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Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens
Journal of Immunological Methods 252 Ž2001. 105–119 www.elsevier.nlrlocaterjim
Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens G.-Halli R. Rajasekariah a,) , Jeffrey R. Ryan b, Scott R. Hillier a , Lisa P. Yi b, John M. Stiteler b, Liwang Cui b, Anthony M. Smithyman a , Samuel K. Martin b a
b
Cellabs Pty Ltd, P.O. Box 421, BrookÕale, NSW 2100, Australia Walter Reed Army Institute of Research, SilÕer Spring, MD 20910-7500, USA
Received 28 June 2000; received in revised form 24 January 2001; accepted 25 January 2001
Abstract An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donoÕani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen Ž50 mgrml. was coated with PBS–methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref y ve sera and higher absorbance with Ref q ve sera. All steps of the ELISA were performed at room temperature. The SrN ratio, the differential absorbance between the negative sample vs. the test or Ref q ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP ŽMAb-conjugate. outperformed a commercially available anti-human polyclonal antibody conjugate ŽPAb-conjugate.. The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 mgrml Ld-ESM mixed in PBS–methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBSrT, were reacted for 1 h at RT. After washing the plate with PBSrT, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the SrN ratio. A batch of n s 22 endemic sera from North Africa were evaluated and
AbbreÕiations: ELISA, enzyme-linked immunosorbent assay; Ld-Ab f-ELISA, L. donoÕani antibody field ELISA; Ld-ESM, excretory, secretory and metabolic antigens released by L. donoÕani promastigotes during in vitro maintenance in serum-free and protein-free medium; MAb-conjugate, mouse anti-human monoclonal IgG–HRP conjugate; NPV, negative predictive value; PAb-conjugate, goat anti-human polyclonal IgG–HRP conjugate; PBS–methyl glyoxal, phosphate buffer saline supplemented with 1% methyl glyoxal; PBSrT, phosphate buffered saline supplemented with 0.05% Tween 20; PPV, positive predictive value; TMB, tetramethylbenzidine; Ref y ve sera, defined negative sera, not exposed to L. donoÕani infection; Ref q ve sera, defined sera from patients infected with L. donoÕani parasites; SrN Ratio, signal noise ratio, i.e., the differential absorbance between the Ref q ve and Ref y ve sera; RT, room temperature, the ambient temperature used to mimic the field situation; VL, visceral leishmaniasis due to L. donoÕani ) Corresponding author. Tel.: q61-2-9905-0133; fax: q61-2-9905-6426. E-mail address: [email protected] ŽG.-H.R. Rajasekariah.. 0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 3 4 1 - 6
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resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Anti-human HRP conjugate; ELISA; Endemic sera; ESM antigen; Field-ELISA; Leishmania donoÕani; Promastigote; Specific antibody reactivity; Signalrnoise ratio; Visceral leishmaniasis
1. Introduction Human leishmaniasis, caused by the protozoan parasite of the genus Leishmania is endemic in tropical and subtropical regions of some 88 different countries. About 12 million cases are estimated to occur worldwide. Of the two million new cases believed to occur each year, several million subclinical infections go undetected amongst 350 million people at risk ŽWorld Health Organization, 1998; Grimaldi and Tesh, 1993.. Human infection with Leishmania parasites is gaining considerable importance due to its relevance in travel medicine, and increased incidence due to urbanisation, agriculture development, deforestation, irrigation and other ecological factors, which led to the vector multiplication and transmission ŽWorld Health Organization, 1998.. Over the last decade, leishmaniasis has become one of the most frequently observed opportunistic infections among AIDS patients in Southern Europe. Clinical disease can also occur with immunosuppressive drugs. Consequently, our inability to detect occult and subclinical infections prior to immunosuppression or blood donation will carry increasingly significant public health implications. Although direct proof is lacking, the possibility exists that parasitized mononuclear cells circulate in individuals with latent infections and may be transmissible to sand flies and other humans. Hence, rapid user-friendly screening tests are needed to identify these silent infections. ELISA has been used successfully as an effective screening test for other diseases ŽVoller, 1993.. They can play a similar role in leishmaniasis if the appropriate antigen and optimal conditions for specific antibody reaction are defined. Immunodiagnostic tests developed to date for leishmaniasis have utilized whole in vitro cultivated promastigotes, native or recombinant antigens from various stages of the parasite ŽKar, 1995.. Field trials with these products frequently yielded inconsistent results, particularly in non-visceral syndromes and populations with co-en-
demic parasitic diseases. The rapid immuno-absorbent antibody-detection dipstick assay based on a recombinant kinesin protein ŽrK39. has made an impact in detecting acute visceral leishmaniasis Žkala azar. ŽBadaro et al., 1986; Reed et al., 1990; Zijlstra et al., 1998; Samuel Raj et al., 1999.. However, it fails to reliably detect the most predominant Leishmania infection, cutaneous leishmaniasis. More importantly, the performance of any diagnostic test in current use against sub-clinical infections, the most challenging problem of the future, is unknown. We recently showed that soluble exo-antigen released by Leishmania donoÕani promastigotes into a proteinfree medium ŽLd-ESM. is suitable for the development of an ELISA assay for the diagnosis of visceral ŽMartin et al., 1998. and cutaneous leishmaniasis ŽRyan et al., 1998.. In an attempt to further develop such tests, studies were undertaken to define the parameters in an ELISA format that affect the specific binding of Ld-ESM to antibodies present in patient sera. Gram quantities of Ld-ESM can be inexpensively and reproducibly generated in any laboratory with routine culture facilities and its reactivity seems to be genus-specific Žpersonal communication.. In this study, assay conditions have been optimised and the ELISA adapted for general use. Keeping the specificity as well as sensitivity as the main objective, we have devised a Leishmania antibody field ELISA ŽLd-Ab f-ELISA. and evaluated its performance using sera samples from an endemic area.
2. Materials and methods 2.1. L. donoÕani ESM antigen (Ld-ESM) Excretory, secretory and metabolic antigens, released by L. donoÕani promastigotes ŽWHO Refer-
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ence Strain 065. in vitro at 268C for 72 h into serum-free and protein-free medium, were collected and used for ELISA assays. Specific details of these in vitro derived antigens are described elsewhere ŽMartin et al., 1998..
2.3.3. Coating ELISA wells The Ld-ESM antigen preparation with defined protein value was used to coat the ELISA plates. The antigen concentration used is indicated under Experiments and Results section.
2.2. SDS-PAGE analysis
2.3.4. Blocking reagents Gelatin ranging in concentration from 0.1% to 0.4% solution and 0.5% casein were tested. BSA and other routinely employed blocking agents were not tested because of non-specific binding noted in previous experiments.
Ld-ESM in serum-free and protein-free medium was separated by SDS-PAGE using 4–12% Novex gradient gels. The proteins were visualized by colloidal Coomassie blue G-250 and silver staining. The molecular weights of the secreted antigens were estimated with reference to a prestained standard ŽNovex.. 2.3. ELISA assay The assay, in brief, involved immobilizing the antigen to the solid phase ELISA wells for 16–18 h at room temperature. Unbound antigen was removed and the wells were blocked. After blocking, the wells were reacted with Leishmania endemic sera and defined Ref y ve and Ref q ve sera. An HRP-conjugated anti-human antibody was used as the detector antibody. Specific binding of antigens on the solid phase and the specific antibodies present in the test sera were measured indirectly by the binding of HRP-labelled detector antibody, which was further detected by using TMBq H 2 O 2 as a chromogenic substrate. The following parameters specified in the Experiments and Results section were investigated. 2.3.1. ELISA plates Two plates viz., Greiner ŽGreiner Labortecnik ELISA plate Cat a 705071. and Labsystem ŽLabsystem combiplate breakable 8 Cat a 95029400. plates with 12 strips of eight wells each fitted to a plastic frame, were evaluated. 2.3.2. Coating buffers Four different coating buffers were used viz., Ža. 0.2 M carbonaterbicarbonate buffer pH 9.6, Žb. 0.01 M phosphate buffer ŽPO4 . pH 7.2, Žc. blank culture medium Žserum-free. pH 7.2, and Žd. 0.1 M phosphate buffered saline ŽpH 7.2. q 1% methyl glyoxal. They were tested for their ability to immobilize the antigen on to the solid-phase ELISA wells.
2.3.5. Wash buffer PBSrT and PBSrTq 0.2 M NaCl were used as the washing buffers. An automatic plate washer ŽDenley Well Wash 04. with four-cycle wash in each step was used to wash the ELISA wells. 2.3.6. Diluent for sera and conjugate In most of the assays, the wash buffer was used as the diluent for sera as well as the conjugated detector antibody. 2.3.7. Reference and endemic sera samples Sera from individual Ž n s 3., well-characterized, culture-positive case patients were used as reference positives. An equal number of sera from North American natives with no travel history to leishmania endemic areas were used as reference negatives. These sera samples were used as reference samples to optimise the assay. The reference sera were not pooled. They were used as individual data points as detailed under Experiments and Results section. After optimising the assay Ždesignated Ld-Ab f-ELISA., a battery of individual sera samples obtained from endemic areas of North Africa were screened and used to show assay performance in terms of specificity and sensitivity. 2.3.8. Detector antibody Two anti-human IgG conjugates ŽHRP-labelled. were tested. A goat anti-human IgG–HRP ŽKirkegaard and Perry Laboratories—KPL , Gaithersburg, MD., a polyclonal conjugate Ždesignated PAb-conjugate., and a mouse anti-human IgG–HRP ŽCellabs, Brookvale, Australia., Ždesignated MAb-conjugate..
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2.3.9. Substrate TMB solutions A and B ŽKPL. were mixed in equal parts 5–10 min before, and transferred an aliquot of 100 ml to each well as per the guidelines provided by the manufacturer. During the developmental phase of the assay, the colour intensity of Ref q ve wells was monitored Žabsorbance OD 620 nm. and stop solution was added when those wells reached an OD of 0.450. In the final optimised assay, incubation time with substrate was fixed at 25 min. 2.3.10. Stop solution and reading After adding the stop solution Ž1 M phosphoric acid prepared in distilled water., the contents were mixed and the plate was read at dual filter Ž450r620 nm. using a plate reader ŽAnthos LabTec Instruments 2001.. 2.4. Assay interpretation The raw data from the plate Ži.e., absorbance at 450r620 nm. were plotted into histograms and graphs. Žb. A signal-to-noise ratio ŽSrN ratio. was calculated by dividing the mean absorbance Ži.e., absorbance at 450r620 nm. of test sera with the mean absorbance of Ref y ve sera, and plotted. The SrN ratio is directly proportional to the specific antibody reactivity in ELISA ŽLocarnini et al., 1979; de Savigny and Voller, 1980; Kurstak, 1985.. 2.5. Final assay protocol After optimising assay steps, a final assay protocol was developed and followed for evaluating sera samples from endemic areas. In short, wells were coated with 50 mgrml Ld-ESM mixed in PBS– methyl glyoxal buffer overnight, and after removing the antigen, wells were blocked with 0.5% casein for 1 h at RT. Patient serum along with control sera diluted to 1:500 in PBSrT was reacted for 1 h, RT. After washing the plate in PBSrT, wells were reacted with MAb-conjugate at 1:8000 dilution for 40 min at RT; after washing, TMB substrate was added and colour development was allowed to proceed for 25 min, and then stopped with the stop solution. Absorbance was read at dual filter Ž450r620 nm. and the results were analysed.
2.6. EÕaluation of sera samples from endemic areas in Ld-Ab f-ELISA Using this protocol, a total of n s 22 test clinical sera obtained from the endemic areas of North Africa were evaluated along with n s 5 reference control negative sera. An optimised final assay format was used. In short, wells were coated with Ld-ESM diluted to 50 mgrml concentration in PBS plus methyl glyoxal overnight at RT. After removing the unbound antigen solution, wells were blocked with 0.5% casein for 1 h at RT. After removing the blocking buffer, wells were reacted with sera diluted to 1:500; 100 ml per well in PBSrT for 1 h, RT. After washing with PBSrT, the wells were reacted with the detector Žconjugate. antibody ŽMAb-conjugate. for 40 min, RT. After further washing, wells were reacted with the substrate TMB, and the colour absorbance was read at OD 450r620 nm. The mean values of raw data were expressed as histograms and subsequently used to calculate the SrN ratio. 2.7. General Wells coated with antigen were fitted into a 96well frame and blocked with 0.5% casein. They were then stored dry in an aluminium foil bag with silica, sealed and kept at 48C until use. This provided a consistent supply of antigen-coated wells for evaluation. All solutions were freshly prepared for the assay. All analytes were added at 100 mlrwell and incubated at ambient room temperature to mimic the situation in the field.
3. Experiments and results 3.1. SDS-PAGE analysis The Ld-ESM antigens were separated on 4–12% gradient gel and visualized by Coomassie staining. Several major bands with estimated molecular weights of 11, 30, 42, 50 and 161 kDa ŽFig. 1. were visualised along with minor bands. This showed that the Ld-ESM employed in the assay was complex and contained a variety of protein antigens.
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tion in PBSrT, and reacted for 1 h at RT. After washing the wells with PBSrT, MAb-conjugate was used as the detector antibody. Other assay conditions were followed as mentioned above. Absorbancies were read at 450r620 nm. The LabSystems plate did not perform well in this study. There was no discrimination between Ref y ve and Ref q ve sera Ždata not shown.. There was a clear distinction between sera in the Greiner plate. Results in Fig. 2 clearly show that PBS plus methyl glyoxal, phosphate buffer and culture medium, respectively, showed higher reactivity with positive sera and relatively less reactivity with negative sera. SrN ratio was analysed; a higher SrN ratio was seen using sera at a 1:500 sera dilution. Phosphate buffer, culture medium and PBS–glyoxal gave higher SrN ratios. Based on this result, the Greiner plate and PBS plus methyl glyoxal were selected as ELISA plate and coating buffer, respectively. 3.3. Conjugate concentration
Fig. 1. SDS-PAGE analysis. Antigens secreted by L. donoÕani promastigotes were concentrated Ž ;10=. and 10 ml Žlane 1. of the antigen concentrates were separated by SDS-PAGE in a 4–12% gradient gel. Protein bands were visualized by colloidal Coomassie G-250 staining. M indicates pre-stained molecular standards ranging from 3 to 188 kDa.
3.2. Selection of ELISA plates and eÕaluation of coating buffer suitable for immobilization of L. donoÕani ESM antigen Two types of ELISA plates ŽGreiner and LabSystems. were used; Ld-ESM antigens diluted to 50 mgrml concentration in four types of coating buffers were used Ž100 ml or about 5 mg to each well.. Here, excess antigen was used to select the coating solution. Antigen was immobilized to the solid phase wells at ambient room temperature ŽRT. for 18 h. The unbound antigen was removed and the wells were blocked with 0.1% gelatin for 40 min. Based on a pilot assay, two sera samples ŽRef y ve and Ref q ve sera. were used at 1:500 and 1:1000 dilu-
Using the Greiner ELISA plate and PBS plus methyl glyoxal as the coating buffer for Ld-ESM, Ref q ve and Ref y ve sera were used at 1:500 dilution. Two anti-human IgG–HRP conjugates were evaluated at four different dilutions. Data in Fig. 3 show that PAb-conjugate is highly reactive with the Ref y ve sera, indicating a high level of non-specific reaction. On the other hand, the same level of reactivity was observed in the blank and Ref y ve sera sample wells with the MAb-conjugate. The reactivity with MAb-conjugate appeared to be more specific with Ref q ve sera as evidenced by higher SrN ratios. 3.4. Influence of washing solution Any non-specific antibody reactivity would reduce the SrN ratio, and consequently, result in lower assay specificity. Here, the influence of wash solution in reducing the non-specific reactivity was examined. Two wash solutions Ži.e., PBSrT and PBSrT supplemented with 0.2 M NaCl. were used to overcome any non-specific attachment of conjugated antibody. Ref y ve and Ref q ve sera were
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Fig. 2. Comparative reactivity of defined sera Ž1:500 and 1:1000. in antibody detection ELISA: wells were coated with identical concentration of Ld-ESM antigen diluted in different coating buffers. Calculated SrN ratio value is indicated above the bar.
reacted at 1:500 dilutions and washed with two different wash solutions. MAb-conjugate was used at 1:8000 dilutions. Plates were read at 450r620 nm. Reactivity of blanks and Ref y ve sera were lower in wells washed with PBSrT q0.2 M NaCl. The data clearly show that PBSrTq 0.2 M NaCl is more effective in removing non-specific binding Žnearly 45% reduction of non-specific signal 0.283 vs. 0.114. and increasing the SrN ratio from 3.7 to 7.2. De-
spite its higher SrN ratio, 0.2 M NaCl was left out of the final wash buffer because it formed a precipitate on standing. 3.5. Concentration of antigen and effectiÕe blocking Having selected the Greiner plate and PBS plus methyl glyoxal as the coating buffer, optimum levels of antigen and appropriate blocking reagents were
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Fig. 3. Influence of conjugate dilution on the antibody detection. MAb-conjugate showed minimal binding in blank and Ref y ve wells, and high in Ref q ve wells. This has generated relatively high SrN ratio.
investigated. Wells were coated with a series of antigen concentration from 1.25 to 40 mgrml. Two blocking reagents, 0.4% gelatin Žto provide maximum blocking effect with higher concentration; Section 3.2. and 0.5% casein prepared in distilled water were evaluated. Defined Ref q ve and Ref y ve sera, diluted to 1:500 in PBSrT, reacted in the Ag-coated
and blocked wells for 1 h at RT. Two conjugates, PAb- and MAb-conjugates, diluted to 1:4000 in PBSrT, were added to wells and incubated for 30 min at RT. The colour was developed for 25 min by adding the substrate and immediately read after addition of stopping solution. Data in Fig. 4a and b show that the reactivity was higher in blank and Ref y ve
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Fig. 4. Ža. Absorbance values are plotted to show how antigen concentration Ž1.25, 2.5, 5, 10, 20, 40 mgrml coded 1 to 6, respectively. and blocking ŽC for 0.5% casein and G for 0.4% gelatin. influence the antibody detection. Žb. How antigen concentration Ž1.25, 2.5, 5, 10, 20, 40 mgrml coded 1 to 6, respectively. and blocking ŽC for 0.5% casein and G for 0.4% gelatin. influence the antibody detection in ELISA with PAb- and MAb-conjugates. Žc. How antigen concentration and blocking with 0.5% casein influence the antibody detection in sera samples when MAb-conjugate was used in ELISA.
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Fig. 4 Ž continued ..
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Fig. 4 Ž continued ..
wells with PAb-conjugate, thereby reducing the differences between samples that resulted with a low SrN ratio. With the MAb-conjugate, the absorbancies of blank and Ref y ve sera wells were almost equal. There was a pattern in the reactivity relative to the antigen concentration. Wells reacted with the positive sera showed a gradual rise in absorbance dependent upon the antigen concentration. On the whole, MAb-conjugate reactivity was relatively lower in control sample ŽRef y ve and blank. wells. The SrN ratios ŽFig. 4b. were higher with MAb-conjugate when casein was used for blocking. MAb-conjugate with casein blocking generated excellent SrN ratios, at 20 and 40 mgrml antigen level. These data ŽFig. 4a and b. clearly demonstrate the relevance of
Ža. higher antigen concentration Ž) 40 mgrml., Žb. casein blocking; and Žc. MAb-conjugate. 3.6. Determination of optimum antigen concentration Additional testing was needed to optimise the antigen concentration. Here, the antigen was titrated from 20 to 90 mgrml concentration. For remaining ELISA steps, favourable parameters ŽSection 3.5. were selected, i.e., blocking antigen-coated wells with 0.5% casein, 1:500-diluted four sera samples and the MAb-conjugate. Assay was performed by using n s 4 sera samples having high, medium and low antibody reactivity. Data in Fig. 4c show how four sera samples reacted with different antigen con-
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centration. A concentration between 50 and 60 mgrml was found to provide a clear distinction between a positive and a negative sample. Ref q ve sera showed higher reactivity at higher antigen concentration, whereas Sera ŽS-1388. showed a flat reactivity. With this data, a concentration of 50 mgrml antigen was found optimal, which was used in future assays. 3.7. Serum dilution Four sera samples Žone Ref y ve, three Ref q ve sera were from endemic area. were used to optimise the assay following identical assay conditions. Fig. 5 shows the relative kinetics of antibody reactivity at
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different sera dilution. A good discrimination was apparent at 1:500 dilutions. This formed the basis for future assays. 3.8. EÕaluation in Ld-Ab f-ELISA A final optimal form of the ELISA ŽLd-Ab fELISA. was adopted after evaluation of all the parameters affecting the assay. This ELISA was then used to test 22 sera samples obtained from an area in North Africa endemic for VL. Reference n s 5 negative sera were reacted, - 0.2 Žrange 0.078–0.186; av 0.130 at absorbance OD 450r620 nm.. Data tabulated ŽTable 1. show some results from field Ži.e., culture, direct agglutination test—DAT, as well as
Fig. 5. Influence of sera dilution. Defined Ref y ve and Ref q ve sera plus two additional sera from field were titrated in serial dilution.
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Table 1 Comparative immunoreactivity of endemic sera from North Africa: results of common tests performed under field conditions are compared with the results of Ld-Ab f-ELISA Sera code
Control sera Ref y Õe sera 1 2 3 4 5 Mean " SD Žnoise level. Ref q Õe sera Code-R Coded test sera 1623 1620 1617 1615 1614 1611 1608 1607 1603 1602 1598 1594 1404 1388 1372 1371 1367 1363 1361 LK61 LK18 MW9710
Culture results
DAT
rK39 dipstick
Absorbance in Ld-Ab f-ELISA, mean " SD
Assessment a
ND ND ND ND ND
ND ND ND ND ND
ND ND ND ND ND
0.078 0.081 0.140 0.186 0.167 0.130 " 0.049
yve yve yve yve yve
qve
ND
ND
2.523 " 0.011
qve
yve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve
BL qve qve qve qve qve qve qve qve qve BL qve qve qve qve BL BL qve qve qve qve ND
yve yve qve qve yve qve qve yve yve qve qve qve qve qve yve yve yve qve yve yve yve ND
0.135 " 0.001 0.336 " 0.014 0.872 " 0.146 1.919 " 0.185 0.741 " 0.001 0.903 " 0.007 2.662 " 0.073 0.487 " 0.004 1.710 " 0.141 2.239 " 0.013 2.628 " 0.127 0.565 " 0.056 0.803 " 0.009 1.304 " 0.008 0.814 " 0.017 0.529 " 0.043 0.718 " 0.018 2.549 " 0.141 0.727 " 0.021 0.810 " 0.007 0.716 " 0.024 0.568 " 0.059
yve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve
BL: border line, neither positive nor negative; ND: not done. a Assessment is based on the cut-off value; see text.
rK39 dipstick test. and the field results were compared with the present Ld-Ab f-ELISA. The cut-off value in the current assay was mean q 3SD of negative Ž n s 5. sera Žmean 0.1304, SD s 0.042., i.e., 0.278 which is rounded off to 0.300. Using an absorbance OD450r620 nm of 0.300 as the cut off, n s 22 test sera from field were categorised as either positive or negative. With the exception of one sample Žserum 1623., all were positive. The sensitiv-
ity, specificity, positive ŽPPV. and negative ŽNPV. predictive values were calculated. The Ld-Ab fELISA has correctly identified n s 21 serum samples as positive. There was no discrepancy between the culture results and the Ld-Ab f-ELISA. With this, we recorded both the sensitivity and specificity as 100%, the PPV as 99.9% and NPV as 95.45%. There was only one sample Žsera 1620 with OD of 0.336. showing low positive value. The resultant
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SrN ratio of these samples suggests that the Ld-Ab f-ELISA is highly sensitive and specific.
4. Discussion ELISA techniques have proven to be extremely useful in the field. Since the original efforts of Hommel et al. Ž1976., there have been a number of reports on serological detection of VL-specific antibodies using a variety of antigens. Most of the earlier works performed on lysate antigen lacked specificity and gave considerable amount of false positives ŽKar, 1995., and on the other hand, sub-unit antigens showed limited scope for active phase of infection ŽZijlstra et al., 1998; Samuel Raj et al., 1999.. Antibody ELISA for VL requires highly specific antigen as the starter reagent in order to capture specific antibody. In the last few years, an increasing number of Leishmania antigens have been characterised; some of which are considered to be species specific proteins such as surface protease gp63, surface glycoproteins gp46 and the lipophosphoglycanassociated protein KMP 11, kinesin, heat shock protein, actin and tubulin ŽSoto et al., 1998.. However, their utility as clinical and epidemiological tools is yet to be determined. Considerable amount of effort is being put to develop appropriate ELISA for field use. The L. chagasi-derived rK39 ŽBurns et al., 1993; Shreffler et al., 1993; Badaro et al., 1996., L. donoÕani surface glycoprotein gp63 ŽBurns et al., 1993; Okong’o-Odera et al., 1995., L donoÕani gene B recombinant product ŽJensen et al., 1999., an MAb ŽD2. which recognizes 70–72-kDa protein of L. donoÕani ŽJaffe and McMahon-Pratt, 1987; Chatterjee et al., 1999., and a 200-kDa protein of L. donoÕani ŽKaul et al., 2000., are prime examples in this direction. L. Immunodiagnosis is traditionally skewed towards parasites of the L donoÕani complex and the visceral syndromes that they engender. In addition, parasites that cause cutaneous diseases are believed to evoke a muted B cell response and low antibody production, in contrast to visceral kala azar. However, the preponderance of non-visceral leishmaniasis cases worldwide and its association with HIV counterbalances the higher morbidity and mortality associated with L. donoÕani infections, and
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calls for a re-examination of the established bias. In that regard, the finding that diagnostic tests based on Ld-ESM are relevant for both visceral and nonvisceral syndromes holds promise. The procedures for producing large quantities of Ld-ESM are standard and inexpensive. At the Walter Reed Institute of Research, the technique of raising Leishmania genus-specific antigens from promastigotes into the protein-free medium, under in vitro condition, was perfected over the years. The antigens have been produced on a large scale with minimum batch-to-batch variation ŽMartin et al., 1998.. This is one of the foremost requirements for the construction of field assays. The SDS-PAGE analyses have indicated multiple immunoreactive antigens in the Ld-ESM preparation. Such novel Leishmania genus-specific antigens formed the basis for the current Ld-Ab f-ELISA. Here, emphasis was given in obtaining a lower or minimal absorbance in wells reacted with Ref y ve sera. The greater the difference in absorbance between Ref y ve and Ref q ve or test samples, the higher the specificity ŽLocarnini et al., 1979; de Savigny and Voller, 1980; Kurstak, 1985.. To achieve this, a number of factors, such as optimum antigen concentration, effective blocking, incubation time, and washing, need to be standardised. Higher SrN ratio seems to be a good indicator for determining the infection level under field conditions. In the current study, we established the optimum antigen concentration, blocking and wash buffer, incubation times, conjugate and serum concentrations needed for the assay. Even though the optimal value for each parameter was incorporated into the final assay, in rare instances, the best value was compromised in order to attain general goals. For example, 0.2 M NaCl was omitted from the final PBSrTween wash buffer despite the fact that it outperformed the competition, to attain increased stability of the reagents. A 50-mgrml antigen concentration was selected due to its ability in discriminating a positive sample from the negative. In our assay, a serum dilution of 1:500 was optimal. This finding is consistent with ELISAs based on the recombinant gp63 ŽReed et al., 1990., and at variance with those based on the recombinant ORFF as antigen; Samuel Raj et al. Ž1999. have used a serum dilution of 1:20 or 1:100. In our experience, lower
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serum dilution Ž1:500. gave higher SrN ratios and this dilution is recommended. A greater specificity and sensitivity is required for field assays to overcome the non-specific reactivity due to polyclonal antibody response. The novel LdESM antigens used here plus the optimised assay conditions did not generate any non-specific reactivity. Moreover, the assay should be able to detect the antibody in infected individuals before the onset of the clinical syndrome. To substantiate this, defined sera samples from field sites are necessary. Analysis of field samples clearly indicated that the Ld-Ab f-ELISA possesses that ability. Those n s 10 sera which were negative in rK39 assay may provide some insight. The current assay on 22 field sera samples showed absolute specificity, very high PPV Ž99.99%. and NPV Ž95.45%.. All positive samples yielded high SrN ratio Ž) 2.0, Kurstak, 1985.. LdAb f-ELISA performed well in a controlled laboratory setting and gave acceptable sensitivity and specificity values. The Ld-Ab f-ELISA embodies all the essential features of the right assay to predict the prevalence of visceral leishmaniasis under field condition. However, the true challenge for the ELISA is against field samples where exposure and infection histories cannot be objectively documented. The lack of a sensitive, Agold standardB diagnostic for leishmaniasis hampers the field evaluation of improved tests, because true positives that are below the level of detection of the Agold standardB are classified as false positives. The impact of this discrepancy is directly proportional to the sensitivity of the new diagnostic test. Unfortunately, polymerase chain reaction ŽPCR. cannot be used to resolve discordant values because of the chronic and occult nature of sub-clinical infections in leishmaniasis, whereby an occult parasite invokes an immune response that includes antibody production, evades detection and compromises the reliability of any antibody detection test. Resolution of this dilemma may emerge with the advent of a sensitive test based on a circulating soluble parasite antigen.
Acknowledgements This work is supported in part by funding received from the United States Department of De-
fence Gulf War Illnesses Research Program ŽPE 0601105D8Z.. References Badaro, R., Reed, S.G., Barral, A., Orge, G., Jones, T.C., 1986. Evaluation of the micro enzyme-linked immunosorbent assay ŽELISA. for antibodies in American visceral leishmaniasis: antigen selection for detection of infection specific responses. Am. J. Trop. Med. Hyg. 35, 72. Badaro, R., Benson, D., Eulalio, M.C. et al., 1996. A cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J. Infect. Dis. 173, 758. Burns Jr., J.M., Shreffler, W.G., Benson, D.R., Ghalib, H.W., Badaro, R., Reed, S.G., 1993. Molecular characterisation of a kinesin-related antigens of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc. Natl. Acad. Sci. U.S.A. 90, 775. Chatterjee, M., Jaffe, C.L., Sundar, S., Basu, D., Sen, S., Mandal, C., 1999. Diagnostic and prognostic potential of a competitive enzyme-linked immunoassay for leishmaniasis in India. Clin. Diagn. Immunol. 6, 550. de Savigny, D., Voller, 1980. The communication of ELISA data from laboratory to clinician. J. Immunoassay 1, 105. Grimaldi Jr., G., Tesh, R.B., 1993. Leishmaniases of the new world; current concepts and implications for future research. Clin. Microbiol. Rev. 6, 230. Hommel, M., Peters, W., Ranque, J. et al., 1976. The micro-ELISA technique in the serodiagnosis of visceral leishmaniasis. Ann. Trop. Med. Parasitol. 72, 213. Jaffe, C.L., McMahon-Pratt, D., 1987. Sero-diagnostic assay for visceral leishmaniasis employing monoclonal antibodies. Trans. R. Soc. Trop. Med. Hyg. 81, 587. Jensen, A.T.R., Gasim, S., Moller, T. et al., 1999. Sero-diagnosis of Leishmania donoÕani infection: assessment of ELISA using recombinant L. donoÕani gene B product ŽrGBP. and a peptide sequence of L. donoÕani ŽGBP.. Trans. R. Soc. Trop. Med. Hyg. 93, 157. Kar, K., 1995. Serodiagnosis of leishmaniasis. Curr. Rev. Microbiol. 21, 123–152. Kaul, P., Malla, N., Kaur, S. et al., 2000. Evaluation of a 200-kDa amastigote-specific antigen of Leishmania donoÕani by enzyme-linked immunosorbent assay ŽELISA. for the diagnosis of visceral leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 94, 173. Kurstak, E., 1985. Progress in enzyme immunoassays; production of reagents, experimental design and interpretation. Bull. WHO 63, 793. Locarnini, S.A., Goulepis, A.G., Stratton, A.M. et al., 1979. Solid-phase enzyme-linked immunosorbent assay for detection of hepatitis a specific immunoglobulin M. J. Clin. Microbiol. 9, 459. Martin, S.K., Thuita-Harun, L., Adoyo-Adoyo, M., Wasunna, K.M., 1998. A diagnostic ELISA for visceral leishmaniasis based on antigen from media conditioned by Leishmania donoÕani promastigotes. Ann. Trop. Med. Parasitol. 92, 571.
G.-H.R. Rajasekariah et al.r Journal of Immunological Methods 252 (2001) 105–119 Okong’o-Odera, E.A., Kurtzhals, J.A.L., Hey, A.S., Kharazmi, A., 1995. Measurement of serum antibodies against native gp63 distinguishes between ongoing and previous L. donoÕani infection. APMIS 101, 642. Reed, S.G., Shreffler, W.G., Burns Jr., J.M. et al., 1990. An improved serological diagnostic procedure for visceral leishmaniasis. Am. J. Trop. Med. Hyg. 43, 632. Ryan, J.R., Smithyman, A.M., Stiteler, J.M., Rowton, E.D., Valli, L., Orndorff, G.R., Thuita-Harun, L., Martin, S.K., 1998. An ELISA based on soluble promastigote antigen detects IgM and IgG antibodies in visceral and cutaneous leishmaniasis. Am. Soc. Trop. Med. Meet. 59, 331. Samuel Raj, V., Ghosh, A., Dole, V.S. et al., 1999. Serodiagnosis of leishmaniasis with recombinant ORFF antigen. Am. J. Trop. Med. Hyg. 61, 482.
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Shreffler, W.G., Burns Jr., J.M., Badaro, R. et al., 1993. Antibody responses of visceral leishmaniasis to gp63, a major surface glycoproteins of Leishmania species. J. Infect. Dis. 167, 426. Soto, M., Requena, J.M., Quijada, L., Alonso, C., 1998. Multicomponent chimeric antigen for sero-diagnosis of canine visceral leishmaniasis. J. Clin. Microbiol. 36, 58. Voller, A., 1993. Immunoassays for tropical parasitic infections. Trans. R. Soc. Trop. Med. Hyg. 87, 497. World Health Organization, 1998. Leishmania and HIV in Gridlock. World Health Organization and Joint United Nations Programme On HIVrAIDS, p. 27. Zijlstra, E.E., Daifalla, N.S., Karger, P.A., Khalil, E.A.G., El-Hassan, A.M., Reed, S.G., Ghalib, H.W., 1998. rK39 enzyme-linked immunosorbent assay for diagnosis of Leishmania donoÕani infection. Clin. Diagn. Lab. Immunol. 5, 717.
Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens G.-Halli R. Rajasekariah a,) , Jeffrey R. Ryan b, Scott R. Hillier a , Lisa P. Yi b, John M. Stiteler b, Liwang Cui b, Anthony M. Smithyman a , Samuel K. Martin b a
b
Cellabs Pty Ltd, P.O. Box 421, BrookÕale, NSW 2100, Australia Walter Reed Army Institute of Research, SilÕer Spring, MD 20910-7500, USA
Received 28 June 2000; received in revised form 24 January 2001; accepted 25 January 2001
Abstract An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donoÕani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen Ž50 mgrml. was coated with PBS–methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref y ve sera and higher absorbance with Ref q ve sera. All steps of the ELISA were performed at room temperature. The SrN ratio, the differential absorbance between the negative sample vs. the test or Ref q ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP ŽMAb-conjugate. outperformed a commercially available anti-human polyclonal antibody conjugate ŽPAb-conjugate.. The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 mgrml Ld-ESM mixed in PBS–methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBSrT, were reacted for 1 h at RT. After washing the plate with PBSrT, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the SrN ratio. A batch of n s 22 endemic sera from North Africa were evaluated and
AbbreÕiations: ELISA, enzyme-linked immunosorbent assay; Ld-Ab f-ELISA, L. donoÕani antibody field ELISA; Ld-ESM, excretory, secretory and metabolic antigens released by L. donoÕani promastigotes during in vitro maintenance in serum-free and protein-free medium; MAb-conjugate, mouse anti-human monoclonal IgG–HRP conjugate; NPV, negative predictive value; PAb-conjugate, goat anti-human polyclonal IgG–HRP conjugate; PBS–methyl glyoxal, phosphate buffer saline supplemented with 1% methyl glyoxal; PBSrT, phosphate buffered saline supplemented with 0.05% Tween 20; PPV, positive predictive value; TMB, tetramethylbenzidine; Ref y ve sera, defined negative sera, not exposed to L. donoÕani infection; Ref q ve sera, defined sera from patients infected with L. donoÕani parasites; SrN Ratio, signal noise ratio, i.e., the differential absorbance between the Ref q ve and Ref y ve sera; RT, room temperature, the ambient temperature used to mimic the field situation; VL, visceral leishmaniasis due to L. donoÕani ) Corresponding author. Tel.: q61-2-9905-0133; fax: q61-2-9905-6426. E-mail address: [email protected] ŽG.-H.R. Rajasekariah.. 0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 3 4 1 - 6
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resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Anti-human HRP conjugate; ELISA; Endemic sera; ESM antigen; Field-ELISA; Leishmania donoÕani; Promastigote; Specific antibody reactivity; Signalrnoise ratio; Visceral leishmaniasis
1. Introduction Human leishmaniasis, caused by the protozoan parasite of the genus Leishmania is endemic in tropical and subtropical regions of some 88 different countries. About 12 million cases are estimated to occur worldwide. Of the two million new cases believed to occur each year, several million subclinical infections go undetected amongst 350 million people at risk ŽWorld Health Organization, 1998; Grimaldi and Tesh, 1993.. Human infection with Leishmania parasites is gaining considerable importance due to its relevance in travel medicine, and increased incidence due to urbanisation, agriculture development, deforestation, irrigation and other ecological factors, which led to the vector multiplication and transmission ŽWorld Health Organization, 1998.. Over the last decade, leishmaniasis has become one of the most frequently observed opportunistic infections among AIDS patients in Southern Europe. Clinical disease can also occur with immunosuppressive drugs. Consequently, our inability to detect occult and subclinical infections prior to immunosuppression or blood donation will carry increasingly significant public health implications. Although direct proof is lacking, the possibility exists that parasitized mononuclear cells circulate in individuals with latent infections and may be transmissible to sand flies and other humans. Hence, rapid user-friendly screening tests are needed to identify these silent infections. ELISA has been used successfully as an effective screening test for other diseases ŽVoller, 1993.. They can play a similar role in leishmaniasis if the appropriate antigen and optimal conditions for specific antibody reaction are defined. Immunodiagnostic tests developed to date for leishmaniasis have utilized whole in vitro cultivated promastigotes, native or recombinant antigens from various stages of the parasite ŽKar, 1995.. Field trials with these products frequently yielded inconsistent results, particularly in non-visceral syndromes and populations with co-en-
demic parasitic diseases. The rapid immuno-absorbent antibody-detection dipstick assay based on a recombinant kinesin protein ŽrK39. has made an impact in detecting acute visceral leishmaniasis Žkala azar. ŽBadaro et al., 1986; Reed et al., 1990; Zijlstra et al., 1998; Samuel Raj et al., 1999.. However, it fails to reliably detect the most predominant Leishmania infection, cutaneous leishmaniasis. More importantly, the performance of any diagnostic test in current use against sub-clinical infections, the most challenging problem of the future, is unknown. We recently showed that soluble exo-antigen released by Leishmania donoÕani promastigotes into a proteinfree medium ŽLd-ESM. is suitable for the development of an ELISA assay for the diagnosis of visceral ŽMartin et al., 1998. and cutaneous leishmaniasis ŽRyan et al., 1998.. In an attempt to further develop such tests, studies were undertaken to define the parameters in an ELISA format that affect the specific binding of Ld-ESM to antibodies present in patient sera. Gram quantities of Ld-ESM can be inexpensively and reproducibly generated in any laboratory with routine culture facilities and its reactivity seems to be genus-specific Žpersonal communication.. In this study, assay conditions have been optimised and the ELISA adapted for general use. Keeping the specificity as well as sensitivity as the main objective, we have devised a Leishmania antibody field ELISA ŽLd-Ab f-ELISA. and evaluated its performance using sera samples from an endemic area.
2. Materials and methods 2.1. L. donoÕani ESM antigen (Ld-ESM) Excretory, secretory and metabolic antigens, released by L. donoÕani promastigotes ŽWHO Refer-
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ence Strain 065. in vitro at 268C for 72 h into serum-free and protein-free medium, were collected and used for ELISA assays. Specific details of these in vitro derived antigens are described elsewhere ŽMartin et al., 1998..
2.3.3. Coating ELISA wells The Ld-ESM antigen preparation with defined protein value was used to coat the ELISA plates. The antigen concentration used is indicated under Experiments and Results section.
2.2. SDS-PAGE analysis
2.3.4. Blocking reagents Gelatin ranging in concentration from 0.1% to 0.4% solution and 0.5% casein were tested. BSA and other routinely employed blocking agents were not tested because of non-specific binding noted in previous experiments.
Ld-ESM in serum-free and protein-free medium was separated by SDS-PAGE using 4–12% Novex gradient gels. The proteins were visualized by colloidal Coomassie blue G-250 and silver staining. The molecular weights of the secreted antigens were estimated with reference to a prestained standard ŽNovex.. 2.3. ELISA assay The assay, in brief, involved immobilizing the antigen to the solid phase ELISA wells for 16–18 h at room temperature. Unbound antigen was removed and the wells were blocked. After blocking, the wells were reacted with Leishmania endemic sera and defined Ref y ve and Ref q ve sera. An HRP-conjugated anti-human antibody was used as the detector antibody. Specific binding of antigens on the solid phase and the specific antibodies present in the test sera were measured indirectly by the binding of HRP-labelled detector antibody, which was further detected by using TMBq H 2 O 2 as a chromogenic substrate. The following parameters specified in the Experiments and Results section were investigated. 2.3.1. ELISA plates Two plates viz., Greiner ŽGreiner Labortecnik ELISA plate Cat a 705071. and Labsystem ŽLabsystem combiplate breakable 8 Cat a 95029400. plates with 12 strips of eight wells each fitted to a plastic frame, were evaluated. 2.3.2. Coating buffers Four different coating buffers were used viz., Ža. 0.2 M carbonaterbicarbonate buffer pH 9.6, Žb. 0.01 M phosphate buffer ŽPO4 . pH 7.2, Žc. blank culture medium Žserum-free. pH 7.2, and Žd. 0.1 M phosphate buffered saline ŽpH 7.2. q 1% methyl glyoxal. They were tested for their ability to immobilize the antigen on to the solid-phase ELISA wells.
2.3.5. Wash buffer PBSrT and PBSrTq 0.2 M NaCl were used as the washing buffers. An automatic plate washer ŽDenley Well Wash 04. with four-cycle wash in each step was used to wash the ELISA wells. 2.3.6. Diluent for sera and conjugate In most of the assays, the wash buffer was used as the diluent for sera as well as the conjugated detector antibody. 2.3.7. Reference and endemic sera samples Sera from individual Ž n s 3., well-characterized, culture-positive case patients were used as reference positives. An equal number of sera from North American natives with no travel history to leishmania endemic areas were used as reference negatives. These sera samples were used as reference samples to optimise the assay. The reference sera were not pooled. They were used as individual data points as detailed under Experiments and Results section. After optimising the assay Ždesignated Ld-Ab f-ELISA., a battery of individual sera samples obtained from endemic areas of North Africa were screened and used to show assay performance in terms of specificity and sensitivity. 2.3.8. Detector antibody Two anti-human IgG conjugates ŽHRP-labelled. were tested. A goat anti-human IgG–HRP ŽKirkegaard and Perry Laboratories—KPL , Gaithersburg, MD., a polyclonal conjugate Ždesignated PAb-conjugate., and a mouse anti-human IgG–HRP ŽCellabs, Brookvale, Australia., Ždesignated MAb-conjugate..
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2.3.9. Substrate TMB solutions A and B ŽKPL. were mixed in equal parts 5–10 min before, and transferred an aliquot of 100 ml to each well as per the guidelines provided by the manufacturer. During the developmental phase of the assay, the colour intensity of Ref q ve wells was monitored Žabsorbance OD 620 nm. and stop solution was added when those wells reached an OD of 0.450. In the final optimised assay, incubation time with substrate was fixed at 25 min. 2.3.10. Stop solution and reading After adding the stop solution Ž1 M phosphoric acid prepared in distilled water., the contents were mixed and the plate was read at dual filter Ž450r620 nm. using a plate reader ŽAnthos LabTec Instruments 2001.. 2.4. Assay interpretation The raw data from the plate Ži.e., absorbance at 450r620 nm. were plotted into histograms and graphs. Žb. A signal-to-noise ratio ŽSrN ratio. was calculated by dividing the mean absorbance Ži.e., absorbance at 450r620 nm. of test sera with the mean absorbance of Ref y ve sera, and plotted. The SrN ratio is directly proportional to the specific antibody reactivity in ELISA ŽLocarnini et al., 1979; de Savigny and Voller, 1980; Kurstak, 1985.. 2.5. Final assay protocol After optimising assay steps, a final assay protocol was developed and followed for evaluating sera samples from endemic areas. In short, wells were coated with 50 mgrml Ld-ESM mixed in PBS– methyl glyoxal buffer overnight, and after removing the antigen, wells were blocked with 0.5% casein for 1 h at RT. Patient serum along with control sera diluted to 1:500 in PBSrT was reacted for 1 h, RT. After washing the plate in PBSrT, wells were reacted with MAb-conjugate at 1:8000 dilution for 40 min at RT; after washing, TMB substrate was added and colour development was allowed to proceed for 25 min, and then stopped with the stop solution. Absorbance was read at dual filter Ž450r620 nm. and the results were analysed.
2.6. EÕaluation of sera samples from endemic areas in Ld-Ab f-ELISA Using this protocol, a total of n s 22 test clinical sera obtained from the endemic areas of North Africa were evaluated along with n s 5 reference control negative sera. An optimised final assay format was used. In short, wells were coated with Ld-ESM diluted to 50 mgrml concentration in PBS plus methyl glyoxal overnight at RT. After removing the unbound antigen solution, wells were blocked with 0.5% casein for 1 h at RT. After removing the blocking buffer, wells were reacted with sera diluted to 1:500; 100 ml per well in PBSrT for 1 h, RT. After washing with PBSrT, the wells were reacted with the detector Žconjugate. antibody ŽMAb-conjugate. for 40 min, RT. After further washing, wells were reacted with the substrate TMB, and the colour absorbance was read at OD 450r620 nm. The mean values of raw data were expressed as histograms and subsequently used to calculate the SrN ratio. 2.7. General Wells coated with antigen were fitted into a 96well frame and blocked with 0.5% casein. They were then stored dry in an aluminium foil bag with silica, sealed and kept at 48C until use. This provided a consistent supply of antigen-coated wells for evaluation. All solutions were freshly prepared for the assay. All analytes were added at 100 mlrwell and incubated at ambient room temperature to mimic the situation in the field.
3. Experiments and results 3.1. SDS-PAGE analysis The Ld-ESM antigens were separated on 4–12% gradient gel and visualized by Coomassie staining. Several major bands with estimated molecular weights of 11, 30, 42, 50 and 161 kDa ŽFig. 1. were visualised along with minor bands. This showed that the Ld-ESM employed in the assay was complex and contained a variety of protein antigens.
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tion in PBSrT, and reacted for 1 h at RT. After washing the wells with PBSrT, MAb-conjugate was used as the detector antibody. Other assay conditions were followed as mentioned above. Absorbancies were read at 450r620 nm. The LabSystems plate did not perform well in this study. There was no discrimination between Ref y ve and Ref q ve sera Ždata not shown.. There was a clear distinction between sera in the Greiner plate. Results in Fig. 2 clearly show that PBS plus methyl glyoxal, phosphate buffer and culture medium, respectively, showed higher reactivity with positive sera and relatively less reactivity with negative sera. SrN ratio was analysed; a higher SrN ratio was seen using sera at a 1:500 sera dilution. Phosphate buffer, culture medium and PBS–glyoxal gave higher SrN ratios. Based on this result, the Greiner plate and PBS plus methyl glyoxal were selected as ELISA plate and coating buffer, respectively. 3.3. Conjugate concentration
Fig. 1. SDS-PAGE analysis. Antigens secreted by L. donoÕani promastigotes were concentrated Ž ;10=. and 10 ml Žlane 1. of the antigen concentrates were separated by SDS-PAGE in a 4–12% gradient gel. Protein bands were visualized by colloidal Coomassie G-250 staining. M indicates pre-stained molecular standards ranging from 3 to 188 kDa.
3.2. Selection of ELISA plates and eÕaluation of coating buffer suitable for immobilization of L. donoÕani ESM antigen Two types of ELISA plates ŽGreiner and LabSystems. were used; Ld-ESM antigens diluted to 50 mgrml concentration in four types of coating buffers were used Ž100 ml or about 5 mg to each well.. Here, excess antigen was used to select the coating solution. Antigen was immobilized to the solid phase wells at ambient room temperature ŽRT. for 18 h. The unbound antigen was removed and the wells were blocked with 0.1% gelatin for 40 min. Based on a pilot assay, two sera samples ŽRef y ve and Ref q ve sera. were used at 1:500 and 1:1000 dilu-
Using the Greiner ELISA plate and PBS plus methyl glyoxal as the coating buffer for Ld-ESM, Ref q ve and Ref y ve sera were used at 1:500 dilution. Two anti-human IgG–HRP conjugates were evaluated at four different dilutions. Data in Fig. 3 show that PAb-conjugate is highly reactive with the Ref y ve sera, indicating a high level of non-specific reaction. On the other hand, the same level of reactivity was observed in the blank and Ref y ve sera sample wells with the MAb-conjugate. The reactivity with MAb-conjugate appeared to be more specific with Ref q ve sera as evidenced by higher SrN ratios. 3.4. Influence of washing solution Any non-specific antibody reactivity would reduce the SrN ratio, and consequently, result in lower assay specificity. Here, the influence of wash solution in reducing the non-specific reactivity was examined. Two wash solutions Ži.e., PBSrT and PBSrT supplemented with 0.2 M NaCl. were used to overcome any non-specific attachment of conjugated antibody. Ref y ve and Ref q ve sera were
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Fig. 2. Comparative reactivity of defined sera Ž1:500 and 1:1000. in antibody detection ELISA: wells were coated with identical concentration of Ld-ESM antigen diluted in different coating buffers. Calculated SrN ratio value is indicated above the bar.
reacted at 1:500 dilutions and washed with two different wash solutions. MAb-conjugate was used at 1:8000 dilutions. Plates were read at 450r620 nm. Reactivity of blanks and Ref y ve sera were lower in wells washed with PBSrT q0.2 M NaCl. The data clearly show that PBSrTq 0.2 M NaCl is more effective in removing non-specific binding Žnearly 45% reduction of non-specific signal 0.283 vs. 0.114. and increasing the SrN ratio from 3.7 to 7.2. De-
spite its higher SrN ratio, 0.2 M NaCl was left out of the final wash buffer because it formed a precipitate on standing. 3.5. Concentration of antigen and effectiÕe blocking Having selected the Greiner plate and PBS plus methyl glyoxal as the coating buffer, optimum levels of antigen and appropriate blocking reagents were
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Fig. 3. Influence of conjugate dilution on the antibody detection. MAb-conjugate showed minimal binding in blank and Ref y ve wells, and high in Ref q ve wells. This has generated relatively high SrN ratio.
investigated. Wells were coated with a series of antigen concentration from 1.25 to 40 mgrml. Two blocking reagents, 0.4% gelatin Žto provide maximum blocking effect with higher concentration; Section 3.2. and 0.5% casein prepared in distilled water were evaluated. Defined Ref q ve and Ref y ve sera, diluted to 1:500 in PBSrT, reacted in the Ag-coated
and blocked wells for 1 h at RT. Two conjugates, PAb- and MAb-conjugates, diluted to 1:4000 in PBSrT, were added to wells and incubated for 30 min at RT. The colour was developed for 25 min by adding the substrate and immediately read after addition of stopping solution. Data in Fig. 4a and b show that the reactivity was higher in blank and Ref y ve
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Fig. 4. Ža. Absorbance values are plotted to show how antigen concentration Ž1.25, 2.5, 5, 10, 20, 40 mgrml coded 1 to 6, respectively. and blocking ŽC for 0.5% casein and G for 0.4% gelatin. influence the antibody detection. Žb. How antigen concentration Ž1.25, 2.5, 5, 10, 20, 40 mgrml coded 1 to 6, respectively. and blocking ŽC for 0.5% casein and G for 0.4% gelatin. influence the antibody detection in ELISA with PAb- and MAb-conjugates. Žc. How antigen concentration and blocking with 0.5% casein influence the antibody detection in sera samples when MAb-conjugate was used in ELISA.
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Fig. 4 Ž continued ..
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Fig. 4 Ž continued ..
wells with PAb-conjugate, thereby reducing the differences between samples that resulted with a low SrN ratio. With the MAb-conjugate, the absorbancies of blank and Ref y ve sera wells were almost equal. There was a pattern in the reactivity relative to the antigen concentration. Wells reacted with the positive sera showed a gradual rise in absorbance dependent upon the antigen concentration. On the whole, MAb-conjugate reactivity was relatively lower in control sample ŽRef y ve and blank. wells. The SrN ratios ŽFig. 4b. were higher with MAb-conjugate when casein was used for blocking. MAb-conjugate with casein blocking generated excellent SrN ratios, at 20 and 40 mgrml antigen level. These data ŽFig. 4a and b. clearly demonstrate the relevance of
Ža. higher antigen concentration Ž) 40 mgrml., Žb. casein blocking; and Žc. MAb-conjugate. 3.6. Determination of optimum antigen concentration Additional testing was needed to optimise the antigen concentration. Here, the antigen was titrated from 20 to 90 mgrml concentration. For remaining ELISA steps, favourable parameters ŽSection 3.5. were selected, i.e., blocking antigen-coated wells with 0.5% casein, 1:500-diluted four sera samples and the MAb-conjugate. Assay was performed by using n s 4 sera samples having high, medium and low antibody reactivity. Data in Fig. 4c show how four sera samples reacted with different antigen con-
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centration. A concentration between 50 and 60 mgrml was found to provide a clear distinction between a positive and a negative sample. Ref q ve sera showed higher reactivity at higher antigen concentration, whereas Sera ŽS-1388. showed a flat reactivity. With this data, a concentration of 50 mgrml antigen was found optimal, which was used in future assays. 3.7. Serum dilution Four sera samples Žone Ref y ve, three Ref q ve sera were from endemic area. were used to optimise the assay following identical assay conditions. Fig. 5 shows the relative kinetics of antibody reactivity at
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different sera dilution. A good discrimination was apparent at 1:500 dilutions. This formed the basis for future assays. 3.8. EÕaluation in Ld-Ab f-ELISA A final optimal form of the ELISA ŽLd-Ab fELISA. was adopted after evaluation of all the parameters affecting the assay. This ELISA was then used to test 22 sera samples obtained from an area in North Africa endemic for VL. Reference n s 5 negative sera were reacted, - 0.2 Žrange 0.078–0.186; av 0.130 at absorbance OD 450r620 nm.. Data tabulated ŽTable 1. show some results from field Ži.e., culture, direct agglutination test—DAT, as well as
Fig. 5. Influence of sera dilution. Defined Ref y ve and Ref q ve sera plus two additional sera from field were titrated in serial dilution.
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Table 1 Comparative immunoreactivity of endemic sera from North Africa: results of common tests performed under field conditions are compared with the results of Ld-Ab f-ELISA Sera code
Control sera Ref y Õe sera 1 2 3 4 5 Mean " SD Žnoise level. Ref q Õe sera Code-R Coded test sera 1623 1620 1617 1615 1614 1611 1608 1607 1603 1602 1598 1594 1404 1388 1372 1371 1367 1363 1361 LK61 LK18 MW9710
Culture results
DAT
rK39 dipstick
Absorbance in Ld-Ab f-ELISA, mean " SD
Assessment a
ND ND ND ND ND
ND ND ND ND ND
ND ND ND ND ND
0.078 0.081 0.140 0.186 0.167 0.130 " 0.049
yve yve yve yve yve
qve
ND
ND
2.523 " 0.011
qve
yve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve
BL qve qve qve qve qve qve qve qve qve BL qve qve qve qve BL BL qve qve qve qve ND
yve yve qve qve yve qve qve yve yve qve qve qve qve qve yve yve yve qve yve yve yve ND
0.135 " 0.001 0.336 " 0.014 0.872 " 0.146 1.919 " 0.185 0.741 " 0.001 0.903 " 0.007 2.662 " 0.073 0.487 " 0.004 1.710 " 0.141 2.239 " 0.013 2.628 " 0.127 0.565 " 0.056 0.803 " 0.009 1.304 " 0.008 0.814 " 0.017 0.529 " 0.043 0.718 " 0.018 2.549 " 0.141 0.727 " 0.021 0.810 " 0.007 0.716 " 0.024 0.568 " 0.059
yve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve qve
BL: border line, neither positive nor negative; ND: not done. a Assessment is based on the cut-off value; see text.
rK39 dipstick test. and the field results were compared with the present Ld-Ab f-ELISA. The cut-off value in the current assay was mean q 3SD of negative Ž n s 5. sera Žmean 0.1304, SD s 0.042., i.e., 0.278 which is rounded off to 0.300. Using an absorbance OD450r620 nm of 0.300 as the cut off, n s 22 test sera from field were categorised as either positive or negative. With the exception of one sample Žserum 1623., all were positive. The sensitiv-
ity, specificity, positive ŽPPV. and negative ŽNPV. predictive values were calculated. The Ld-Ab fELISA has correctly identified n s 21 serum samples as positive. There was no discrepancy between the culture results and the Ld-Ab f-ELISA. With this, we recorded both the sensitivity and specificity as 100%, the PPV as 99.9% and NPV as 95.45%. There was only one sample Žsera 1620 with OD of 0.336. showing low positive value. The resultant
G.-H.R. Rajasekariah et al.r Journal of Immunological Methods 252 (2001) 105–119
SrN ratio of these samples suggests that the Ld-Ab f-ELISA is highly sensitive and specific.
4. Discussion ELISA techniques have proven to be extremely useful in the field. Since the original efforts of Hommel et al. Ž1976., there have been a number of reports on serological detection of VL-specific antibodies using a variety of antigens. Most of the earlier works performed on lysate antigen lacked specificity and gave considerable amount of false positives ŽKar, 1995., and on the other hand, sub-unit antigens showed limited scope for active phase of infection ŽZijlstra et al., 1998; Samuel Raj et al., 1999.. Antibody ELISA for VL requires highly specific antigen as the starter reagent in order to capture specific antibody. In the last few years, an increasing number of Leishmania antigens have been characterised; some of which are considered to be species specific proteins such as surface protease gp63, surface glycoproteins gp46 and the lipophosphoglycanassociated protein KMP 11, kinesin, heat shock protein, actin and tubulin ŽSoto et al., 1998.. However, their utility as clinical and epidemiological tools is yet to be determined. Considerable amount of effort is being put to develop appropriate ELISA for field use. The L. chagasi-derived rK39 ŽBurns et al., 1993; Shreffler et al., 1993; Badaro et al., 1996., L. donoÕani surface glycoprotein gp63 ŽBurns et al., 1993; Okong’o-Odera et al., 1995., L donoÕani gene B recombinant product ŽJensen et al., 1999., an MAb ŽD2. which recognizes 70–72-kDa protein of L. donoÕani ŽJaffe and McMahon-Pratt, 1987; Chatterjee et al., 1999., and a 200-kDa protein of L. donoÕani ŽKaul et al., 2000., are prime examples in this direction. L. Immunodiagnosis is traditionally skewed towards parasites of the L donoÕani complex and the visceral syndromes that they engender. In addition, parasites that cause cutaneous diseases are believed to evoke a muted B cell response and low antibody production, in contrast to visceral kala azar. However, the preponderance of non-visceral leishmaniasis cases worldwide and its association with HIV counterbalances the higher morbidity and mortality associated with L. donoÕani infections, and
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calls for a re-examination of the established bias. In that regard, the finding that diagnostic tests based on Ld-ESM are relevant for both visceral and nonvisceral syndromes holds promise. The procedures for producing large quantities of Ld-ESM are standard and inexpensive. At the Walter Reed Institute of Research, the technique of raising Leishmania genus-specific antigens from promastigotes into the protein-free medium, under in vitro condition, was perfected over the years. The antigens have been produced on a large scale with minimum batch-to-batch variation ŽMartin et al., 1998.. This is one of the foremost requirements for the construction of field assays. The SDS-PAGE analyses have indicated multiple immunoreactive antigens in the Ld-ESM preparation. Such novel Leishmania genus-specific antigens formed the basis for the current Ld-Ab f-ELISA. Here, emphasis was given in obtaining a lower or minimal absorbance in wells reacted with Ref y ve sera. The greater the difference in absorbance between Ref y ve and Ref q ve or test samples, the higher the specificity ŽLocarnini et al., 1979; de Savigny and Voller, 1980; Kurstak, 1985.. To achieve this, a number of factors, such as optimum antigen concentration, effective blocking, incubation time, and washing, need to be standardised. Higher SrN ratio seems to be a good indicator for determining the infection level under field conditions. In the current study, we established the optimum antigen concentration, blocking and wash buffer, incubation times, conjugate and serum concentrations needed for the assay. Even though the optimal value for each parameter was incorporated into the final assay, in rare instances, the best value was compromised in order to attain general goals. For example, 0.2 M NaCl was omitted from the final PBSrTween wash buffer despite the fact that it outperformed the competition, to attain increased stability of the reagents. A 50-mgrml antigen concentration was selected due to its ability in discriminating a positive sample from the negative. In our assay, a serum dilution of 1:500 was optimal. This finding is consistent with ELISAs based on the recombinant gp63 ŽReed et al., 1990., and at variance with those based on the recombinant ORFF as antigen; Samuel Raj et al. Ž1999. have used a serum dilution of 1:20 or 1:100. In our experience, lower
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serum dilution Ž1:500. gave higher SrN ratios and this dilution is recommended. A greater specificity and sensitivity is required for field assays to overcome the non-specific reactivity due to polyclonal antibody response. The novel LdESM antigens used here plus the optimised assay conditions did not generate any non-specific reactivity. Moreover, the assay should be able to detect the antibody in infected individuals before the onset of the clinical syndrome. To substantiate this, defined sera samples from field sites are necessary. Analysis of field samples clearly indicated that the Ld-Ab f-ELISA possesses that ability. Those n s 10 sera which were negative in rK39 assay may provide some insight. The current assay on 22 field sera samples showed absolute specificity, very high PPV Ž99.99%. and NPV Ž95.45%.. All positive samples yielded high SrN ratio Ž) 2.0, Kurstak, 1985.. LdAb f-ELISA performed well in a controlled laboratory setting and gave acceptable sensitivity and specificity values. The Ld-Ab f-ELISA embodies all the essential features of the right assay to predict the prevalence of visceral leishmaniasis under field condition. However, the true challenge for the ELISA is against field samples where exposure and infection histories cannot be objectively documented. The lack of a sensitive, Agold standardB diagnostic for leishmaniasis hampers the field evaluation of improved tests, because true positives that are below the level of detection of the Agold standardB are classified as false positives. The impact of this discrepancy is directly proportional to the sensitivity of the new diagnostic test. Unfortunately, polymerase chain reaction ŽPCR. cannot be used to resolve discordant values because of the chronic and occult nature of sub-clinical infections in leishmaniasis, whereby an occult parasite invokes an immune response that includes antibody production, evades detection and compromises the reliability of any antibody detection test. Resolution of this dilemma may emerge with the advent of a sensitive test based on a circulating soluble parasite antigen.
Acknowledgements This work is supported in part by funding received from the United States Department of De-
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