Therefore, the antibody content-antibody reactivity standard curve plotted using a standard sample can be used to accurately quantify the antibody content (expressed as IU/ml) in a sample.

(19) FIG. 11 shows the change in the content of an antibody against pp150 in a certain natural population as described in Example 8 over 12 months (one year), wherein, abscissa represents a baseline antibody level (antibody content), and ordinate represents the probability that the antibody level increases or decreases by 4 folds one year later. The results show that individuals with a lower baseline antibody level, have a higher probability that the level of an antibody against pp150 increases by 4 folds or more one year later (i.e., the antibody level obtained by the second detection is at least 4 folds higher than the antibody level obtained by the first detection, indicating that the individual has HCMV active infection within the interval of 12 months); while, individuals with a higher baseline antibody level, has a lower probability that the level of an antibody against pp150 increases by 4 folds or more one year later (i.e., developing HCMV active infection). The risk of developing HCMV active infection in an individual is negatively correlated with the baseline antibody level in serum.

(20) FIG. 12 shows ROC curve analytic results of the method described in Example 8, wherein, the method predicts HCMV active infection based on the content/absolute quantity of an antibody against pp150 in serum, wherein abscissa represents (100%specificity % of the method), and ordinate represents the sensitivity % of the method. The results in FIG. 12 show that the method of the invention can be used to assess the risk of developing human cytomegalovirus (HCMV) active infection in a subject accurately, reliably and effectively.

SEQUENCE INFORMATION

(21) The information of the sequences involved in the invention are provided in the following Table 1.

(22) TABLE-US-00001 TABLE1 Sequenceinformation SEQ IDNO Name Sequenceinformation5-3 1 HCMV MSLQFIGLQRRDVVALVNFLRHLTQKPDVDLEAHPKILKKCGEKRLHR pp150 RTVLFNELMLWLGYYRELRFHNPDLSSVLEEFEVRCVAVARRGYTYPF GDRGKARDHLAVLDRTEFDTDVRHDAEIVERALVSAVILAKMSVRETL VTAIGQTEPIAFVHLKDTEVQRIEENLEGVRRNMFCVKPLDLNLDRHAN TALVNAVNKLVYTGRLIMNVRRSWEELERKCLARIQERCKLLVKELRM CLSFDSNYCRNILKHAVENGDSADTLLELLIEDFDIYVDSFPQSAHTFLG ARSPSLEFDDDANLLSLGGGSAFSSVPKKHVPTQPLDGWSWIASPWKG HKPFRFEAHGSLAPAAEAHAARSAAVGYYDEEEKRRERQKRVDDEVV QREKQQLKAWEERQQNLQQRQQQPPPPARKPSASRRLFGSSADEDDDD DDDEKNIFTPIKKPGTSGKGAASGGGVSSIFSGLLSSGSQKPTSGPLNIPQ QQQRHAAFSLVSPQVTKASPGRVRRDSAWDVRPLTETRGDLFSGDEDS DSSDGYPPNRQDPRFTDTLVDITDTETSAKPPVTTAYKFEQPTLTFGAGV NVPAGAGAAILTPTPVNPSTAPAPAPTPTFAGTQTPVNGNSPWAPTAPLP GDMNPANWPRERAWALKNPHLAYNPFRMPTTSTASQNTVSTTPRRPST PRAAVTQTASRDAADEVWALRDQTAESPVEDSEEEDDDSSDTGSVVSL GHTTPSSDYNNDVISPPSQTPEQSTPSRIRKAKLSSPMTTTSTSQKPVLGK RVATPHASARAQTVTSTPVQGRLEKQVSGTPSTVPATLLQPQPASSKTT SSRNVTSGAGTSSASSARQPSASASVLSPTEDDVVSPATSPLSMLSSASP SPAKSAPPSPVKGRGSRVGVPSLKPTLGGKAVVGRPPSVPVSGSAPGRL SGSSRAASTTPTYPAVTTVYPPSSTAKSSVSNAPPVASPSILKPGASAALQ SRRSTGTAAVGSPVKSTTGMKTVAFDLSSPQKSGTGPQPGSAGMGGAK TPSDAVQNILQKIEKIKNTEE 2 HCMV DDVVSPATSPLSMLSSASPSPAKSAPPSPVKGRGSRVGVPSLKPTLGGKA pp150-2 VVGRPPSVPVSGSAPGRLSGSSRAASTTPTYPAVTTVYPPSSTAKSSVSN APPVASPSILKPGASAALQSRRSTGTAAVGSPVKSTTGMKTVAFDLSSP QKSGTGPQPGSAGMGGAKTPSDAVQNILQKIEKIKNTEE 3 HCMV MGAELCKRICCEFGTTPGEPLKDALGRQVSLRSYDNIPPTSSSDEGEDDD pp28 DGEDDDNEERQQKLRLCGSGCGGNDSSSGSHREATHDGSKKNAVRSTF REDKAPKPSKQSKKKKKPSKFIHHHQQSSIMQETDDLDEEDTSIYLSPPP VPPVQVVAKRLPRPDTPRTPRQKKISQRPPTPGTKKPAASLPF 4 HCMV MESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRV pp65 SQPSLILVSQYTPDSTPCHRGDNQLQVQHTYFTGSEVENVSVNVHNPTG RSICPSQEPMSIYVYALPLKMLNIPSINVFIHYPSAAERKHRHLPVADAVI HASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVAL RHVVCAHELVCSMENTRATKMQVIGDQYVKVYLESFCEDVPSGKLFM HVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHIM LDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQ YDPVAALFFFDIDLLLQRGPQYSEHPTFTSQYRIQGKLEYRHTWDRHDE GAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGASTSAGRKR KSASSATACTSGVMTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPP WQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFAELEGVWQP AAQPKRRRHRQDALPGPCIASTPKKHRG 5 HCMV MDRKTRLSEPPTLALRLKPYKTAIQQLRSVIRALKENTTVTFLPTPSLILQ gp52 TVRSHCVSKITFNSSCLYITDKSFQPKTINNSTPLLGNFMYLTSSKDLTKF YVQDISDLSAKISMCAPDFNMEFSSACVHGQDIVRESENSAVHVDLDFG VVADLLKWIGPHTRVKRNVKKAPCPTGTVQILVHAGPPAIKFILTNGSE LEFTSNNRVSFHGVKNMRINVQLKNFYQTLLNCAVTKLPCTLRIVTEHD TLLYVASRNGLFAVENFLTEEPFQRGDPFDKNYVGNSGKSRGGGGGGG SLSSLANAGGLHDDGPGLDNDLMNEPMGLGGLGGGGGGGGKKHDRG GGGGSGTRKMSSGGGGGDHDHGLSSKEKYEQHKITSYLTSKGGSGGG GGGGGGGLDRNSGNYFNDAKEESDSEDSVTFEFVPNTKKQKCG 6 HCMV MSHPLSAAVPAATAPPGATVAGASPAVSSLAWPHDGVYLPKDAFFSLL pp38 GASRSAVPVMYPGAVAAPPSASPAPLPLPSYPASYGAPVVGYDQLAAR HFADYVDPHYPGWGRRYEPAPSLHPSYPVPPPPSPAYYRRRDSPGGMD EPPSGWERYDGGHRGQSQKQHRHGGSGGHNKRRKETAAASSSSSDED LSFPGEAEHGRARKRLKSHVNSDGGSGGHAGSNQQQQQRYDELRDAIH ELKRDLFAARQSSTLLSAALPSAASSSPTTTTVCTPTGELTSGGGETPTA LLSGGAKVAERAQAGVVNASCRLATASGSEAATAGPSTAGSSSCPASV VLAAAAAQAAAASQSPPKDMVDLNRRIFVAALNKLE 7 UL48a MSNTAPGPTVANKRDEKHRHVVNVVLELPTEISEATHPVLATMLSKYT RMSSLFNDKCAFKLDLLRMVAVSRTRR 8 primer GGATCCATGAGTTTGCAGTTTATCGGT 9 primer GCTAGCTTCCTCCGTGTTCTTAATCTT 10 primer GGATCCATGGAGTCGCGCGGTCGCCGT 11 primer GCTAGCACCTCGGTGCTTTTTGGGCGT 12 primer GGATCCATGGATCGCAAGACGCGCCTC 13 primer GCTAGCGCCGCACTTTTGCTTCTTGGT 14 primer GAATTCATGTCGCACCCTCTGAGTGCT 15 primer GCTAGCCTCGAGCTTATTGAGCGCAGC 16 primer GGATCCATGGGTGCCGAACTCTGCAAA 17 primer GAATTCAAAGGGCAAGGAGGCGGCGGG 18 primer GGATCCATGTCTAACACCGCGCCGGGA 19 primer GAATTCGCGCCGGGTGCGCGACAC

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

(23) The present invention is illustrated by reference to the following examples (which are not intended to limit the protection scope of the present invention).

(24) Unless indicated otherwise, the molecular biological experimental methods and immunological assays used in the present invention are carried out substantially in accordance with the methods as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3.sup.rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes are used under the conditions recommended by manufacturers of the products. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the protection scope of the present invention.

Example 1. Cloning and Expression of Proteins

(25) In the Example, the inventor obtained 7 proteins by recombinant expression, i.e., HCMV pp150 (UL32), HCMV pp150-2 (a truncated protein of pp150), HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99) and UL48a. The information of the proteins for recombinant expression is shown in Table 2. The primers for use in PCR amplification of the genes encoding the target proteins are listed in Table 3.

(26) TABLE-US-00002 TABLE 2 Information of 7 proteins for recombinant expression Sequence Sequence accession Bacterial Protein name information No. Vector strain HCMV pp150 SEQ ID NO: 1 ACL51112 B11 BL21 HCMV pp150-2 SEQ ID NO: 2 ACL51112 B11 BL21 HCMV pp28 SEQ ID NO: 3 ACL51167.1 Pet- er competent GST HCMV pp65 SEQ ID NO: 4 ACL51152.1 B6 er competent HCMV gp52 SEQ ID NO: 5 ACL51123.1 B6 er competent HCMV pp38 SEQ ID NO: 6 ACL51150.1 Pet- er competent GST UL48a SEQ ID NO: 7 ACL51128.1 Pet- er competent GST

(27) TABLE-US-00003 TABLE3 InformationoftheprimersforuseinPCRamplificationofthe genesencodingthetargetproteins SEQID NO: Targetprotein Primer Sequenceinformation5-3 8 pp150(UL32) upstream GGATCCATGAGTTTGCAGTTTATCGGT 9 downstream GCTAGCTTCCTCCGTGTTCTTAATCTT 10 pp65(UL83) upstream GGATCCATGGAGTCGCGCGGTCGCCGT 11 downstream GCTAGCACCTCGGTGCTTTTTGGGCGT 12 gp52(UL44) upstream GGATCCATGGATCGCAAGACGCGCCTC 13 downstream GCTAGCGCCGCACTTTTGCTTCTTGGT 14 pp38(UL80a) upstream GAATTCATGTCGCACCCTCTGAGTGCT 15 downstream GCTAGCCTCGAGCTTATTGAGCGCAGC 16 pp28(UL99) upstream GGATCCATGGGTGCCGAACTCTGCAAA 17 downstream GAATTCAAAGGGCAAGGAGGCGGCGGG 18 UL48a upstream GGATCCATGTCTAACACCGCGCCGGGA 19 downstream GAATTCGCGCCGGGTGCGCGACAC

Example 2. Establishment of an Antigen Protein-Based ELISA Assay Platform for Detecting an Antibody

(28) In the Example, based on HCMV pp150 & pp150-2 (as a combination coating antigen), HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99) or UL48a, respectively, the inventor established the ELISA assay platforms for detecting an antibody against pp150, an antibody against pp65, an antibody against gp52, an antibody against pp38, an antibody against pp28 and an antibody against UL48a, respectively (called pp150 assay platform, pp65 assay platform, gp52 assay platform, pp38 assay platform, pp28 assay platform, and UL48a assay platform for short, respectively), each comprising: a microwell plate coated with an antigen protein (i.e., a coating antigen), a coating buffer, a blocking solution, a washing solution, an enzyme-labelled anti-human IgG antibody, an enzyme-labelled antibody diluent, negative/positive control, a chromogenic solution and a stop solution.

(29) Coating antigen: HCMV pp150 and pp150-2, HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99) or UL48a.

(30) Sample diluent: Tris-Base buffer (pH 7.8-8.3), comprising 1-5% (mass/volume ratio) of bovine serum albumin, 5-10% (mass/volume ratio) of sucrose and 2-7% (mass/volume ratio) of casein; and 7-12% (volume/volume ratio) of fetal bovine serum.

(31) Concentrated washing solution: comprising a phosphate buffer (pH 7-7.6) and a surfactant Tween20.

(32) Enzyme-labelled anti-human IgG antibody: Horseradish peroxidase (HRP)-labelled mouse anti-human IgG monoclonal antibody.

(33) Diluent for enzyme-labelled antibody: a phosphate buffer (pH 6.8-7.3), wherein each 1000 ml phosphate buffer comprises 0.1-1 M NaCl and 0.3-1% (mass/volume ratio) of casein, 0.1-0.4% (mass/volume ratio) of TritonX-100, 7-12% (volume/volume ratio) of fetal bovine serum, and 0.2-0.5% (mass/volume ratio) of Geltin.

(34) Chromogenic solution: A solution comprising trisodium citrate, citric acid, sodium acetate, glacial acetic acid and hydrogen peroxide; and, B solution comprising absolute ethyl alcohol, ethylene glycol, dimethyl formamide, and 3,3,5,5-tetramethylbenzidine.

(35) Stop solution: 0.1-1 M sulphuric acid.

(36) The pp150 antibody assay platform was used as an example below to show the effect of the ELISA assay platform established in the Example. The pp150 & pp150-2-based ELISA assay platform and purified virus-based ELISA assay platform were used to determine 288 randomly selected serums in parallel. The results are shown in FIG. 1A. The results show that the results determined by the two ELISA assay platforms are highly identical, the coincidence rate of them is 99.3%, and the correlation of response intensity is good (correlation coefficient r=0.85). In addition, the pp150 & pp150-2-based ELISA assay platform and a commercially available CMV-IgG reagent (Diasorin-IgG)-based ELISA assay platform were used to determine 36 randomly selected serums in parallel. The results are shown in FIG. 1B. The results show that the reactivity of the pp150 & pp150-2-based ELISA assay platform is significantly stronger than the reactivity of the Diasorin-based ELISA assay platform, wherein the latter has a relatively low level of reactivity for most of the positive serums. The results in FIGS. 1A and 1B show that the ELISA assay platforms established in the Example can be used to determine the level of an antibody against a CMV protein (an antibody against pp150) in a serum sample accurately, reliably and effectively.

Example 3. Detection of an Anti-HCMV-IE1 Antibody by Elispot Assay Platform

(37) In the Example, the inventor established an IE1 protein-based Elispot assay platform for detecting an anti-HCMV-IE1 antibody.

(38) IE1 is a HCMV immediate early protein that is present in the nucleus of an infected cell one hour after the infection. The Elispot assay established in the Example is a method for quickly determining cytomegalovirus titer that was established based on a traditional enzyme-linked immunospot assay in combination with Elispot automated spot counter. Compared with the traditional method for detecting TCID50, the Elispot assay established in the Example obtains the experimental results by the specific binding between a monoclonal antibody and a virus immediate early protein, rather than obtaining the experimental results by cytopathogenic counting or plaque formation. Therefore, since the cells infected by a virus can be detected in the Elispot assay before cytopathogeny, it greatly shortens the time for detection (20 h). In addition, in the Elispot assay, the results are read by automated image collection and spot counting program of the Elispot assay instrument, which greatly improve the stability and accuracy of the detection.

(39) FIG. 2 shows the correlation between the HCMV infection dose and the Elispot detection value, in the IE1 protein-based Elispot assay platform as established in the Example. The results show that there is a significant linear relationship (R.sup.2=0.9988) between the HCMV infection dose and the Elispot detection value in the Elispot assay platform. This indicates that the Elispot assay platform established in the Example can be used to determine the level of an antibody against a CMV protein (an antibody against IE1 protein) in a serum sample accurately, reliably and effectively.

(40) Furthermore, the results obtained by the Elispot assay platform established in the Example were compared with the results obtained by the commercially available HCMV IgG antibody assay kit (Diasorin-IgG agent, DIASORIN, P002033). The results are shown in FIG. 3. The results show that among the 61 serum samples detected in parallel, the 31 serum samples, which were determined by the kit to be positive, were also positive in the results determined by the Elispot assay platform; and the 30 serum samples, which were determined by the kit to be negative, were also negative in the results determined by the Elispot assay platform; their results were completely identical to each other. This indicates the accuracy and reliability of the Elispot assay platform established in the Example.

(41) Furthermore, the results determined by the Elispot assay platform established in the Example were compared with the results determined by HCMV IRD neutralization assay platform as established by Aimin Tang et al. (Aimin Tang, Fengsheng Li, Daniel C. Freed, Adam C. Finnefrock, Danilo R. Casimiro, Dai Wang, Tong-Ming Fu. A novel high-throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines. Vaccine. 2011 Oct. 26; 29(46): 8350-6). The results are shown in FIG. 4. The results show that the results determined by the Elispot assay platform (Elispot NT50) are in a good correlation (R.sup.2=0.8960) with the results determined by the IRD neutralization assay platform (IRD NT50). This indicates the accuracy and reliability of the Elispot assay platform established in the Example.

Example 4. Detection of Serum with a Known Background by ELISA Assay Platform

(42) In the Example, the inventor used the ELISA assay platform established in Example 2 to detect the antibodies in 61 serums with known background, so as to confirm the reliability and effectiveness of the ELISA assay platform, wherein the backgrounds of the serum samples had been determined by the commercially available HCMV IRD assay reagent (Aimin Tang, Fengsheng Li, Daniel C. Freed, Adam C. Finnefrock, Danilo R. Casimiro, Dai Wang, Tong-Ming Fu. A novel high-throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines. Vaccine. 2011, 29:8350-6) and the commercially available HCMV IgG antibody assay kit (Diasorin-IgG agent, DIASORIN, P002033). The detection method comprised the following steps:

(43) step 1: collecting a sample

(44) the serum to be tested was centrifuged for 5-10 min (10000 rpm/min), for use in detection;

(45) step 2: loading a sample for detection

(46) the ingredients in the kit were equilibrated to room temperature; to the sample well of the coated microwell plate in the ELISA assay platform established in Example 2, a sample diluent (90 ul) and the serum to be tested (10 ul) were added; and meanwhile, a negative control and a positive control were set in the microwell plate, wherein to each of the negative control wells, serum with a negative background (10 ul) and a sample diluent (90 ul) were added; to each of the positive control wells, serum with a positive background (10 ul) and a sample diluent (90 ul) were added; the plate was then shaken to mix the solutions homogeneously on a plate vibrator; the plate was then covered with a sealing film, and the reaction was carried out in a 37 C. incubator/thermostat water bath for 1 h;

(47) step 3: after the reaction, the sealing film was removed, and the wells were washed with a washing solution for 5 times, and dried upside down;

(48) step 4: to each of the wells, an enzyme-labelled anti-human IgG antibody solution (100 ul) was added, and the plate was covered with a sealing film; the reaction was then carried out in a 37 C. incubator/thermostat water bath for 30 min;

(49) step 5: after the reaction, the sealing film was removed, and the wells were washed with a washing solution for 5 times, and dried upside down;

(50) step 6: to each of the wells, a substrate solution A (50 ul) and a substrate solution B (50 ul) were added, and mixed homogeneously; and the reaction was then carried out in a 37 C. incubator/thermostat water bath for 15 min; and

(51) step 7: to each well, a stop solution (50 ul) was added, and the plate was then read by a Microplate Reader at OD450, thereby obtaining the OD values for the antibody reactions in the wells. The results are shown in FIG. 5.

(52) FIG. 5 shows the results of 61 serum samples with known backgrounds as determined by six ELISA assay platforms (which are based on pp150 & pp150-2, HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99), and UL48a, respectively) as established in Example 2, wherein pp150 refers to the ELISA assay platform based on a mixture of pp150 and pp150-2. The results show that the ELISA assay platforms established in Example 2 (in particular, the pp150 & pp150-2-based ELISA assay platform and the pp28-based ELISA assay platform) can determine the level of an antibody against a CMV protein in a serum sample and determine whether the serum sample is negative or positive, accurately, reliably and effectively.

Example 5. Detection of the Antibody Titer in a Sample by ELISA Assay Platform

(53) In the Example, the inventor used the ELISA assay platforms established in Example 2 to determine the antibody titer in a serum sample, so as to confirm the reliability and effectiveness of the ELISA assay platforms.

(54) In brief, in the Example, pp150 was used as an example, and the pp150 & pp150-2-based ELISA assay platform was used to detect HCMV IgG antibody (an antibody against pp150) in parallel in the serial dilution samples of two serums (Serum 1 and Serum 2) (two repeated experimentations were performed for each sample), to determine the antibody titer of an antibody against pp150 in the two serums. During the detection, negative control wells and positive control wells were set, and the detection method comprised the following steps:

(55) step 1: collecting a sample

(56) the serum to be tested was centrifuged for 5-10 min (10000 rpm/min), for use in detection;

(57) step 2: loading a sample for detection

(58) the ingredients in the kit were equilibrated to room temperature; to the first sample well of the coated microwell plate, a sample diluent (180 ul) was added, and to the second to the tenth sample wells, a sample diluent (100 ul per well) was added; to the first sample well, the serum to be test (20 ul) was then added, and the plate was shaken for 30 s-60 s to mix the solution homogeneously on a plate vibrator; and then, 100 ul solution was drawn from the first sample well and added to the second sample well, and the solution was mixed homogeneously under shaking; 100 ul solution was drawn from the second well and added to the third well, and the solution was mixed homogeneously under shaking; the serial dilution was performed until the tenth well; 100 ul solution was drawn from the tenth well and discarded; the process was repeated to each of the two serums;

(59) meanwhile, negative control wells, positive control wells, and blank control wells were set in the microwell plate, wherein to each of the negative control wells, serum with a negative background (10 ul) and a sample diluent (90 ul) were added; to each of the positive control wells, serum with a positive background (10 ul) and a sample diluent (90 ul) were added; and to each of the blank control wells, a sample diluent (100 ul) was added;

(60) the plate was then covered with a sealing film, and the reaction was carried out in a 37 C. incubator/thermostat water bath for 1 h;

(61) step 3: after the reaction, the sealing film was removed, and the wells were washed with a washing solution for 5 times, and dried upside down;

(62) step 4: to each of the wells, an anti-human IgG antibody solution (100 ul) was added, and the plate was covered with a sealing film; the reaction was then carried out in a 37 C. incubator/thermostat water bath for 30 min;

(63) step 5: after the reaction, the sealing film was removed, and the wells were washed with a washing solution for 5 times, and dried upside down;

(64) step 6: to each of the wells, a substrate solution A (50 ul) and a substrate solution B (50 ul) were added, and mixed homogeneously; and then the reaction was carried out in a 37 C. incubator/thermostat water bath for 15 min; and

(65) step 7: to each well, a stop solution (50 ul) was added, and the plate was then read by a Microplate Reader at OD450, thereby obtaining the OD values for the antibody reactions in the wells. The results are shown in Table 4.

(66) In Table 4, Serum 1 and Serum 2 are two independent serum samples, and Group 1 and Group 2 represent two repeated experimentations, and the antibody titer is defined as the maximum dilution fold of serum when OD450 reaches above 0.2. The detection results show that the antibody titer of HCMV IgG (i.e., an antibody against pp150) is 80 in Serum 1, and the antibody titer is 320 in Serum 2.

(67) TABLE-US-00004 TABLE 4 Determination of antibody titers in samples by ELISA assay platform OD values determined at different dilution folds Antibody Sample 10 20 40 80 160 320 640 1280 titer Serum 1 1.46 0.886 0.379 0.21 0.024 0.014 0.01 0.011 80 (Group 1) Serum 1 1.38 0.768 0.367 0.186 0.063 0.032 0.025 0.021 160 (Group 2) Serum 2 3.288 2.289 1.533 0.783 0.406 0.201 0.087 0.042 320 (Group 1) Serum 2 3.926 2.656 1.773 1.043 0.525 0.239 0.102 0.062 320 (Group 2) Positive 3.589 2.503 1.614 0.918 0.441 0.163 0.061 0.02 320 control Negative 0.016 0.015 0.013 0.009 0.012 0.002 0.02 0.009 <10 control Blank 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 control

(68) The results in Table 4 show that the ELISA assay platform established in Example 2 (for example, pp150 & pp150-2-based ELISA assay platform) can determine the antibody titer of an antibody against a CMV protein (for example, an antibody against pp150) in a sample accurately, reliably and effectively.

Example 6. Evaluation of the Risk of Developing HCMV Active Infection by ELISA Assay Platform

(69) The proteins used in the Example were the proteins obtained in Example 1, and the detection method used was the method described in Example 5.

(70) In brief, in the Example, the ELISA assay platform established in Example 2 (including, pp150 & pp150-2-based ELISA assay platform (called pp150 assay platform for short, the same below), pp28 assay platform, pp38 assay platform, UL48a assay platform, gp52 assay platform, pp65 assay platform) and IE1 antibody assay platform (i.e., the Elispot assay platform for detecting an anti-HCMV-IE1 antibody as described in Example 3) were used to detect the 202 pairs of serums collected from a certain natural population of Guangxi over an interval of 12 months (1 year), which were well preserved.

(71) For each pair of serums, if compared with the serum collected first, the serum collected later has the antibody titer increased by 4 folds or more, it is defined that the detection result of the pair of serums (which are from the same individual, and therefore are regarded as one sample) was positive.

(72) As defined above, among the 202 pairs of serum samples, the number of the samples determined to be positive by IE1 antibody assay platform, pp150 assay platform, pp28 assay platform, pp38 assay platform, UL48a assay platform, gp52 assay platform and pp65 assay platform (i.e., the number of the samples in which the antibody titer in the latter collected serum increased by 4 folds or more) was 8, 77, 21, 29, 30, 24, and 9, respectively.

(73) Furthermore, in order to evaluate the efficacy of pp150 assay platform for assessing the risk of developing HCMV active infection, the results determined by 6 other assay platforms are used to define an individual having a virus event (i.e., developing HCMV active infection). In brief, the criterion for indicating that an individual has a virus event (i.e., developing HCMV active infection) is that among the 6 detection results of the serum in the individual as obtained by the 6 other assay platforms, at least 2, at least 3 or at least 4 detection results are positive simultaneously.

(74) Table 5 shows the statistical information of the results determined by other 6 assay platforms.

(75) TABLE-US-00005 Detection results Number of samples at least 5 results being positive simultaneously 5 (pentuple positive and more) at least 4 results being positive simultaneously 11 (quadruple positive and more) at least 3 results being positive simultaneously 19 (triple positive or more) at least 2 results being positive simultaneously 33 (double positive or more) at least 1 result being positive simultaneously 54 (single positive or more) all the 6 results being positive 148

(76) Table 6a shows the results of AUC curve parameter analysis of the pp150 assay platform under different criteria for defining a virus event. Table 6b shows the results of LOGISTIC regression analysis of the pp150 assay platform under different criteria for defining a virus event.

(77) TABLE-US-00006 TABLE 6a AUC curve parameter of the pp150 assay platform under different criteria for defining a virus event lower upper Criterion for a virus event AUC limit limit Baseline level of double positive or more 0.792 0.702 0.882 pp150 antibody triple positive or more 0.849 0.768 0.929 quadruple positive or more 0.890 0.830 0.951

(78) TABLE-US-00007 TABLE 6b LOGISTIC regression analysis of the pp150 assay platform under different criteria for defining a virus event Criterion for a Predict- virus event B sig. ivity (%) Baseline level of double positive or more 0.001 0.003 83.7 pp150 antibody triple positive or more 0.006 0.006 90.6 quadruple positive or 0.016 0.014 94.6 more

(79) The ROC curve analysis of the pp150 assay platform under different criteria for defining a virus event is also shown in FIG. 6A-C, wherein,

(80) FIG. 6A show the analysis on sensitivity and specificity of the pp150 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the circumstance where the results determined by other assay platforms are double positive or more is defined as the occurrence of a virus event (HCMV active infection);

(81) FIG. 6B show the analysis on sensitivity and specificity of the pp150 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the circumstance where the results determined by other assay platforms are triple positive or more is defined as the occurrence of a virus event (HCMV active infection); and

(82) FIG. 6C show the analysis on sensitivity and specificity of the pp150 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the circumstance where the results determined by other assay platforms are quadruple positive or more is defined as the occurrence of a virus event (HCMV active infection).

(83) The results in Table 6a-6b and FIG. 6A-C show that under all the three circumstances, the accuracy of pp150 assay platform for predicting the occurrence of a virus event (HCMV active infection) is above 80%, and therefore can be used to assess the risk of developing human cytomegalovirus (HCMV) active infection in a subject accurately, reliably and effectively.

(84) Table 7a shows the results of AUC curve parameter analysis of the pp28 assay platform under different criteria for defining a virus event. Table 7b shows the results of LOGISTIC regression analysis of the pp28 assay platform under different criteria for defining a virus event.

(85) TABLE-US-00008 TABLE 7a AUC curve parameter of the pp28 assay platform under different criteria for defining a virus event lower Upper Criterion for a virus event AUC limit limit Baseline level of double positive or more 0.823 0.751 0.895 pp28 antibody triple positive or more 0.860 0.796 0.924 quadruple positive or more 0.831 0.740 0.923

(86) TABLE-US-00009 TABLE 7b LOGISTIC regression analysis of the pp28 assay platform under different criteria for defining a virus event Criterion for a Predict- virus event B sig. ability (%) Baseline level of double positive or more 3.258 0.000 78.2 pp28 antibody triple positive or more 7.787 0.000 87.1 quadruple positive or 7.753 0.002 92.6 more

(87) ROC curve analysis of the pp28 assay platform under different criteria for defining a virus event is also shown in FIGS. 7A-C, wherein,

(88) FIG. 7A show the analysis on sensitivity and specificity of the pp28 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the circumstance where the results determined by other assay platforms are double positive or more is defined as the occurrence of a virus event (HCMV active infection);

(89) FIG. 7B show the analysis on sensitivity and specificity of the pp28 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the circumstance where the results determined by other assay platforms are triple positive or more is defined as the occurrence of a virus event (HCMV active infection); and

(90) FIG. 7C show the analysis on sensitivity and specificity of the pp28 assay platform for assessing the occurrence of a virus event (HCMV active infection), when the circumstance where the results determined by other assay platforms are quadruple positive or more is defined as the occurrence of a virus event (HCMV active infection).

(91) The results in Table 7a-7b and FIG. 7A-C show that under all the three circumstances, the pp28 assay platform has an accuracy of above 78% for predicting a virus event (HCMV active infection), and therefore can be used to assess the risk of developing human cytomegalovirus (HCMV) active infection in a subject accurately, reliably and effectively.

Example 7. Detection Results of a Certain Natural Population of Guangxi by the pp150 Assay Platform

(92) In the Example, the inventor employed the ELISA assay platform (pp150 assay platform) established in Example 2 to assess the risk of developing HCMV active infection in a certain natural population of Guangxi. The detection method and the method for calculating an antibody titer were as described in Example 5.

(93) In brief, the inventor used the pp150 assay platform to detect the two serums obtained from each individual of a certain natural population of Guangxi (1659 persons) before and after an interval of 12 months (1 year). The results show that in the population, the antibody positive rate was 98.7% and 98.9%, and the average antibody titer was 1:269 and 1:260, respectively, for the two detections before and after the interval. This indicates that most of the individuals in the population have been infected with HCMV. The inventor further compared the results of the population obtained by the two detections before and after the interval. The comparative results are shown in FIG. 8-9 and Tables 8a-8b.

(94) In particular, FIG. 8 shows the change in the antibody titer of an antibody against pp150 in the certain natural population over 12 months (one year). The results show that individuals with a higher baseline antibody level (i.e., the antibody titer obtained by the first detection), has a lower probability that the level of an antibody against pp150 increases by 4 folds or more one year later (i.e., the antibody level obtained by the second detection is at least 4 folds higher than the antibody level obtained by the first detection, indicating that the individual has HCMV active infection within the interval of 12 months); while, individuals with a lower baseline antibody level, have a higher probability that the level of an antibody against pp150 increases by 4 folds or more one year later. The results show again that the risk of developing HCMV active infection in an individual is negatively correlated with the baseline antibody level. The lower the level of an antibody against a CMV protein (for example, the titer of an antibody against pp150) is, the higher the risk of infecting CMV in the individual is higher.

(95) FIG. 9 shows the correlation between the baseline level of an antibody against pp150 and the risk of developing HCMV active infection in the natural population, wherein, abscissa represents the baseline level of an antibody against pp150 (antibody titer), and ordinate represents the risk of developing HCMV active infection (ratio). The results show that individuals with a lower baseline antibody level, have a higher risk of developing HCMV active infection later (i.e., the level of an antibody against pp150 increases by 4 folds or more). The risk of HCMV active infection is negatively correlated with the baseline antibody level. When the baseline antibody level (antibody titer) is lower than 10, the risk of developing HCMV active infection is up to 82.6%; and when the baseline antibody level (antibody titer) is no more than 160, the risk of developing HCMV active infection is up to 23.66%. The results in FIG. 9 are also specifically described in Tables 8a-8b.

(96) TABLE-US-00010 TABLE 8a Statistical analysis of the results determined before and after an interval of 12 months in a certain natural population of Guangxi (one year) (I) Baseline Number of individuals antibody Number of having a virus event Infection ratio level individuals during the period (%) relative risk (95% CI) <10 23 19 82.6 1.0 10 26 18 69.2 0.838 (0.610-1.151) 20 42 22 52.4 0.634 (0.450-0.894) 40 86 39 45.3 0.549 (0.407-0.740) 80 161 27 16.8 0.203 (0.137-0.300) 160 317 30 9.5 0.115 (0.078-0.169) 320 404 12 3.0 0.034 (0.020-0.064) 640 359 3 0.8 0.010 (0.003-0.032) 1280 170 1 0.6 0.007 (0.001-0.051) >2560 71 0 0.0 0.0 total 1659 171 10.3

(97) TABLE-US-00011 TABLE 8b Statistical analysis of the results determined before and after an interval of 12 months (one year) in a certain natural population of Guangxi (II) Number of individuals Baseline having a virus relative antibody Number of event during the Infection risk (95% Youden level individuals period ratio (%) CI) Sensitivity Specificity index 40 177 98 55.37 11.2 (8.7-14.6) 57.3% 94.7% 0.52 >40 1482 73 4.93 80 338 125 36.98 10.6 (7.7-14.6) 73.1% 85.7% 0.59 >80 1321 46 3.48 160 655 155 23.66 14.8 (9.0-24.6) 90.6% 66.4% 0.57 >160 1004 16 1.59 320 1059 167 15.77 23.7 (8.8-63.4) 97.7% 40.1% 0.37 >320 600 4 0.67

(98) The results in Table 8a-8b and FIG. 9 also show: (1) for individuals having negative base antibody level (antibody titer<10), the percentage of developing HCMV active infection (primary infection) is up to 82.6% (19/23); while, for individuals having the base antibody level (antibody titer)1:2560, the percentage of developing HCMV active infection (recurrent infection) is 0% (0/71) (p<0.0001); (2) the HCMV active infection ratio is in significantly negative correlation with the base antibody level: the higher the base antibody level is, the lower the HCMV active infection ratio is. For the individuals having an antibody titer>1:80 (accounting for about 80%), the virus active infection ratio is 3.48% within 1 year, while for the individuals having an antibody titer1:80 (accounting for about 20%), the virus active infection ratio is 36.98% within 1 year. Similarly, for individuals having an antibody titer1:40, the virus active infection ratio is 4.93% within 1 year, while for individuals having an antibody titer1:40, the virus active infection ratio is 55.37% within 1 year. For individuals having an antibody titer>1:160, the virus active infection ratio is 1.59% within 1 year, while for individuals having an antibody titer1:160, the virus active infection ratio is 23.66% within 1 year.

(99) The results in Table 8b also show: the methods of the invention can be used to determine the relative risk of infection in a subject, wherein, the reference value of the base antibody level (i.e., antibody titer) for determining the relative risk can be set as an antibody titer in a range of 40-320, for example, 40, 80, 160 or an antibody titer of 320. If the antibody titer determined in a sample from a subject is below or equal to the reference value, the subject can be determined to have a high relative risk of developing HCMV active infection. It can be seen from Table 8b that when the reference value is between 40 and 320, the subjects having an antibody titer below the reference value, have a relative risk of more than 10 for developing HCMV active infection, and the lower limit of 95% CI is more than 7. This indicates that there is a strong or a very strong correlation between the parameter (antibody titer) and HCMV active infection.

(100) For example, if the antibody titer determined in a sample from a subject is below or equal to 40, the subject has a relative risk of 11.2 for developing HCMV active infection, and 95% CI is 8.7-14.6 (that is, the risk of developing HCMV active infection is significantly enhanced), compared to a subject having an antibody titer above 40. If the antibody titer determined in a sample from a subject is below or equal to 80, the subject has a relative risk of 10.6 for developing HCMV active infection, and 95% CI is 7.7-14.6, compared to a subject having an antibody titer above 80. If the antibody titer determined in a sample from a subject is below or equal to 160, the subject has a relative risk of 14.8 for developing HCMV active infection, and 95% CI is 9.0-24.6, compared to a subject having an antibody titer above 160. If the antibody titer determined in a sample from a subject is below or equal to 320, the subject has a relative risk of 23.7 for developing HCMV active infection, and 95% CI is 8.8-63.4, compared to a subject having an antibody titer above 320.

(101) In addition, Table 8b also shows that the sensitivity, specificity and Youden index of the methods of the invention for predicting HCMV active infection. It can be seen from Table 8b that when the reference value is set as an antibody titer of 40, the methods of the invention for predicting HCMV active infection have a sensitivity of 57.3%, a specificity of 94.7%, and a Youden index of 0.52; when the reference value is set as an antibody titer of 80, the methods of the invention for predicting HCMV active infection have a sensitivity of 73.1%, a specificity of 85.7%, and a Youden index of 0.59; when the reference value is set as an antibody titer of 160, the methods of the invention for predicting HCMV active infection have a sensitivity of 90.6%, a specificity of 66.4%, and a Youden index of 0.57; when the reference value is set as an antibody titer of 320, the methods of the invention for predicting HCMV active infection have a sensitivity of 97.7%, a specificity of 40.1%, and a Youden index of 0.37. These results show that when the reference value is set as an antibody titer of 80, the methods of the invention have the best predictive effect (i.e, the highest Youden index, which is 0.59); and when the reference value is set as an antibody titer of 160 and 40, the methods of the invention have a good predictive effect (i.e., the Youden index is higher than 0.5).

(102) The multi-factor analysis of the natural population also show that the HCMV active infection rate is independent of gender, age, occupation, degree of education, health habit, etc.

Example 8. Calibration of an Antibody Content in Serum

(103) In the Example, the inventor utilized a standard sample to calibrate the antibody content in a serum sample. The standard sample used was a CMV IgG standard sample (Paul-Ehrlich-Instltut, Referenz-CMV-IgG, Juli 1996, 110 IU/ml), and the target antibody to be calibrated was an antibody against pp150.

(104) For this purpose, the inventor subjected the serial diluents of the standard sample to ELISA assay (the ELISA assay used was as described in Example 5), and plotted the antibody content-antibody reactivity standard curve according to the results determined by ELISA. The standard curve is shown in FIG. 10; wherein, abscissa represents an antibody content (expressed as IU/ml); and ordinate represents antibody reactivity (expressed by the OD value obtained by ELISA). The results show that in a range of 0.06-0.7 IU/ml, there is a significant linear relationship between the antibody content and antibody reactivity (y=3.163*x0.073, R.sup.2=0.9984), and the linearity range is more than an order of magnitude. Therefore, the absolute quantification of the antibody content in a serum sample can be carried out by the following solution: (1) subjecting a serum sample to 10-fold gradient dilution; (2) subjecting each diluted serum sample to ELISA assay; and (3) selecting the detection results falling into the linear range, and calculating the antibody content of the initial serum sample according to the linear curve and the dilution fold.

(105) According to the solution above, two serums, collected from each individual of a certain natural population (726 persons) of Guangxi before and after an interval of 12 months (1 year), were subjected to the assay. The detection results show that in the population, the antibody positive rate was 100% as determined before and after an interval of 12 months (1 year), and the average antibody titer was 6.19 IU/ml and 5.08 IU/ml, respectively. The inventor further compared the results determined before and after the interval. The comparative results are shown in FIG. 11 and Table 9.

(106) In particular, FIG. 11 shows the change in the content of an antibody against pp150 in the natural population over 12 months (one year). The results are substantively identical to the results in FIG. 8. Individuals with a lower baseline antibody level, have a higher probability that the level of an antibody against pp150 increases by 4 folds or more one year later (i.e., the antibody level obtained by the second detection is at least 4 folds higher than the antibody level obtained by the first detection, indicating that the individuals have HCMV active infection within the interval of 12 months); while, individuals with a higher baseline antibody level, have a lower probability that the level of an antibody against pp150 increases by 4 folds or more one year later (i.e., developing HCMV active infection). The results show again that the risk of developing HCMV active infection in an individual is negatively correlated with the baseline antibody level in serum. The lower the level of an antibody against HCMV protein (such as the content of an antibody against pp150) in serum is, the higher the risk of infecting HCMV in an individual is.

(107) The results in Tables 9a-9b also show that in the natural population, individuals with a lower baseline antibody level, have a higher risk of developing HCMV active infection later (i.e., the level of an antibody against pp150 increases by 4 folds or more); the baseline level of an antibody against pp150 is in a negative correlation with the risk of HCMV active infection, wherein, an individual, in which the base antibody level is not more than 0.2 IU/ml, has a risk/infection rate of up to 80% for developing HCMV active infection within one year; an individual, in which the base antibody level is not more than 0.8 IU/ml, has a risk/infection rate of up to 60.0% for developing HCMV active infection within one year; an individual, in which the base antibody level is not more than 1.6 IU/ml, has a risk/infection rate of up to 50.0% for developing HCMV active infection within one year; an individual, in which the base antibody level is not more than 3.2 IU/ml, has a risk/infection rate of up to 31.3% for developing HCMV active infection within one year; an individual, in which the base antibody level is not more than 6.4 IU/ml, has a risk/infection rate of up to 19.0% for developing HCMV active infection within one year. In contrast, an individual, in which the base antibody level is above 0.8 IU/ml, has a risk/infection rate of 5.2% for developing HCMV active infection; an individual, in which the base antibody level is above 1.6 IU/ml, has a risk/infection rate of 3.3% for developing HCMV active infection; an individual, in which the base antibody level is above 3.2 IU/ml, has a risk/infection rate of 1.6% for developing HCMV active infection; an individual, in which the base antibody level is higher than 6.4 IU/ml, has a risk/infection rate of 0.8% for developing HCMV active infection; and an individual, in which the base antibody level is above 25.6 IU/ml, has a risk/infection rate of 0 for developing HCMV active infection.

(108) TABLE-US-00012 TABLE 9a Statistical analysis of the results determined before and after an interval of 12 months in a certain natural population of Guangxi (one year) (I) Number of Baseline individuals having a antibody Number of virus event during Infection content (IU/ml) individuals the period ratio Relative risk (95% CI) <=0.2 15 12 80% 100% 0.2-0.4 7 5 71% 89.3% (52.4%-152.1%) 0.4-0.8 28 13 46% 58.0% (36.2%-93.0%) 0.8-1.6 38 14 37% 46.1% (28.3%-75.0%) 1.6-3.2 91 12 13% 16.5% (9.2%-29.6%) 3.2-6.4 148 6 4% 5.1% (2.2%-11.6%) 6.4-12.8 186 2 1% 1.3% (0.3%-5.5%) 12.8-25.6 136 1 1% 0.9% (0.1%-6.6%) 25.6-51.2 77 0 0% 0.8% (0.05%-13.2%) Total 726 65 9%

(109) TABLE-US-00013 TABLE 9b Statistical analysis of the results determined before and after an interval of 12 months in a certain natural population of Guangxi (one year) (II) Number of Baseline individuals antibody having a virus relative content Number of event during the Infection risk (95% Youden (IU/ml) individuals period ratio CI) Sensitivity Specificity index <=0.8 50 30 60.0% 11.6 (7.8-17.2) 46.15% 96.97% 0.43 >0.8 676 35 5.2% <=1.6 88 44 50.0% 15.2 (9.5-24.3) 67.69% 93.34% 0.61 >1.6 638 21 3.3% <=3.2 179 56 31.3% 19.0 (9.6-37.7) 86.15% 81.39% 0.68 >3.2 547 9 1.6% <=6.4 327 62 19.0% 25.2 (8.0-79.6) 95.38% 59.91% 0.55 >6.4 399 3 0.8%

(110) The results in Table 9b also show: the methods of the invention can be used to determine the relative risk of infection in a subject, wherein, the reference value of the baseline antibody level (i.e., an antibody content/an antibody absolute quantity) for determining the relative risk can be set as an antibody absolute quantity in a range of 0.8-6.4 IU/ml, for example, an antibody absolute quantity of 0.8, 1.6, 3.2 or 6.4 IU/ml. If the antibody content determined in a sample from a subject is below or equal to the reference value, the subject can be regarded as having a high relative risk of developing HCMV active infection. It can be seen from Table 9b that when the reference value is between 0.8 and 6.4 IU/ml, the subjects with an antibody content is below the reference value, all have a relative risk of above 11 for developing HCMV active infection exceeding 11, and the lower limit of 95% CI exceeds 7. These indicate that there is a strong correlation or a very strong correlation between the parameter (an antibody content/an antibody absolute quantity) and HCMV active infection.

(111) For example, if the antibody content determined in a sample from a subject is below or equal to 0.8 IU/ml, the subject has a relative risk of 11.6 for developing HCMV active infection, and 95% CI is 7.8-17.2 (that is, the risk of developing HCMV active infection is significantly enhanced), compared to a subject with an antibody content above 0.8 IU/ml. if the antibody content determined in a sample from a subject is below or equal to 1.6 IU/ml, the subject has a relative risk of 15.2 for developing HCMV active infection, and 95% CI is 9.5-24.3, compared to a subject with an antibody content above 1.6 IU/ml. If the antibody content determined in a sample from a subject is below or equal to 3.2 IU/ml, the subject has a relative risk of 19.0 for developing HCMV active infection, and 95% CI is 9.6-37.7, compared to a subject with an antibody content above 3.2 IU/ml. If the antibody content determined in a sample from a subject is below or equal to 6.4 IU/ml, the subject has a relative risk of 25.2 for developing HCMV active infection, and 95% CI is 8.0-79.6, compared to a subject with an antibody content above 6.4 IU/ml.

(112) In addition, Table 9b also shows the sensitivity, specificity and Youden index of the methods of the invention for predicting HCMV active infection when the reference value is set as an antibody content in a range of 0.8-6.4 IU/ml. It can be seen from Table 9b that when the reference value is set as an antibody absolute quantity of 0.8 IU/ml, the methods of the invention for predicting HCMV active infection have a sensitivity of 46.15%, a specificity of 96.97%, and a Youden index of 0.43; when reference value is set as an antibody absolute quantity of 1.6 IU/ml, the methods of the invention for predicting HCMV active infection have a sensitivity of 67.69%, a specificity of 93.34%, and a Youden index of 0.61; when the reference value is set as an antibody absolute quantity of 3.2 IU/ml, the methods of the invention for predicting HCMV active infection have a sensitivity of 86.15%, a specificity of 81.39%, and a Youden index of 0.68; when the reference value is set as an antibody absolute quantity of 6.4 IU/ml, the methods of the invention for predicting HCMV active infection have a sensitivity of 95.38%, a specificity of 59.91%, and a Youden index of 0.55. The results show that when the reference value is set as an antibody absolute quantity of 3.2 IU/ml, the methods of the invention have the best predictive effect (i.e., the highest Youden index, which is 0.68); and, when the reference value is set as an antibody absolute quantity of 0.8, 1.6 and 6.4 IU/ml, the methods of the invention also have good predictive effect (i.e., the Youden index is close to or above 0.5).

(113) In addition, the results in Table 9b also show that with the increase in the set reference value, the methods of the invention for predicting HCMV active infection have the sensitivity increased gradually (increased from 46.15% to 95.38%), but have the specificity decreased gradually (decreased from 99.97% to 59.91%). It is substantively identical to the result of ROC curve analysis. The ROC curve analytic results of the methods are shown in FIG. 12, wherein, the area under the curve is 0.913. It can be seen from FIG. 12 that with the increase in sensitivity, the specificity of the methods of the invention for predicting HCMV active infection decreases gradually. Moreover, the results in FIG. 12 show that the methods of the invention can be used to assess the risk of developing human cytomegalovirus (HCMV) active infection in a subject accurately, reliably and effectively.

(114) Although the embodiments of the invention have been described in detail, a person skilled in the art would understand that according to all the disclosed teachings, details can be amended and modified, and these alterations all fall into the protection scope of the invention. The whole scope of the invention is defined by the attached claims and any equivalent thereof.

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