Application of recombinant Leptospiral outer membrane protein in ELISA-based serodiagnosis Thareerat Kalambaheti 1
Background to study Leptospirosis : Caused by bacteria of the genus Leptospira Humans infected by contacting to infected urine of carrier animals Common in temperate (0.1-10/100000) or tropical climates (10-100/100000) 2
Clinical manifestation Incubation periods: 5-14 days Subclinical Symptomatic 2 phase: biphasic fever 1. Leptospiremic phase fever, headache, myalgia, conjunctival suffusion 2. Immune phase (Weil disease) liver/renal fail, aseptic meningitis 3
STANDARD CRITERIA FOR DIAGNOSIS Patients fulfilling any of the following criteria were considered as confirmed cases of leptospirosis; Positive blood culture Seroconversion in MAT with a minimum titer of 1:100 in the second sample Four fold rise in titer in MAT Microagglutination test (MAT) is inadequate for rapid case identification, as it can only be performed in reference laboratory. 4
Leptospiremia Immune phase 5
Benefit of recombinant protein The recombinant leptospiral proteins are possible to be express and purify those fusion proteins in a form suitable for diagnostic formats such as ELISA assay, Western blot. Recombinant protein-based serologic tests achieve high sensitivity and specificity because of the high concentration of immunoreactive antigens were used in assays and the lack of nonspecific moieties presented in whole-cell preparations. 6
Recent application of recombinant protein in human Leptospirosis Flannery et al.( 2001) evaluated 3 recombinant protein; rhsp58, rlipl32, rompl1, using IgG-based ELISA. rlipl32 had the highest sensitivity; 56% in acute and 94% in convalescent, in comparative to MAT. Srimanote et al. (2007) applied rliga based ELISA for serodiagnosis with specificity greater than 95%, in comparative to MAT. Croda et al (2007) employed rligb in immunoblot assay using both IgG and IgM conjugate to detect acute phase of disease 7
An ideal test will need to discriminate between leptospirosis and a broad spectrum of diseases that cause acute febrile illnesses and have overlapping clinical presentations. This study will include the following serum samples; Leptospirosis with MAT positive Scrub typhus (ST) Dengue fever (DHF) Melioidosis (melioid) Human serum from endemic area (HE) Human serum from non-endemic area (HOE) MAT were negative 8
Proteome study revealed the presence of LigA, LipL41,LipL32, LipL21,Loa 22, in host tissue Leptospira (HTL) probe with chronic rat serum (CRS) 9
prset-b Cloning vector N-terminal 6x Histidine fusion peptide Ampicillin resistance gene 10
Prediction of Lipoprotein from L. interrogans serovar Copenhageni Protein MW; kd spii score splip MW; kd Obtained expressed - protein LipL41 (Lic12966) 38.93 10.63 probable lipoprotein 27 LipL32 (Lic11352) 29.61 15.73 probable lipoprotein 35 LipL21 (Lic10011) 19.66 21.70 probable lipoprotein 27 Loa22 (Lic10191) 20.91 18.70 not lipoprotein 27 11
Translation of LipL41 clone Lip41 concencus Universal code Strand RF plus 1 1 START STOP 1145 plus 2 plus 3 minus 1 minus 2 minus 3 There is a stop codon within LipL41 clone, so the predicted MW of expressed protein is 24.34 kd. 12
Validation of Leptospiral recombinant protein Prepare the antibody to recombinant protein, and use it to react with native antigen of Leptospira Determine the reactivity to human serum 13
Mice immunization Intraperitoneal route with 5-105 µg with Alum adjuvant per dose. Three doses with 2 weeks interval 14
Reactivity of anti Loa22 to leptospiral lysate panel-2 MW (kd) 55 40 35 25 15 10 27 kd Loa 22 His tag Loa22 mice sera BL21 24 25 26 28 29 31 32 26 kd 22 kd Panel of Leptospiral serovar 24 New 25 Ranarum 26 Sarmin 28 Mini 29 Cynopteri Louisiana 31 Panama 32 Shermani 15
MW (kd) 55 40 35 25 15 10 Reactivity of anti LipL32 to leptospiral lysate panel-2 35 kd LipL32 His taq LipL32 mice sera BL21 24 25 26 28 29 31 32 32 kd Panel of Leptospiral serovar 24 New 25 Ranarum 26 Sarmin 28 Mini 29 Cynopteri Louisiana 31 Panama 32 Shermani 16
Anti LipL21 vs Lepto cell panel 2 27 kd LipL21 LipL21 His mice taq sera 24 25 26 28 29 31 32 BL21 55 40 35 25 15 10 23 kd Panel of Leptospiral serovar 24 New 25 Ranarum 26 Sarmin 28 Mini 29 Cynopteri Louisiana 31 Panama 32 Shermani 17
Anti LipL41 vs Lepto cell panel 1 27 kd MW kd 70 55 40 35 25 15 LipL41 His taq LipL41 mice sera BL21 9 11 16 18 19 22 23 17 41 kd Panel of Leptospiral serovar 9 Copenhageni 11 Djasiman 16 Javanica 18 Pomona 19 Pyrogenes 22 Sejroe 23 Wolffi 17 Patoc Negative color 18
Reactivity to L wolffii kd 70 55 40 35 25 15 Anti LipL41 Anti LipL32 Anti LipL21 Anti Loa22 19
Reactivity of mice anti recombinant protein to Leptospiral whole cell lysate Leptospira species Lab no./ serovar Serogroup Anti Loa 22 Anti LipL32 Anti LipL21 Anti LipL41 L. interrogans 9 Copenhageni Icterohaemoragiae L. interrogans 11 Djasiman Djasiman L. borgpetersen 16 Javanica Javanica L. interrogans 18 Pomona Pomona - - - L. interrogans 19 Pyrogenes Pyrogenes L. borgpetersen 22 Sejroe Sejroe - - - L. interrogans 23 Wolffi Sejroe - - L. biflexa 17 Patoc Patoc - - - - L. interrogans 24 New Autumnalis L. meyeri 25 Ranarum Ranarum - - - - L. weilli 26 Sarmin Sarmin - L. borgpeterseni 28 Mini Mini L. kirshneri 29 Cynopteri Cynopteri L. noguchii Saigon Louisiana L. noguchii 31 Panama Panama L. santarosai 32 Shermani Shermani - - - - L. wolffii Khorat Khorat 20
ELISA assay employing Total Igs conjugate HRP (predominant IgG class was conjugated to HRP) 21
Optimization of ELISA-based assay OD 1.2 1 0.8 0.6 Optimization of rloa22 antigen conc Dengue MAT pos The following protein concentration of Ag were used; rlipl21 = 15 ng/ml 0.4 0.2 0 15.6 31.2 62.5 125 rlipl41 = ng/ml rlipl32 = ng/ml protein (ng/ml) rloa22 = ng/ml Assay on rloa Ag ng/ml Den Serum dilution 1:1000 was selected. OD 1.4 1.2 1 0.8 0.6 0.4 0.2 0 MAT Conjugation anti Total Ig or anti IgM with HRP of 1:4000 dilution was used. Utilized the ABTS substrate 500 1000 2000 4000 8000 serial serum dilution 22
2.5 LipL21 OD T-Ig 1:1000 OD profile of anti LipL21 2.0 1.5 No significant difference between Mean - 2 SE ODLIP21 1.0.5 0.0 N = 19 16 39 Lepto MAT pos Lepto cul pos Lepto Dip stick pos HOE 31 HE ST 31 DHF mellioid Lepto MAT pos VS HOE ST DHF And Melioid group were even higher group by disease 95% Confidence Interval (I) GROUP group by disease (J) GROUP group by disease Mean Difference (I-J) Std. Error Sig. Lower Bound Upper Bound 1 Lepto MAT pos 2 Lepto Dip stick pos -.06127.122178 1.000 -.46355.34100 3 Lepto cul pos.533(*).075062.006.06162.54904 4 HOE -.06574.111752.999 -.420.28881 5 HE -.40244(*).078750.000 -.65619 -.14868 6 ST -.19191.082628.8 -.45660.07278 7 DHF 8 mellioid.03724 -.98181(*).092447.081571 1.000.000 -.25638-1.24349.386 -.72013 Dependent Variable: ODLIP21 Games-Howell 23
1.0.9 Anti Loa22 OD T-Ig 1:1000 OD profile of anti Loa22.8 Mean - 2 SE ODLOA22.7.6.5.4.3 N = 16 38 31 X 1SD = 0.581 Lepto MAT pos Lepto cul pos Lepto Dip stick pos HOE group by disease HE ST DHF mellioid Dependent Variable: ODLOA22 Games-Howell 95% Confidence Interval There were significant difference between (I) GROUP group by disease 1 Lepto MAT pos (J) GROUP group by disease 2 Lepto Dip stick pos Mean Difference (I-J).20360 Std. Error.080017 Sig..207 Lower Bound -.05231 Upper Bound.45950 Lepto MAT pos VS Lepto cul pos HOE HE ST DHF melioid 3 Lepto cul pos 4 HOE 5 HE 6 ST 7 DHF 8 mellioid.36824(*).35227(*).25821(*).24793(*).26133(*).31190(*).061783.062099.059408.065316.080625.062594.000.000.003.009.039.000.17072.15382.06677.04064.00783.11213.56577.55071.44965.45523.51483 24.51167
1.0 Anti LipL32 OD T-Ig 1:1000 OD profile of anti LipL32.8.6 X1S D =0.621 Mean - 2 SE ODLIP32.4.2 0.0 N = 16 38 31 Dependent Variable: ODLIP32 Games-Howell Lepto MAT pos Lepto cul pos Lepto Dip stick pos HOE group by disease HE There were significant difference between ST DHF mellioid (I) GROUP group by diseas e 1 Lepto MAT pos (J) GROUP group by disease 2 Lepto Dip stick pos Mean Difference (I-J).18822 Std. Error.082969 Sig..336 95% Confidence Interval Lower Bound -.07748 Upper Bound.45391 Lepto MAT pos VS Lepto cul pos HOE HE ST DHF melloid 3 Lepto cul pos 4 HOE 5 HE 6 ST 7 DHF 8 mellioid.48323(*).41407(*).26310(*).22373(*).27670(*).54703(*).069747.065624.060968.069080.066385.062786.000.000.003.041.003.000.26320.20548.06698.005.06598.34618.70326.62265.45921.44217.48742 25.74789
1.2 1.0 Anti LipL41 OD T-Ig 1:1000 OD profile of anti LipL41 Mean - 2 SE OD LIP41.8.6.4 X1SD 0.622.2 N = 16 38 Lepto MAT Lepto cul pos pos Lepto Dip stick pos HOE 31 HE ST DHF mellioid Dependent Variable: ODLIP32 Games-Howell group by disease 95% Confidence Interval There were significant difference between (I) GROUP group by disease 1 Lepto MAT pos (J) GROUP group by disease 2 Lepto Dip stick pos Mean Difference (I-J).28907 Std. Error.093639 Sig..064 Lower Bound -.009 Upper Bound.58745 Lepto MAT pos VS Lepto cul pos HOE HE ST DHF melloid 3 Lepto cul pos 4 HOE 5 HE 6 ST 7 DHF 8 mellioid.48602(*).31620(*).29065(*).320(*).40197(*).29617(*).075792.077003.073505.077442.083348.075994.000.004.008.004.000.008.24291.06985.053.07548.13803.05247.72912.56255.52800.57052.66590 26.53986
Setting up the cut off value as differential diagnosis 1.0 Anti Loa22 OD T-Ig 1:1000.9.8 Mean - 2 SE ODLOA22.7.6.5.4.3 N = 16 38 31 X 2 SD = 0.625 Lepto MAT pos Lepto cul pos Lepto Dip stick pos HOE HE ST DHF mellioid group by disease Mean ± SD of anti Loa22 Lepto MAT pos = 0.823 ± 0.310 Control group = 0.537 ± 0.044 (HOE, HE, ST, DHF, Melioid) Cut off value = mean 1 SD = 0.537 0.044 = 0.581 mean 2 SD = 0.537 0.088 = 0.625 27
Percent positive of anti rlipl32 1SD-anti rlipl32 100 2SD-anti LipL32 90 80 70 60 50 40 20 10 0 10.5 5.1 Acutephase sera (38) 54.8 39.2 MAT positive sera (124) 25.8 6.4 6.6 6.6 28.1 20 40 18.7 0 0 HE (31) HOE () DHF (32) ST () Melloid () Number of positive case (%) based on the cut off value derived from 1SD and 2SD among each studied group was determined; 100 90 80 70 60 50 40 20 10 21 12.8 63.7 59.2 Percent positive anti-rloa22 38.7 25.8 16.6 6.6 28.1 36.6 50 33.3 25 23.3 1SD-anti rloa22 2SD-anti rloa22 LipL32 was able to differentiate melioidosis patient and normal human in non-endemic area, from Leptospirosis patient well. 0 Acutephase sera (38) MAT positive sera (124) HE (31) HOE () DHF (32) ST () Melloid () 28
Percent positive of anti rlipl32 1SD-anti rlipl32 100 90 80 70 60 50 40 20 10 0 10.5 5.1 Acutephase sera (38) 54.8 39.2 MAT positive sera (124) 25.8 6.4 6.6 6.6 28.1 20 40 18.7 0 0 HE (31) HOE () DHF (32) ST () Melloid () 2SD-anti LipL32 Number of positive case (%) based on the cut off value derived from 1SD and 2SD among each studied group was determined; Percent positive of anti rlipl41 1SD-anti rlipl41 100 2SD-anti rlipl41 90 80 70 60 50 40 20 13.1 49.1 44.8 45.1 46.6 32.2 20 33.3 31.2 36.6 21.8 50 40 10 0 2.5 Acutephase sera (38) MAT positive sera (124) HE (31) HOE () DHF (32) ST () Melloid () 29
Efficacy of ELISA-based recombinant protein (MAT as gold standard) Percentage rloa22 rlipl32 rlipl41 Sensitivity 76.60 56.60 70.0 Specificity 76.58 88.70 74.6 Accuracy 76.59 84.60 74.0
Comparative to previous study Whole-cell Leptospira-based serologic assays using enzyme-linked immunosorbent assay (ELISA) demonstrated the sensitivities and specificities of these tests ranged from 28 to 72% and 10 to 99%, respectively (McBride et al.,2007). The major limitation of whole-cell Leptospira-based serologic assays is the low sensitivity (<67%) to samples obtained from patients in the first week of illness. Low sensitivity was revealed among residence of endemic area. 31
Efficacy of ELISA-based recombinant protein to detect suspected Leptospirosis cases The cut off value derived from mean 2 SD of OD of control group Group by disease Total Number Positive anti rlip41 Positive anti rlipl32 Positive anti rloa22 of Cases OD 0.666 % positive OD 0.754 % positive OD 0.625 % positive 1 MAT pos 125 56 44.8 49 39.2 74 59.2 2 Dip stick pos 16 6 37.5 5 31.2 7 43.7 3 Lepto cul pos 39 1 2.5 2 5.1 5 12.8 4 West ern pos 29 12 41.3 16 55.1 14 48.2 Total 209 75 35.8 72 34.4 100 47.8 32
IgM-based ELISA was proposed to use to indicate acute infection. 33
OD of anti LipL32 (IgM) 1:1000 Anti LipL32 response in acute sera OD 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 10 20 40 50 60 Patient ID Total Ig 1:1000 IgM 1:1000 When cut off was set as; OD X 1SD = 0.662 Positive 7 out of 39 (17.9%) IgM 1000 LipL32 group by disease Mean N Std. Deviation Lepto MAT pos 0.52367 0.273459 Lepto cul pos 0.48808 39 0.296085 HOE HE ST DHF melioid 0.51088 0.65963 0.576 0.55463 0.26437 24 0.245864 0.333736 0.280228 0.264361 0.164975 Cut off value was derived from control group X 1SD=0.51.149 =0.662 Total 0.51007 213 0.290658 34
OD of anti Loa22 (IgM) 1:1000 Anti Loa 22 response in acute phase sera OD 1.4 1.2 1 0.8 0.6 0.4 0.2 Total Ig 1:1000 IgM 1:1000 When cut off was set as; OD X 1SD = 0.650 0 0 10 20 40 50 60 patient ID Positive 5 out of 39 = 12.8% IgM 1:1000 Loa22 group by disease Mean N Std. Deviation Lepto MAT pos 0.4288 0.271103 Lepto cul pos 0.40638 39 0.2903 HOE 0.53733 24 0.262881 HE 0.68613 0.343947 ST DHF 0.4984 0.44793 0.271616 0.243178 X 1SD=0.4740.176 = 0.650 melioid 0.20233 0.167954 Total 0.45377 213 0.298404 35
OD of anti LipL41 (IgM) 1:1000 Anti LipL41 response in acute phase sera 1.6 1.4 1.2 1 Total Ig 1:1000 IgM 1:1000 When cut off was set as; OD X 1SD = 0.649 OD 0.8 0.6 0.4 0.2 0 0 10 20 40 50 60 Patient ID Positive 6 out of 39 (15.3%) IgM1:1000 LipL41 group by disease Mean N Std. Deviation Lepto MAT pos 0.2887 0.225791 Lepto cul pos 0.40272 39 0.322333 HOE 0.54221 24 0.267474 HE 0.7145 0.363705 ST DHF 0.3735 0.38283 0.282647 0.2962 X 1SD = 0.424 0.225 =0.649 melioid 0.10703 0.133968 Total 0.39773 213 0.327354 36
IgM positivity among studied group N group by disease (N) Lepto MAT pos () Lepto cul pos (39) HOE (24) HE () Anti LipL32 9 7 4 15 IgM 1:1000 Anti loa22 7 6 6 15 Anti LipL41 2 6 6 16 Interpretation of healthy individuals of endemic area was concerned, as these people may exposed to antigen recently, with no infection. ST () DHF () Melioid () 11 7 1 8 5 0 5 5 0 Patient s symptom should also be considered. Total (213) 54 47 40 Co-infection of Leptospirosis with ST and DHF, could elevate Ab to Leptospira. 37
As IgM antibodies become detectable during 5 7 days after the onset of symptoms The low percentage of detection of the acute sera, indicate the less amount of IgM at that point. The second sera was thus required, as at first time point of sera collection, IgM was not raised up. 38
Relation of MAT titer to ELISA assay (MATtiter) total cases AntiLipL32 Positive (%) AntiLipL41 Positive (%) Anti Loa22 Positive (%) (100) 25 (46) 29 (53) 38 (70) 54 (200-800) 22 (34) 24 (37) (46) 64 ( 1000) 7 (35) 9 (45) 14 (70) 20 The MAT cut off titer at 1:100 as positive, was in question? 39
In summary rlipl32 gave the most accurate result (84.6%)in discrimination among other febrile illness. rloa22 gave the moderately accurate (76%), while the sensitivity was higher (76%) than rlipl32 (56.6%) ELISA assay was able to indicate the positivity among MAT negative samples, that gave positive results by Lepto Dipstick and Western blot. 40
Acknowledgement Dr. Sharon Peacock Vanaporn Wuthiekanun MORU (Mahidol-Oxford Tropica Medicine Research unit) Assoc Prof Wichit Fungladda Fac. Trop. Med Assoc Prof Varee Wongchotikul Assoc Prof Wipawee Jampangern Assist Prof Usanee Sutthisuntrakul Lecturer Akanit Jitmittraparp 41
Acknowledgement Phanita Chanket Siriporn Chattanadee Staffs Piyanart Chalayon Toungporn Boonchawalit MSc students Dr. Potjanee Srimanote 42
Acknowledgement 43