Peptides and methods for the detection of Leishmaniasis

Abstract

The present invention relates to peptides and methods for the detection of anti-leishmanial antibodies in individuals suspected of infection with the protozoan parasite of the genus Leishmania, especially infection with a South American strain causing the American Tegumentary Leishmaniasis (ATL).

Claims

1. In vitro diagnostic method for the detection of the presence or absence of antibodies indicative of a South American Leishmania strain responsible for the American Tegumentary Leishmaniasis, comprising the steps of: a) contacting H2A-P9 (SEQ. ID. NO. 9) and P2a-P6 (SEQ. ID. NO. 67) with a biological sample for a time and under conditions sufficient to form a first immune complex comprising a first antibody bound to SEQ. ID. NO. 9 and a second immune complex comprising a second antibody bound to SEQ. ID. NO. 67; and b) detecting the presence or absence of the first immune complex and second immune complex formed in a) wherein detecting the presence of the first immune complex and the second immune complex indicates the presence of antibodies indicative of a South American Leishmania strain.

2. In vitro diagnostic method according to claim 1, wherein said South American Leishmania strain is selected in the group consisting of L. braziliensis, L. mexicana, L. major, L. amazonensis or L. infantum.

3. In vitro diagnostic method according to claim 1, wherein said method comprises two additional steps c) and d) consisting in: c) contacting said biological sample with the peptide H2A-P9 of sequence KGGKKGKATPSA (SEQ. ID. NO. 9) for a time and under conditions sufficient to form a third immune complex comprising SEQ. ID. NO. 9 and a third antibody; and d) detecting the presence or absence of the immune complex formed in c), wherein detecting the presence of the third immune complex indicates the presence of antibodies indicative of a South American Leishmania strain.

Description

(1) FIG. 1 represents four graphs that show the reactivity of the CL (open bars) and MCL (solid bars) sera against the synthetic peptides, 20-mer overlapping by five residues (H2A) and ten residues (H2B, H3, H4), covering the entire sequence of the proteins. The OD450 mean values of a pool of two to six sera against each one of the peptides are represented. Sera were used at a dilution of 1:150. The amino acid sequences of the synthetic peptides are provided in the Sequence Listing along with their respective sequence identifiers.

(2) FIG. 2 represent four graphs that show the reactivity of the CL (open bars) and MCL (solid bars) sera against the synthetic peptides, 20-mer overlapping by five residues, covering the entire sequence of LiP2a, LiP2b and P0. The OD450 mean values of a pool of two to six sera against each one of the peptides are represented. Sera were used at a dilution of 1:150. The amino acid sequences of the synthetic peptides are provided in the Sequence Listing along with their respective sequence identifiers.

(3) The present invention will be more readily understood by referring to the following example. This example is illustrative of the wide range of applicability of the present invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any method and material similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.

EXAMPLE 1—IDENTIFICATION OF IMMUNODOMINANT PEPTIDES FROM L. INFANTUM HISTONES AND ARP

(4) I.A. Methods

(5) I.A.1. Human Sera

(6) The serum samples used were divided into three groups:

(7) Group I: Sera from twenty ML and twenty one CL patients were obtained from the bank of sera of the Microbiology Laboratory, Faculty of Biology, San Antonio Abad University of Cuzco, Peru. All samples were reactive to at least one conventional laboratory test for ATL, such as parasite culture, IFA or microscopy. This group was tested against recombinant histones and ARP and two to five hyper reactive sera from each clinical manifestation were pooled for epitope mapping.
Group II: Sera from fifteen CL, ten negative endemic controls (NEC) from Cusco, Peru were obtained from the bank of sera of FP7 RAPSODI Project. Samples from patients with Chagas (7) and Sporotrichosis (6) from Lima, Peru were also included. These samples were individually tested against the selected ten synthetic peptides.
Group III: Eighteen CL and thirteen NEC sera from the bank of sera of the FP7 RAPSODI Project were individually assayed with the two diagnostic candidate epitopes.
I.A.2. Epitope Mapping

(8) For the epitope mapping, a library of overlapping peptides covering the whole sequence of the L. infantum LiH2A, LiH2B, LiH3, LiH4, LiP0, LiP2a and LiP2b was employed The screening was carried out with a pool of sera obtained from ATL patients.

(9) Peptides were synthesized by the simultaneous multiple-peptide solid-phase synthetic method using a polyamide resin and FMOC chemistry [41]. Purity was checked by amino acid analysis and HPLC. For LiH2A [26], LiH3 [27], LiP2a and LiP2b [25], and LiP0 [28] peptides overlapped by 5 amino acids. For LiH2B and LiH4, peptides overlapped by 10 amino acids [24]. A total of 75 peptides were assayed, all peptides were 20-mer long, except H2A-P9 (12-mer), P2a-P7 (16-mer), H3-P12 (19-mer) and H2B-P10 and P2b-P7 (21-mer).

(10) I.A.3. ELISA Measurements

(11) To select both the most reactive sera against recombinant antigens and to carry out the epitope mapping, sera from Group I were tested using the Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA; Becton Dickinson) [27].

(12) The antigen concentration for recombinant proteins was 2 μg/ml and 100 μg/ml for synthetic peptides. Antigen-coated lids were incubated for 1 h with the blocking solution and pools of the most reactive two to six serum samples for each recombinant protein were used for the epitope mapping.

(13) Serum samples were diluted 1:200 for recombinant proteins and 1:150 for synthetic peptides in blocking solution and incubated for 2 h at room temperature with shaking. As secondary antibody, horseradish peroxidase-labelled anti-IgG antibodies (dilution 1:2000, Nordic Immunology) were used. After 1 h of incubation at room temperature, lids were washed and the immune complexes were revealed with orthophenylenediamine as the chromogenic substrate. Absorbance was read at 450 nm.

(14) I.B. Results—B-Cell Epitope Mapping Using Synthetic Peptides

(15) To select the most reactive serum samples from a panel of CL and ML sera corresponding to Group I, recombinant proteins were individually tested (data not shown). It was possible to combine at least 3 sera for the epitope mapping of H2A, H2B, H4, P0 and P2b. For P2a and H3 only two sera were pooled for ML and CL respectively. A collection of synthetic peptides spanning the whole protein sequences, were tested by FAST-ELISA, with exception of LiP0 protein, where positions between 170 and 180 were not available.

(16) Peptides located at the N-terminal region of histones (H2A-P1, H2B-P1, H3-P2, H3-P3, H4-P1), at the C-terminal region of LiH2A (H2A-P9), LiP2a (P2a-P6) and LiP2b (P2b-P7) and at the middle of LiH4 (H4-P7) and LiP0 (P0-P8) demonstrated to be immunodominant (FIGS. 1 to 7). A total of ten linear epitopes were selected like peptide candidates for the determination of their diagnostic value: H2A-P1 (SEQ. ID. No 1), H2A-P9 (SEQ. ID. No 9), H2B-P1 (SEQ. ID. No 10), H3-P2 (SEQ. ID. No 21), H3-P3 (SEQ. ID. No 22), H4-P1 (SEQ. ID. No 32), H4-P7 (SEQ. ID. No 38), P0-P8 (SEQ. ID. No 48), P2a-P6 (SEQ. ID. No 67) and P2b-P7 (SEQ. ID. No 75).

EXAMPLE 2—ASSESSMENT OF THE DIAGNOSTIC VALUE OF PEPTIDE CANDIDATES: INDIVIDUAL ASSAY OF PEPTIDE CANDIDATES

(17) The ten selected peptides were tested with serum samples from Group II to confirm their highly antigenic nature; their potential interest for diagnostic purpose has been evaluated using a first set of diagnostic parameters defined by Inventors. Considering that the diagnostic test needs to be sensitive and with a minimal rate of false positive because false positive will lead to the application of unnecessary treatment which is costly, risky and painful, peptides recognized by more than 70% of CL sera and with less than 15% of cross-reactivity were arbitrarily considered as potential diagnostic candidates.

(18) II.A. Method

(19) Ten immunodominant peptides, selected after the epitope mapping were provided by Bio-Synthesis, Inc. (612 East Main Street Lewisville, Tex. 75057, USA). Peptides were further analyzed using conventional ELISA following the conditions described above and sera from Group II. After the selection of the two diagnostic candidate peptides, they were analyzed using serum samples from Group III. All samples were processed in duplicates.

(20) Cut-off values were defined by the area under the receiver-operating characteristic curve.

(21) Statistical Analysis

(22) The results obtained for each serum sample tested were used to construct 2×2 contingency tables where the sera were further classified according to the disease's presence or absence, as positive or negative.

(23) II.B. Results

(24) Results are presented in Table 1 below.

(25) TABLE-US-00002 Others All no NEGATIVE OTHER PATHOLOGIES pathologies Leishmaniasis CL CONTROLS Chagas Sporotrichosis combined sera Number of samples tested 15 10 7 6 13 23 N. N. N. N. N. N. % positives % positives % positives % positives % positives % positives H2A-P1 60.00 9 10.00 1 42.86 3 66.67 4 53.85 7 34.78 8 H2A-P9 73.33 11 0.00 0 0.00 0 16.87 1 7.69 1 4.35 1 H2B-P1 93.33 14 20.00 2 14.29 1 33.33 2 23.08 3 21.74 5 H3-P2 80.00 12 10.00 1 28.57 2 16.67 1 23.08 3 17.39 4 H3-P3 46.67 7 30.00 3 28.57 2 33.33 2 30.77 4 30.43 7 H4-P1 66.67 10 20.00 2 28.57 2 66.67 4 46.15 6 34.78 8 H4-P7 60.00 9 30.00 3 0.00 0 16.67 1 7.69 1 17.39 4 P0-P8 66.67 10 10.00 1 0.00 0 0.00 0 0.00 0 4.35 1 P2a-P6 80.00 12 0.00 0 14.29 1 0.00 0 7.69 1 4.35 1 P2b-P7 86.67 13 50.00 5 71.43 5 0.00 0 38.46 5 43.48 10

(26) Under these criteria, peptides H2A-P9 and P2a-P6 were selected.

(27) II.C. Confirmation of the Diagnostic Value of Peptides H2A-P9 and P2a-P6

(28) The purpose is here to confirm that the use of the combination of the two selected peptides H2A-P9 and P2a-P6 allows a very reliable diagnostic of American Tegumentary Leishmaniasis.

(29) Diagnostic parameters used to describe the accuracy of the diagnostic test were calculated; they included: accuracy: number and proportion of all the observations in the table which have been classified correctly by the test; kappa: this parameter takes on the value 1 if there is perfect agreement; i.e. the test always correctly predicts the outcome (1 perfect, >0.75 excellent, 0.4-0.75 fair, <0.4 poor). Kappa is a measure of agreement and takes on the value zero if there is no more agreement between test and outcome then can be expected on the basis of chance. Kappa takes on the value 1 if there is perfect agreement; i.e. the test always correctly predicts the outcome. It is considered that Kappa values lower than 0.4 represent poor agreement, values between 0.4 and 0.75 fair to good agreement, and values higher than 0.75 excellent agreement. Negative Kappa indicates a problem in the application of the test. Kappa is dependent not only on the quality of the test, i.e., the inside of the table, but also on the prevalence of the disease in the population in which the test is applied, kappa is also sensitive to the distribution of cases in the table margin. Basically what Kappa shows is that for the same sensitivity and specificity the agreement between test and outcome will decrease with a decreasing prevalence. In Kappa terms a test will perform worse in low prevalence populations.
Mode of Calculation:

(30) $\mathrm{Kappa}=\frac{\left(\mathrm{Observed}\mathrm{agreement}-\mathrm{expected}\mathrm{agreement}\right)}{1-\mathrm{expected}\mathrm{agreement}}$$(\begin{array}{c}\mathrm{see}\\ \underset{\_}{\mathrm{http}\text{:}//\mathrm{epiville}.\mathrm{ccnmtl}.\mathrm{columbia}.\mathrm{edu}\text{/}\mathrm{popup}\text{/}\mathrm{how\_to}\mathrm{\_calculate}\mathrm{\_kappa}.\mathrm{html}}\end{array})$ sensitivity (S): it is the probability that an individual which is diseased is indeed tested as diseased. The level of sensitivity to be applied in a diagnostic test is defined taking into account the seriousness of the “disease” and the cost and availability of the treatment; i.e. the sensitivity of a test is to be high if the “disease” is relatively serious and the “cure” is relatively inexpensive and easily available. specificity (E): it is the probability that an individual which is not diseased is tested as not diseased. The level of specificity is usually high if the disease is not so serious and the “cure” is relatively expensive in money and other terms. There is a tradeoff between specificity and sensitivity, high specificity mostly means low sensitivity, and vice versa. positive predictive value (PPV): indicates how much more likely it is to get a positive test in the diseased as opposed to the non-diseased group. negative predictive value (NPV): indicates how much more likely it is to get a negative test in the non-diseased as opposed to the diseased group. diagnostic odds: this parameter is often used as a measure of the discriminative power of the test; it has the value one if the test does not discriminate between diseased and not diseased individuals. Very high values above one means that a test discriminates well. Values lower than one mean that there is something wrong in the application of the test. error odds: this parameter indicates if the probability of being wrongly classified is highest in the diseased or in the non-diseased group. If the error odds is higher than one the probability is highest in the diseased group (and the specificity of the test is better than the sensitivity), if the value is lower than one the probability of an incorrect classification is highest in the non-diseased group (and the sensitivity of the test is better than the specificity). Youden's J: this parameter is used to study the overall performance of a test; it takes on the value 1 if a diagnostic test discriminates perfectly and without making any mistakes. As a consequence, if the purpose is to minimize the probability of making an error, the Youden's J has to be maximized; that, of course, is of only theoretical importance, such decisions should be taken on the basis of a cost-benefit analysis of treatment as opposed to no treatment. positive predictive accuracy: in a table representative of the population, this parameter gives: 1) the post-test probability, the probability for an individual in the population who tested positive of having the disease; 2) of those who tested positive the fractions who were correctly and who were not-correctly classified. negative predictive accuracy: In a table representative of the population this parameter gives: 1) the post-test probability, the probability for an individual in the population who tested negative of not having the disease; 2) of those who tested negative the fractions who were correctly and who were not-correctly classified.

(31) Based on the extended clinical experience of the American Tegumentary Leishmaniasis of the Inventors, they have selected the parameters, sensitivity and specificity and the two predictive accuracies (accuracy and kappa), that are considered as the most valuable of the indicators.

(32) The threshold value for each peptide is 3 standard deviations of the value obtained when the peptides were tested with true negative control sera.

(33) Sensitivity and specificity give a good view of the quality of the test relatively independent of circumstances. The predictive accuracies give a view of what happens in different practical situations in terms of numbers and proportions tested with correct and incorrect results. Predictive accuracies also give the post test probability of having the disease, an essential piece of information to communicate to the patient together with his or her test result.

(34) Results

(35) To determine the above-mentioned diagnostic parameters of H2A-P9 and P2a-P6, a new panel of serum samples is assayed (group III) and analyzed in combination with the results obtained for group II. All peptides showed high levels of specificity (>80%) but their individual sensitivity were below 80% (Table II). Nonetheless, when the results obtained for each of the three synthetic peptides were analyzed altogether, thirty one out of thirty three of confirmed CL patients were correctly diagnosed. This combination provided 94% sensitivity, 83% specificity, 84% PPV and 94% NPV.

(36) TABLE-US-00003 H2A-P9CT H2A-P9 Ct + P2a-P6 Ct P2a-P6 Cases tested: 69 Cases tested: 69 Cases tested: 69  23 + 1 = 24 tested positive 28 + 2 = 30 tested positive 26 + 1 = 27 tested positive 10 + 35 = 45 tested negative 5 + 34 = 39 tested negative 7 + 35 = 42 tested negative 23 + 10 = 33 were positive 28 + 5 = 33 were positive 26 + 7 = 33 were positive  1 + 35 = 36 were negative 2 + 34 = 36 were negative 1 + 35 = 36 were negative True positives True positives True positives 23/69 = 0.333 28/69 = 0.406 26/69 = 0.377 variance: 0.00322; Std. Err: variance: 0.00349; Std. Err: variance: 0.0034; Std. Err: 0.05675 0.05911 0.05834 95% Cl: 0.191 < Tp < 0.475 95% Cl: 0.258 < Tp < 0.554 95% Cl: 0.231 < Tp < 0.523 Wilson Cl: 0.205 < Tp < 0.491 Wilson Cl: 0.266 < Tp < 0.562 Wilson Cl: 0.241 < Tp < 0.534 True negatives True negatives True negatives 35/69 = 0.507 34/69 = 0.493 35/69 = 0.507 variance: 0.00362; Std. Err: variance: 0.00362; Std. Err: variance: 0.00362; Std. Err: 0.06019 0.06019 0.06019 95% Cl: 0.357 < Tn < 0.658 95% Cl: 0.342 < Tn < 0.643 95% Cl: 0.357 < Tn < 0.658 Wilson Cl: 0.356 < Tn < 0.657 Wilson Cl: 0.343 < Tn < 0.644 Wilson Cl: 0.356 < Tn < 0.657 False positives False positives False positives 1/69 = 0.014 2/69 = 0.029 1/69 = 0.014 variance: 0.00021; Std. Err: variance: 0.00041; Std. Err: variance: 0.00021; Std. Err: 0.01439 0.0202 0.01439 95% Cl: −0.021 < Fp < 0.05 95% Cl: −0.022 < Fp < 0.079 95% Cl: −0.021 < Fp < 0.05 Wilson Cl: 0.001 < Fp < 0.119 Wilson Cl: 0.004 < Fp < 0.141 Wilson Cl: 0.001 < Fp < 0.119 False negatives False negatives False negatives 10/69 = 0.145 5/69 = 0.072 7/69 = 0.101 variance: 0.0018; Std. Err: variance: 0.00097; Std. Err: variance: 0.00132; Std. Err: 0.04238 0.03121 0.03635 95% Cl: 0.039 < Fn < 0.251 95% Cl: −0.006 < Fn < 0.15 95% Cl: 0.011 < Fn < 0.192 Wilson Cl: 0.064 < Fn < 0.289 Wilson Cl: 0.022 < Fn < 0.2 Wilson Cl: 0.037 < Fn < 0.237 Accuracy Accuracy Accuracy (23 + 35)/69 = 0.841 (23 + 35)/69 = 0.899 (26 + 35)/69 = 0.884 variance: 0.00194; Std. Err: variance: 0.00132; Std. Err: variance: 0.00149; Std. Err: 0.04407 0.03635 0.03854 95% Cl: 0.73 < Acc < 0.951 95% Cl: 0.808 < Acc < 0.989 95% Cl: 0.788 < Acc < 0.98 Wilson Cl: 0.695 < Acc < 0.926 Wilson Cl: 0.763 < Acc < 0.963 Wilson Cl: 0.746 < Acc < 0.954 Kappa agreement measure = Kappa agreement measure = Kappa agreement measure = (58 − (2412/69))/ . . . /(69 − (62 − (2394/69))/ . . . /(69 − (61 − (2403/69))/ . . . /(69 − (2412/69)) = 0.677 94/69)) = 0.796 (2403/69)) = 0.766 variance = 0.01348; Std. Err: variance = 0.01438; Std. Err: variance = 0.01405; Std. Err: 0.1161 0.11992 0.11852 95% Cl: 0.387 < Kappa < 0.967 95% Cl: 0.496 < Kappa < 1.096 95% Cl: 0.47 < Kappa < 1.062 Sensitivity Sensitivity Sensitivity 23/33 = 0.697 23/33 = 0.848 26/33 = 0.788 variance: 0.0064; Std. Err: 0.08 variance: 0.0039; Std. Err: variance: 0.00506; Std. Err: 0.06242 0.07116 95% Cl: 0.497 < Sens < 0.897 95% Cl: 0.692 < Sens < 1.005 95% Cl: 0.61 < Sens < 0.966 Wilson Cl: 0.465 < Sens < 0.862 Wilson Cl: 0.622 < Sens Wilson Cl: 0.557 < Sens < 0.921 Specificity Specificity Specificity 35/36 = 0.972 34/36 = 0.944 35/36 = 0.972 variance: 0.00075; Std. Err: variance: 0.00146; Std. Err: variance: 0.00075; Std. Err: 0.02739 0.03818 0.02739 95% Cl: 0.904 < Spec < 1.041 95% Cl: 0.849 < Spec < 1.04 95% Cl: 0.904 < Spec < 1.041 Wilson Cl: 0.904 < Spec < 0.999 Wilson Cl: 0.75 < Spec < 0.993 Wilson Cl: 0.788 < Spec < 0.999 Positive Likelihood Positive Likelihood Positive Likelihood 0.697/(1 − 0.972)) = 25.091 0.848/(1 − 0.944)) = 15.273 0.788/(1 − 0.972)) = 28.364 95% Cl: 2.098 < PL < 300.121 95% Cl: 2.714 < PL < 85.957 95% Cl: 2.386 < PL < 337.128 Negative Likelihood Negative LikeIihood Negative Likelihood (1 − 0.697)/0.972 = 0.312 (1 − 0.848)/0.944 = 0.16 (1 − 0.788)/0.972 = 0.218 95% Cl: 0.16 < NL < 0.605 95% Cl: 0.057 < NL < 0.452 95% Cl: 0.094 < NL < 0.506 Diagnostic Odds Diagnostic Odds Diagnostic Odds 0.697/(1 − 0.697))/(0.972/(1 − 0.848/(1 − 0.848))/(0.944/(1 − 0.788/(1 − 0.788))/(0.972/(1 − 0.972) = 80.5 0.944) = 95.2 0.972) = 130 variance: 7595.175; Std. Err: variance: 6934.368; Std. Err: variance 20447.14286; 87.1503 83.27285 Std. Err: 142.99351 95% Cl: −137.376 < 95% Cl: −112.982 < 95% Cl: −227.484 < Dor < 298.376 Dor < 303.382 Dor < 487.484 Wald's Cl: Wald's Cl: Wald's Cl: 5.375 < Eor < 1205.667 10.689 < Eor < 847.904 8.312 < Eor < 2033.288 Error Odds Error Odds Error Odds 0.697/(1 − 0.697))/(0.972/(1 − 0.848/(1 − 0.848))/(0.944/(1 − 0.788/(1 − 0.788))/(0.972/(1 − 0.972) = 0.066 0.944) = 0.329 0.972) = 0.106 variance: 0.00506; Std. Err: variance: 0.08303; Std. Err: variance: 0.01363; Std. Err: 0.07114 0.28814 0.11673 95% Cl: −0.112 < Eor < 0.244 95% Cl: −0.391 < Eor < 1.05 95% Cl: −0.186 < Eor < 0.398 Wald's Cl: 0.004 < Eor < 0.984 Wald's Cl: 0.037 < Eor < 2.934 Wald's Cl: 0.007 < Eor < 1.66 Youden's J Youden's J Youden's J 0.972 + 0.697 − 1 = 0.669 0.944 + 0.848 − 1 = 0.793 0.972 + 0.788 − 1 = 0.76 variance: 0.00715; Std. Err: variance: 0.00535; Std. Err: variance: 0.00581; Std. Err: 0.08456 0.07317 0.07625 95% Cl: 0.458 < J < 0.881 95% Cl: 0.61 < J < 0.976 95% Cl : 0.569 < J < 0.951 Prevalence = (23 + 10)/69 = 0.478 Prevalence = (28 + 5)/69 = 0.478 Prevalence = (26 + 7)/69 = 0.478 variance: 0.00362; Std. Err: variance: 0.00362; Std. Err: variance: 0.00362; Std. Err: 0.06014 0.06014 0.06014 95% Cl: 0.328 < Pr < 0.629 95% Cl: 0.328 < Pr < 0.629 95% Cl : 0.328 < Pr < 0.629 Wilson Cl: 0.33 < Pr < 0.631 Wilson Cl: 0.33 < Pr < 0.631 Wilson Cl: 0.33 < Pr < 0.631 Positive predictive accuracy Positive predictive accuracy Positive predictive accuracy 23/24 = 0.958 28/30 = 0.933 26/27 = 0.963 variance: 0.00166; Std. Err: variance: 0.00207; Std. Err: variance: 0.00132; Std. Err: 0.04079 0.04554 0.03634 95% Cl: 0.856 < pp < 1.06 95% Cl: 0.819 < pp < 1.047 95% Cl: 0.872 < pp < 1.054 Wilson Cl: 0.705 < pp < 0.999 Wilson Cl: 0.709 < pp < 0.992 Wilson Cl: 0.731 < pp < 0.999 Negative predictive accuracy Negative predictive accuracy Negative predictive accuracy 35/45 = 0.778 34/39 = 0.872 35/42 = 0.833 variance: 0.00384; Std. Err: variance: 0.00287; Std. Err: variance: 0.00331; Std. Err: 0.06197 0.05353 0.05751 95% Cl: 0.623 < np < 0.933 95% Cl: 0.738 < np < 1.006 95% Cl: 0.69 < np < 0.977 Wilson Cl: 0.583 < np < 0.9 Wilson Cl: 0.671 < np < 0.962 Wilson Cl : 0.636 < np < 0.938 Chi squares Chi squares Chi squares (All with 1 degree of freedom): (All with 1 degree of freedom): (All with 1 degree of freedom): Pearson's = 33.989 (p = 0) Pearson's = 44.05 (p = 0) Pearson's = 41.763 (p = 0) LRX = 39.536 (p = 0) LRX = 50.957 (p = 0) LRX = 49.123 (p = 0) Yate's = 31.103 (p = 0) Yate's = 40.883 (p = 0) Yate's = 38.633 (p = 0) M-Haenszel = 33.497 (p = 0) M-Haenszel = 43.412 (p = 0) M-Haenszel = 41.158 (p = 0) Pearson's correlation: 0.70185 Pearson's correlation: 0.79901 Pearson's correlation: 0.77799

EXAMPLE 3—DIAGNOSTIC TEST FOR NEW WORLD LEISHMANIASIS (IgG)

(37) Materials: The ELISA Kit to measure anti-Leishmania IgG contains components required to perform an enzyme-linked immunoassay for the specific measurement of human IgG. Sufficient quantities of reagents are provided to yield 4 plates of 96 wells if there commended assay procedure and recommended storage and handling of materials are followed as specified on this insert.
Control: Human Serum Control (Monoclonal)
Form: Liquid, 1 vial×0.2 mL; Storage: Prolonged store at or below −20° C.
Use: Gently agitate to dissolve completely prior to use. Human Serum
Control is diluted in glycerol [1/2] and the recommended working dilution is 1/150 in Blocking Buffer.
Secondary Antibody: Horseradish Peroxidase Anti-Human IgG
Form: Powder, 1 vial×0.3 mg; Storage: Lyophilized conjugate may be stored at +4° C.; prolonged storage at or below −20° C.
Use: Reconstitute reagent by adding 0.1 ml sterile distilled water; dissolve it and add an equal volume of glycerol (final concentration of 0.5 mg/ml). Divide into small aliquots, freeze and store at or below −20° C. Prior to use, an aliquot is thawed slowly at ambient temperature and used to prepare working dilutions by adding Blocking Buffer at a ratio of 1:200. Do not prepare more diluted Anti-Human IgG solution than is needed. Repeated thawing and freezing should be avoided. Working dilutions should be stored at +4° C., not refrozen, and preferably used the same day.
Chromogen: OPD Tablets from Sigma Cod. P6662
Form: 33 OPD Tablets, 1 mg each; Storage: Store tablets at 2-8° C. Protect from heat, light and moisture. Allow to reach room temperature 10 minutes before use.
Use: Dissolve one tablet in 2.5 ml of 0.05M phosphate-citrate buffer, pH 5.0 to a final concentration of 0.4 mg/ml to prepare the Developing Solution. Add 1 μL of fresh 30% hydrogen peroxide per 2.5 ml of substrate buffer solution, immediately prior to use.
Stop Solution: 3M H2SO4 Merck
Form: Liquid, 1 vial×20 mL; Storage: Store at room temperature.
Use: To stop the reaction, add 50 μL of 3M H2SO4 per well.
Blocking Agent: Casein (Commercial Powder Milk)
Form: Powder, 1 vial×8 g; Storage: Store at 4-8° C.
Use: Dissolve casein in Wash Buffer to obtain a solution of Wash Buffer 3% casein. Stir until all the powder is dissolved. Blocking solution must be preferably used the same day.
Wash Buffer: Wash Buffer Concentrate (30λ)
Form: Liquid, 1 vial×10 mL; Storage: Store at 4-8° C.
Use: Dilute 1 volume of the 10× Wash Buffer concentrate with 9 volumes of deionized water to obtain the Working Wash Buffer 1× (ie. 1 mL may be diluted up to 10 mL).
Plate:
Form: One microplate with selected peptide Coated eight-stripwells for each antigen (A, B, C); Storage: Store at 4-8° C.
Use: To perform the analysis of one problem sample, take one well from each microplate and store the remaining wells.
Protocol
Additional Materials Required: pipettes and timer; microplate reader with a detector that can measure absorbance at 450 nm or color scale; 1 L graduated cylinder; plate washer or wash bottle; polypropylene tubes for standards and sample dilutions, if needed.
Principle of the Assay
This kit is an indirect type ELISA using a horseradish peroxidase detection system. A microtiter plate coated with specific antigens which are recognized by specific human anti-Leishmania IgG. The antigens in turn bind to the human IgG. The anti-Leishmania IgG is then labeled by a horseradish-peroxidase anti-human IgG reagent. The detection signal is then generated in proportion to the amount of human antibody.
Assay Procedure
Prior to use, allow the kit to warm to room temperature. Remove the number of stripwells according to your design plan.
Sample Dilution Procedure: tested samples and Human Control Serum should be diluted at 1:150 in Blocking Buffer.
Wash the wells 3 times with 100 μL Wash Buffer for 5 minutes before using.
1. Block the wells with 100 μL of Blocking Solution for 2 hours.
2. Discard the Blocking Buffer by tapping.
3. Add 100 μl of the appropriate human serum sample dilution to each well. For the positive control wells, add 100 μl of diluted Human Serum Control serum sample. All serum samples should be diluted in Blocking Buffer. Incubate at room temperature for 2 hours.
4. Remove contents inverting the plate into the sink. Add 200 μL of Working Wash Buffer 1× into each well and remove by inverting the plate into the sink and tap on absorbent paper to remove access liquid. Repeat washes, three times, five minutes each wash.
5. Add 100 μL of diluted Horseradish Peroxidase Anti-Human IgG conjugate solution into each well. Incubate at room temperature for 1 hour.
6. Remove contents inverting the plate into the sink. Repeat washes as in Step 4, three times, five minutes each wash.
7. Add 100 μl of the Developing Solution into each well. Incubate at room temperature for 10-15 min.
8. Quickly add 50 μL of Stop Solution into each well and shake for a few seconds. A dramatic color change from yellow to dark orange should occur.
9. Measure absorbance at 490 nm within 1 hour of adding the Stop Solution.
Verify the assay: The assay can be considered valid if the protocol has been followed correctly; the Positive Control optical density is greater than 0.8 and the ratio of the Cut-off Calibrator to the Negative Control is greater than 2.0.
Interpret the Results:
Score results with an optical density greater than Cut-off 0.3 as positive.
Score results with an optical density less than Cut-off 0.1 as negative.
Results between these values, that is 0.1<Cut-off<0.3, are equivocal and should be repeated to confirm the status.

(38) If the result is uncertain, additional steps are added to the method to increase accuracy and sensitivity:

(39) c) contacting said biological sample with one the peptide H2A-P9 of sequence KGGKKGKATPSA (SEQ. ID. No 9) and

(40) d) detecting the presence or absence of the immune complex formed in c).

REFERENCES

(41) 1. Braz R F, Nascimento E T, Martins D R, et al. The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection. Am J Trop Med Hyg 2002; 67:344-8 2. Reithinger R, Dujardin J C, Louzir H, Pirmez C, Alexander B, Brooker S. Cutaneous leishmaniasis. Lancet Infect Dis. 2007 September; 7(9):581-96. Review. PMID: 17714672 3. Alvar J, Croft S and Olliaro P. Chemotherapy in the treatment and control of leishmaniasis. Adv Parasitol 2006; 61:223-74 4. Boggild A K, Ramos A P, Espinosa D, et al. Clinical and demographic stratification of test performance: a pooled analysis of five laboratory diagnostic methods for American cutaneous leishmaniasis. Am J Trop Med Hyg 2010; 83:345-50 5. Cuba C A C. Diagnostico Parasitologico de la Leishmaniasis Tegumentaria Americana. Revista Peruana de Medicina Experimental y Salud Publica 2000:39-42 6. 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Diagnostic performance indices for immunofluorescent tests and enzyme immunoassays of leishmaniasis sera from northern and north-eastern Brazil. Bull World Health Organ 1990; 68:39-43 15. Guimaraes M C, Celeste B J, Franco E L, Cucé L C and Belda W J. Evaluation of serological diagnostic indices for mucocutaneous leishmaniasis: immunofluorescence tests and enzyme-linked immunoassays for IgG, IgM and IgA antibodies. Bull World Health Organ 1989; 67:643-8 16. Carmelo E, Martinez E, Gonzales A C, et al. Antigenicity of Leishmania braziliensis Histone H1 during Cutaneous Leishmaniasis: Localization of Antigenic Determinants Clin Diagn Lab Immunol 2002; 9:808-11 17. Webb J R, Campos-Neto A, Ovendale P J, et al. Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family. Infect Immun 1998; 66:3279-89 18. Montoya Y, Leon C, Talledo M, et al. 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(42) TABLE-US-00004 SEQUENCES LISTING SEQ. ID. No 1 H2A P1 MATPRSAKKAVRKSGSKSAK SEQ. ID. No 2 H2A P2 SKSAKCGLIFPVGRVGGMMR SEQ. ID. No 3 H2A P3 GGMM RRGQYARRIGASGAVY SEQ. ID. No 4 H2A P4 SGAVYLAAVLEYLTAELLEL SEQ. ID. No 5 H2A P5 ELLELSVKAAAQSGKKRCRL SEQ. ID. No 6 H2A P6 KRCRLNPRTVMLAARHDDDI SEQ. ID. No 7 H2A P7 HDDDIGTLLKNVTLSHSGVV SEQ. ID. No 8 H2A P8 HSGVVPNISKAMAKKKGGKK SEQ. ID. No 9 H2A P9 KGGKKGKATPSA SEQ. ID. No 10 H2B P1 MASSRSAPRKASHAHKSHRK SEQ. ID. No 11 H2B P2 ASHAHKSHRKPKRSWNVYVG SEQ. ID. No 12 H2B P3 PKRSWNVYVGRSLKAINAQM SEQ. ID. No 13 H2B P4 RSLKAINAQMSMSHRTMSIV SEQ. ID. No 14 H2B P5 SMSHRTMSIVNSYVNDVMER SEQ. ID. No 15 H2B P6 NSYVNDVMERICMEAASIVR SEQ. ID. No 16 H2B P7 ICMEAASIVRANKKRTLGAR SEQ. ID. No 17 H2B P8 ANKKRTLGAREVQTAVRIVL SEQ. ID. No 18 H2B P9 EVQTAVRIVLPAELAKHAMA SEQ. ID. No 19 H2B P10 PAELAKHAMAEGTKAVSSASA SEQ. ID. No 20 H3 P1 MSRTKETARAKRTITSKKSK SEQ. ID. No 21 H3 P2 KRTITSKKSKKAPSGASGVK SEQ. ID. No 22 H3 P3 KAPSGASGVKRSHRRWRPGT SEQ. ID. No 23 H3 P4 RSHRRWRPGTCAIREIRKFQ SEQ. ID. No 24 H3 P5 CAIREIRKFQKSTSLLIQCA SEQ. ID. No 25 H3 P6 KSTSLLIQCAPFQRLVRGVE SEQ. ID. No 26 H3 P7 PFQRLVRGVERQKEGLRFQS SEQ. ID. No 27 H3 P8 RQKEGLRFQSSAIMALQEAT SEQ. ID. No 28 H3 P9 SAIMALQEATEAYIVSLMAD SEQ. ID. No 29 H3 P10 EAYIVSLMADTNLACIHAKR SEQ. ID. No 30 H3 P11 TNLACIHAKRVTIQPKDIQL SEQ. ID. No 31 H3 P12 VTIQPKDIQLALRLRGERH SEQ. ID. No 32 H4 P1 MAKGKRSTDAKGSQRRQKKV SEQ. ID. No 33 H4 P2 KGSQRRQKKVLRDNIRGITR SEQ. ID. No 34 H4 P3 LRDNIRGITRGCVRRMARRG SEQ. ID. No 35 H4 P4 GCVRRMARRGGVKRISTEVY SEQ. ID. No 36 H4 P5 GVKRISTEVYEEVRRVLKAY SEQ. ID. No 37 H4 P6 EEVRRVLKAYVEDIVRCSTA SEQ. ID. No 38 H4 P7 VEDIVRCSTAYTEYARKKTV SEQ. ID. No 39 H4 P8 YTEYARKKTVTACDVVTALR SEQ. ID. No 40 H4 P9 TACDVVTALRKQGHILYGYA SEQ. ID. No 41 P0 P1 MPSITTAKREYEERLVDCLT SEQ. ID. No 42 P0 P2 VDCLTKYSCVLFVGMDNVRS SEQ. ID. No 43 P0 P3 DNVRSQQVHDVGRALRAKAE SEQ. ID. No 44 P0 P4 RAKAEFMMGKKTLQGKIVEK SEQ. ID. No 45 P0 P5 KIVEKRAQAKDASPEAKHFN SEQ. ID. No 46 P0 P6 AKHFNDQCEEYNLVTRNTGL SEQ. ID. No 47 P0 P7 RNTGLIFTNNAVQEITSVLD SEQ. ID. No 48 P0 P8 TSVLDAHRVKRAARVGAISP SEQ. ID. No 49 P0 P9 GAISPCDVIVAAGSTGMEPT SEQ. ID. No 50 P0 P10 GMEPTQTSFFQALMIATKIA SEQ. ID. No 51 P0 P11 ATKIAKGMVEIVTEKKVLSV SEQ. ID. No 52 P0 P12 LLQKLNISPFYYQVNVLSVW SEQ. ID. No 53 P0 P13 VLSVWDRGDLFTREDLMMTE SEQ. ID. No 54 P0 P14 LMMTEDMVEKMLMEGLSNVA SEQ. ID. No 55 P0 P15 LSNVAAMALGAGIPTSSTIG SEQ. ID. No 56 P0 P16 SSTIGPMLVDAFKNLLAVSV SEQ. ID. No 57 P0 P17 LAVSVATSYEFEEHNGKELR SEQ. ID. No 58 P0 P18 GKELREAAIMGLLAGSCSAA SEQ. ID. No 59 P0 P19 SCSAAAEPAAAAPAAPSAAA SEQ. ID. No 60 P0 P20 PSAAAKEEPEESDEDDFGMG SEQ. ID. No 61 P0 P21 AAKEEPEESDEDDFGMGGLF SEQ. ID. No 62 P2a P1 MQYLAAYALVALSGKTPSKA SEQ. ID. No 63 P2a P2 TPSKADVQAVLKAAGVAVDA SEQ. ID. No 64 P2a P3 VAVDASRVDAVFQEVEGKSF SEQ. ID. No 65 P2a P4 EGKSFDALVAEGRTKLVGSG SEQ. ID. No 66 P2a P5 LVGSGSAAPAGAVSTAGAGA SEQ. ID. No 67 P2a P6 AGAGAGAVAEAKKEEPEEEE SEQ. ID. No 68 P2a P7 PEEEEADDDMGFGLFD SEQ. ID. No 69 P2b P1 MSTKYLAAYALASLSKASPS SEQ. ID. No 70 P2b P2 KASPSQADVEAICKAVHIDV SEQ. ID. No 71 P2b P3 VHIDVDQATLAFVMESVTGR SEQ. ID. No 72 P2b P4 SVTGRDVATLIAEGAAKMSA SEQ. ID. No 73 P2b P5 AKMSAMPAASSGAAAGVTAS SEQ. ID. No 74 P2b P6 GVTASAAGDAAPAAAAAKKD SEQ. ID. No 75 P2b P7 AAKKDEPEEEADDDMGFGLFD