CHAPTER 4 RESULTS 42

Transcription

1 CHAPTER 4 RESULTS 42

2 4.1 Introduction This chapter is divided in two main sections. The first section deals with results obtained for the point-of-care tests, which encompass results of optimization final performance testing of the immunofiltration (flow-through) and immunochromatographic (lateral flow) dual test for syphilis. The application and performance of the nontreponemal micelle and the treponemal Tp r-17 antigens to the nitrocellulose membrane of both the immunofiltration and immunochromatographic devices were measured by treating the devices with a panel of positive and negative syphilitic sera. The second section deals with the modification of the cardiolipin molecule by the process of oxidation of the double bonds of the fatty acid moieties and their conversion to carboxyl groups. The resulting compounds can readily be attached to solid support media such as latex beads as in the chemiluminescence assay and micro plates as in the ELISA test. Both these platforms are laboratory based systems, offering the advantage of high throughput where large numbers of samples are simultaneously tested for treponemal and nontreponemal antibodies. Another advantage is numerical reading of results, which avoids subjectivity in interpretation. 4.2 Determination of suitability of sample panel A representative serum sample panel (described in section 3.2), collected from the WHO Collaborative Center for Syphilis Serology were characterized by testing by the quantitative rapid plasma reagin (RPR) assay and the distribution and number of RPR positive samples is shown in (Figure 4.1). 121

3 Number of samples RPR titer Figure 4.1 Distribution of the number of RPR positive samples from a representative WHO panel by the quantitative test. (R represent RPR titer of individual serum). 4.3 Evaluation of micelles for use in poit-of-care tests Lipoidal micelle antigen In order to determine the sensitivity of the lipoidal micelle antigens, prepared either by differential centrifugation and filtration (3.4.2) or by sonication and filtration (3.4.3), preparations were tested quantitatively with reference serum samples following the method described in the USR test given in Appendix 3 Table 4.1 Comparison of the sensitivity of the micelle antigen preparations by (a) differential centrifugation and filtration and (b) Sonication and filtration procedure a) Serum Dilutions Micelle Undiluted Concentration (%) Serum No. R R R R W N N 2.0% L R R R R W(-) N N 1.0% R R R(-) W N N N 0.5% Serum No. N N N N N N N 2.0% U1465 N N N N N N N 1.0% N N N N N N N 0.5% 122

4 b) Serum Dilutions Micelle Undiluted Concentration (%) Serum No. R R R R W N N 2.0% L R R R R W(-) N N 1.0% R R R(-) W N N N 0.5% Serum No. N N N N N N N 2.0% U1465 N N N N N N N 1.0% N N N N N N N 0.5% R(-) = large and medium clumps. W = Medium clumps. W(-) = Medium and small clumps. As can be seen in by comparing the results presented in (Table 4.1a andb), micelle antigens prepared by differential centrifugation and filtration and by sonication and filtration procedures indicated that these preparations were essentially identical in sensitivity and could therefore both be used as the nontreponemal antigen for direct attachment to the nitrocellulose membrane in the immuno-filtration and immuno-chromatographic devices. The direct attachment of the micelle to the nitrocellulose membrane is possible because the micelles from these preparations are 5 µm in size, which increases the contact surface area and allows the negative charge of the cardiolipin head group to react with the positive charge of the nitrocellulose ester present on the nitrocellulose membrane. However, the concept of attaching Fab (1) fragments from hyperimmune syphilitic sera to the surface of the micelles was explored in order to improve the attachment of micelles to the nitrocellulose membrane. 4.4 Separation of papain digested IgG fragments from hyperimmune syphilitic human sera by Protein A column and the effectiveness of Fab (1) fragments In order to separate the fractions of digested IgG from hyperimmune serum, the papain digest was applied to the protein A column as described in section and the fractions collected. The first and second peaks containing the Fab (1) and Fc fragments respectively (Figure 4.2) were collected and concentrated. The purity of the preparations was 123

5 subsequently confirmed by SDS- Page gel electrophoresis (Figure 4.3) as described in Appendix 2. Peak 2 Fc Peak 1 Fab Figure 4.2 Fab (1) and Fc fragments after papain digestion using protein A column. Std Std 216, ,000 78,000 45,700 32,500 32,500 18,400 Figure 4.3 Gel electrophoresis pattern after protein A and enzymatic digestion of IgG. All lanes were applied at a concentration of 2 mg/ml. (1) Ammonium sulfate globulin fraction. (2) Protein A column peak 1. (3) Protein A column peak 2. (4) IgG from Protein A before papain digestion. (5) IgG from protein A column after papain digestion. (6) IgG peak 1 after papain digestion (Fab 1 ) fraction. (7) IgG peak 2 after papain digestion (Fc) fraction. 124

6 4.4.1 Determination of the effectiveness of the papain digestion for the reparation of the Fab (1) fragments The efficacy of the papain digestion for preparation of the Fab (1) fragments was determined by a simple immunochromatographic lateral flow test as described in section Results of the experiments indicated that the Protein A gold conjugate detected the Fc fragments and IgG fractions but not the Fab (1) fragments (Figure 4.4). Because of the monovalent nature of the Fab (1) fragments structure, they were able to attach to the micelle antigen without agglutinating the micelles. Figure 4.4 (1) (2) (3) (4) (1) Duplicate nitrocellulose strip spotted with Fab (1) fragments, showing no reaction with Protein A gold conjugate. (2) Duplicate Fc fragments showing reaction with the conjugate. (3) Duplicate IgG fraction showing reaction with the conjugate. (4) Procedural control with no antigen showing no reaction. 125

7 Table 4.2 Sensitivity of the micelle antigen coated with Fab (1) fragments from enzymatic separation. Micelle from procedure % concentration Serum No. Antibody titer Fab(1) Undiluted Concent. R R R R W(-) N N 250 µg/ml L R R R R(-) W N N 125 µg/ml R R R R(-) W N N 62.5 µg/ml R R R R(-) W N N µg/ml Micelle from procedure % concentration R R R R W(-) N N 250 µg/ml L R R R R W(-) N N 125 µg/ml R R R R W N N 62.5 µg/ml R R R R W N N µg/ml Micelle from procedure % concentration L R R W W(-) N N N 250 µg/ml R R R W(-) N N N 125 µg/ml R R R W(-) N N N 62.5 µg/ml R R R W(-) N N N µg/ml Micelle from procedure % concentration L R R R R W(-) N N 250 µg/ml R R R R(-) W(-) N N 125 µg/ml R R R R(-) W N N 62.5 µg/ml R R R R(-) W N N µg/ml Micelle from procedure % concentration L R R R R W(-) N N 250 µg/ml R R R R W(-) N N 125 µg/ml R R R R W N N 62.5 µg/ml R R R R W N N µg/ml Micelle from procedure % concentration L R R R W(-) N N N 250 µg/ml R R R W(-) N N N 125 µg/ml R R R W(-) N N N 62.5 µg/ml R R R W(-) N N N µg/ml R= Large clumps, R (-) = large and medium W= Medium, W (-) = Medium and small. N= Negative 126

8 The concentrations of the Fab (1) fragments coating the surface of the micelle prepared by differential centrifugation and by filtration and sonication and filtration did not interfere with the available head groups of the cardiolipin antigen, at various micelle concentrations (0.5-2%) and Fab (1) concentrations ranging from µg/ml to 250 µg/ml (Table 4.2). There were sufficient cardiolipin antigenic sites on the surface of the micelles to allow the reference syphilitic sera to react with the coated micelle. Because of the protein nature of the Fab (1) fragments coating the outer layer of the micelles, they will readily be attached to the nitrocellulose membrane of the rapid test devices. In this case the Fab (1) fragments serves as a bridge between the micelle antigen and the nitrocellulose membrane. It was then established that any of the three micelle preparations were suitable for the flow-through and lateral flow devices. However, taking into account commercial considerations while using these preparations, it would merit in choosing the least expensive one for the application to the nitrocellulose membrane as those in the preparations by differential centrifugation and filtration and sonication and filtration 4.5 Determination of the use of goat anti-human IgG and IgM for preparation of immunogold conjugates This section describes the results of the assays used to determine the usefulness of the goat serum obtained from goat immunized with human IgG and IgM. In addition the fractionation and purification of the goat anti-human immunoglobulins obtained and attachment to immunogold for use in the POC devices Capillary precipitin test results This test was performed in order to determine the response to the immunization protocol from section (3.6.1) and the suitability of goat serum for affinity purification. A capillary precipitin test tube procedure (3.6.2) was performed on whole serum samples of the immunized goats # 548 and # 544 diluted to 2 mg/ml in PBS. Commercial preparations of (Pierce) affinity purified goat anti-human IgG (2 mg/ml) and goat anti-human IgM (1.5 mg/ml) and (Jackson) human IgG (2 mg/ml) and IgM (2 mg/ml) purified fractions were used as reference controls (Table 4.3). 127

9 The capillary precipitin test results indicated that serum of goat # 548 was suitable for affinity purification while the serum from goat # 544 required further immunizations in order to achieve a sufficient degree of reactivity. Both goat sera were later fractionated using ammonium sulphate. Table 4.3 Comparison of capillary precipitin test of immunized goats with commercial sera. Antiserum Antigen Degree Precipitin Time Goat Anti-human IgG (Pierce) Human IgG (Jackson) seconds Goat Anti-human IgG (Pierce) Human IgM (Jackson) N N Goat # 548 Human IgG (Jackson) seconds Goat # 548 Human IgM (Jackson) 2+ 5 minutes Goat Anti-human IgM (Pierce) Human IgM (Jackson) seconds Goat Anti-human IgM (Pierce) Human IgG (Jackson) N N Goat # 544 Human IgG (Jackson) minutes Goat # 544 Human IgM (Jackson) ± 22 minutes N = No precipitin test reaction; 1+, 2+, 4+ =Degree of reaction Affinity purification of goat anti-human serum Following the ammonium sulfate fractionation described in section , 84 ml of the globulin fraction from goat # 548 and 93 ml from goat # 544 were applied to a protein G column as per the procedures described in 3.6.3, resulting in 20 ml and 24 ml respectively of Peak I (IgM plus other immunoglobulins) and 50 ml and 60 ml, respectively of Peak II (IgG) (Figure 4.5). The peak 1containing IgM and other immunoglobulins were discarded. The peak 2 from the protein G columns were concentrated at 34 mg/ml or 340 mg total for goat # 548 and 38 mg/ml or 456 mg total for goat #

10 Peak 1 Peak 2 Figure 4.5 Affinity purification of IgG from globulin fraction of hyper immune goat serum using a protein G column. Peak 1 IgM and other immunoglobulins. Peak 2 IgG. The purity of the preparations was confirmed using the Bio-Rad, Experion Automated Electrophoresis Station (Figure 4.6). Figure 4.6 The effectiveness of goat anti-human serum separation using protein G column affinity purification (L) Molecular weight standard (1) IgG standard (2) BSA standard (3) Goat # 548 globulin fraction (4) Goat # 548 following IgG protein G purification (5) Goat # 544 globulin fraction (6) Goat # 544 following IgG protein G purification (7) PBS blank. 129

11 Four µl of 20 µg/ml of each of the goat globulin fractions, after protein G purification, molecular weight standards, IgG and BSA standards were applied to each of the corresponding lanes of a Bio-Rad, Experion electrophoresis unit. The results obtained confirmed those of the capillary precipitin test (Table 4.3) indicating that goat #548 had a higher IgG reactivity than goat #544 after purification. This was demonstrated in the affinity purification density of the electrophoretic patterns obtained in lane (3) for goat # 548 as opposed to lanes (5) of goat # Application of goat anti human IgG to human IgG affinity column The anti-human IgG from peak II, of the protein G column was further purified by applying 5 ml of goat #548 to the human IgG affinity column as described in sections and The specific goat anti-human IgG was retained in the column and subsequently eluted using a 100 mm glycine, with 300 mm NaCl ph 2.7. The purity of the preparation was confirmed by electrophoresis as before (Figure 4.7) Figure 4.7 The effectiveness of goat anti-human IgG serum separation using human IgG affinity column (L) Molecular weight reference standard (1) IgG standard (2) BSA standard (3) Peak II of Goat #548 human IgG affinity purification (4) Peak I of goat #548 (5) Peak II of goat #544 human IgG affinity column (6) peak I of goat #544 (7) PBS blank. The results obtained with the specific affinity purified goat anti-human IgG fractions also confirmed the results of the capillary precipitin tests. 130

12 4.5.4 Application of goat anti human IgG to human IgM affinity column Five ml of goat #548 anti-human IgG was subsequently applied to a human IgM affinity column as described in section (3.6.6). The first peak containing nonspecific goat IgG was discarded. The specific goat anti-human IgM was retained in the column and was eluted using a 100 mm glycine, with 300 mm NaCl (ph 2.7). L Figure 4.8 The effectiveness of goat anti-human IgG serum separation using human IgM affinity column (L) Molecular weight reference standard (1) IgG standard (2) Peak II of goat #548 human IgM affinity Purification (3) Peak II of goat #544 human IgM affinity column (4) PBS blank. A similar pattern of anti-human IgM activity was obtained with goats #548 and # 544 (Figure 4.8). The results of the electrophoretic bands suggest that the concentration of specific anti-human IgM in the two goats was similar and thus they could be used in making a goat anti-human IgM colloidal gold conjugate. However, since the anti-human IgG activity of goat #548 was greater than that of goat #544, the selection of goat # 548 was more appropriate when making an anti-human IgG/IgM colloidal gold conjugate. 131

13 4.6 Determination of optimum protein content and ph for the goat antihuman IgG, IgM (affinity purified serums) and protein A/G for coupling to colloidal gold The optimum protein content and ph of affinity purified goat anti-human IgG and IgM (goat #548) and protein A/G coupled with colloidal gold were determined by applying checker board titration as described in section Table 4.4 Checker board titration for optimum protein and ph content for goat antihuman IgG. Goat-anti Column 1 Column 2 Column 3 Column 4 Column 5 IgG ph 6.0 ph 7.0 ph 8.0 ph 9.0 ph 10.0 A* R R R R R 10.0 µg B R R R R R 5.0 µg C R R R R R 2.5 µg D R R R R R 1.25 µg E ± R R R R µg F B ± ± R R µg G B B B ± ± µg H B B B B B µg human *Goat Anti-Human IgG (1 mg/ml) (or 10 µg) R = Dark pink colloidal gold particles fully coated with affinity purified goat anti-human IgG. ± = Dark purple colloidal gold particles partially coated with affinity purified goat antihuman IgG. B = Dark blue metallic gold particles not coated with affinity purified goat anti-human IgG. 132

14 A concentration of µg per 100 µl or 3.12 µg/ml at ph 9.0 was suitable for conjugation to colloidal gold. Table 4.5 Checker board titration for optimum protein content and ph for the goat anti-human IgM. Column 1 Column 2 Column 3 Column 4 Column 5 Goat-anti human IgM ph 6.0 ph 7.0 ph 8.0 ph 9.0 ph 10.0 A* R R R R R 10.0 µg B R R R R R 5.0 µg C R R R R R 2.5 µg D R R R R R 1.25 µg E ± ± R R R µg F B B ± R R µg G B B B ± ± µg H B B B B B µg *Goat Anti-Human IgM (1 mg/ml) (or 10 µg) R = Dark pink colloidal gold particles fully coated with affinity purified goat anti-human IgM. ± = Dark purple colloidal gold particles partially coated with affinity purified goat antihuman IgM. B = Dark blue metallic gold particles not coated with affinity purified goat anti-human IgM. A concentration of µg per 100 µl or 3.12 µg/ml at ph 9.0 was suitable for conjugation to colloidal gold. 133

15 Table: 4.6 Checker board titration for optimum protein content and ph for Protein A/G for colloidal gold coupling reagent. Protein Column 1 Column 2 Column 3 Column 4 Column 5 A/G ph 6.0 ph 7.0 ph 8.0 ph 9.0 ph 10.0 A* R R R R R 10.0 µg B R R R R R 5.0 µg C R R R R R 2.5 µg D R R R R R 1.25 µg E R R R R R µg F ± ± R R B µg G B B ± ± ± µg H B B B B B µg *Protein A/G (1 mg/ml) (or 10 µg) R = Dark pink colloidal gold particles fully coated with protein A/G. ± = Dark purple colloidal gold particles partially coated with protein A/G. B = Dark blue metallic gold particles not coated with protein A/G. A concentration of µg/100 µl or 3.12 µg/ml at ph 9.0 was suitable for conjugation to colloidal gold. 134

16 4.7 Evaluation of the affinity purified goat anti-human IgG, anti-human IgM and protein A/G coupled to colloidal gold Affinity purified goat anti-human IgG, goat anti-human IgM and protein A/G coupled with colloidal gold (conjugates) were tested in the immunofiltration assay using nontreponemal, micelle antigens as described in section 3.7. A panel of 18 reactive and 7 nonreactive serum samples was used to evaluate the conjugate preparations. Each serum sample was tested quantitatively for nontreponemal antibodies using the RPR test and qualitatively with the TP-PA for treponemal antibodies test. The results of the quantitative RPR test were expressed as the reciprocal of the end point. The conjugates were evaluated using the immunofiltration test in duplicate and the results obtained are shown in Table 4.7. Serum samples varied as to the content of antinontreponemal IgG and IgM and this is reflected in the ability of the test to detect the antibody. 135

17 Table 4.7 Evaluation of the affinity purified goat anti-human IgG, anti-human IgM and protein A/G attached to colloidal gold. IgG IgM Protein A/G No conjugate conjugate conjugate Sample ID RPR TP-PA R64 R R f R f R s R s R f R f R32 R R f R f R s R s R f R f R4 R R w R w R w R w R f R f R2 R R w R w N N R f R f R1 R R f R f N N R f R f R8 R N N R w R w N N R1 R R f R f N N N N R16 R R f R f R s R s R f R f R1 R R w R w N N R w R w R2 R N N R w R w N N R16 R R w R w R s R s R w R w R16 R R w R f N N R f R f R4 R R w R w R w R w R f R f R16 R R w R w R s R s R w R w R1 R R f R f N N R f R f R8 R R s R s R w R w R w R w R4 R R w R w R f R f R w R w R1 R R f R f N N R f R f N N N N ± ± N N N N N N N N N N N N N N N ± N N N N N N ± N N N N N N N ± ± N N N N N N N ± N N R s = Reactive Strong. R w = Reactive Weak. R f = Reactive Faint. ± = Equivocal. N=Negative 136

18 In Table 4.7 samples 4485 (R 64) and 4489 (R 32) appear to have a high RPR titer because the RPR test is able to detect both IgG and IgM antibodies. However, it was noticed that in the immunofiltration test the IgG conjugate was little less sensitive than the IgM and protein A/G conjugates in detecting the antibody. Samples 2070 (R8) and 2074 (R2) showed no reactivity with the IgG and Protein A/G conjugates but showed reactivity with the IgM, suggesting a newly-infected patient in the process of seroconversion. Since protein A/G detects IgG and, lesser degree, IgM. Samples 2080 (R16) and 3536 (R1) showed reactivity with both the IgG and Protein A/G conjugates but no reactivity with the IgM conjugate. This would suggest an old or treated case of syphilis. It should be noted that IgM conjugates have the tendency to yield equivocal reactions with negative sera. Taken into account that the concentration of anti-igg and anti-igm antibodies in patients with syphilis varies according to the stages of the infection, it was necessary to test a mixture of the two conjugates proportions to ensure that all positive samples could be detected by the test and that all negative samples would not give equivocal or false positive reaction. Since the IgG antibodies predominante in most of serum samples, the ratios of IgG to IgM conjugates were empirically selected to 4 + 2, 4 + 1, and The results of testing various anti human IgG/IgM conjugate ratios against six RPR positive and three RPR negative sera are shown in Table 4.8. The ratio of 4 volumes of IgG to one volume of IgM conjugates gave the most acceptable results for the preparation of a conjugate capable of detecting various concentrations of anti-igg and anti-igm antibodies in patients with syphilis. At the same time the nonspecific characteristics associated with IgM in serum appears to be reduced by the addition of a higher concentration ratio of anti-human IgG conjugate. 137

19 Table 4.8 Ratio of concentration of anti human IgG and IgM tested in the immunofiltration test. Anti human IgG : IgM conjugate ratio Sample No. RPR TP-PA 4 : 2 4 : 1 4 : : R64 R R s R s R s R s R f R f R f R f 4489 R32 R R s R s R s R s R f R f R f R f 2070 R8 R R w R w R w R f R f ± N N 2074 R2 R R w R w R w R w R f ± N N 2080 R2 R ± N R f R f R f R w R w R w 3536 R1 R N N N N R f R f R f R f 8021 N N ± ± N N N N N N 8039 N N ± ± N N N N N N 8047 N N N ± N N N N N N R s = Reactive Strong. R w = Reactive Weak. R f = Reactive Faint. ± = Equivocal. N=Negative. Test was done in duplicate Evaluation of the immunofiltration device using affinity purified goat anti-human IgG and IgM conjugated to colloidal gold The evaluation of the immunofiltration test involving the affinity purified goat anti-human IgG and IgM gold conjugate was performed using 100 clinical samples, tested with the nontreponemal laboratory based RPR and treponemal TP-PA tests for syphilis and the results are shown in Table 4.9 The comparison of the laboratory based RPR and the nontreponemal spot of the immunofiltration test indicated that of the 42 samples reactive for RPR, 2 samples were found nonreactive with the nontreponemal spot of the immunofiltration test giving a sensitivity of 95.4 %. 138

20 Fifty eight samples nonreactive for the RPR test were also found negative for the nontreponemal spot of the immunofiltration test giving a specificity of 100 %. The comparison of the laboratory based TP-PA and the treponemal spot of the immunofiltration test indicated that of the 48 samples reactive for the laboratory based TP-PA test, one sample was found nonreactive with the treponemal spot of the immunofiltration test giving a sensitivity of 97.9 %. Fifty two samples nonreactive for the laboratory based TP-PA test, one sample was found reactive with treponemal spot of the immunofiltration test giving a specificity of 98.1 %. The overall sensitivity and specificity of the immunofiltration treponemal and nontreponemal spots when compared to the laboratory based RPR and TP-PA test demonstrated the efficacy of the immunofiltration test developed in this work. Table 4.9 Evaluation of the immunofiltration device using affinity purified goat anti- human IgG and IgM conjugated to colloidal gold compared to the laboratory based RPR and TP-PA tests. Description RPR TP-PA Pos Neg Total Pos Neg Total Immunofiltration Test Pos Neg Total The comparison of the laboratory based RPR and the nontreponemal spot of the immunofiltration test according to the following formula: Sensitivity = TP x 100 = 42 x 100 = 95.4 % TP + FN TP = True Positive FN = False Negative 139

21 Specificity = TN x 100 = 58 x 100 = 100 % TN + FP The comparison of the laboratory based TP-PA and the treponemal spot of the immunofiltration test according to the following formula: Sensitivity = TP x 100 = 48 x 100 = 97.9 % TP + FN Specificity = TP x 100 = 52 x 100 = 98.1 % TP + FN Evaluation of the immunofiltration device using Protein A/G conjugated to colloidal gold The evaluation of the immunofiltration test involving Protein A/G conjugate was performed using a panel of 376 clinical samples tested with the nontreponemal laboratory based RPR and treponemal TP-PA tests for syphilis and the results are shown in Table The results obtained with the immunofiltration test were compared to those obtained in the comparator tests for both nontreponemal and treponemal tests. The immunofiltration test gave a sensitivity of 96.6% and specificity of 97.7% for the nontreponemal test spot and a sensitivity of 96.6% and specificity 99.1% for the treponemal test spot, respectively. 140

22 Table 4.10 Comparison of the performance of the immunofiltration test with the RPR and the TP-PA tests. Description RPR TP-PA Pos Neg Total Pos Neg Total Immunofiltration Test Pos Neg Total The comparison of the laboratory based RPR and the nontreponemal spot of the immunofiltration test according to the following formula: Sensitivity = TP x 100 = 115 x 100 = 96.6 % TP + FN TP = True Positive FN = False Negative Specificity = TN x 100 = 261 x 100 = 97.7 % TN + FP The comparison of the laboratory based TP-PA and the treponemal spot of the immunofiltration test according to the following formula: Sensitivity = TP x 100 = 151 x 100 = 97.4 % TP + FN Specificity = TP x 100 = 225 x 100 = 99.1 % TP + FN

23 Number of Samples The distribution of RPR titers obtained following quantitative RPR testing of these sera is shown in Figure 4.9 Seventeen of 20 samples classified as R1 (neat) following RPR titration were found to be reactive in immunofiltration test, yielding a sensitivity of 85.0%. All 14 sera with an end-point titer of R2 were positive in immunofiltration test giving a sensitivity of 100%, while 14 of 15 sera with a titer of R4 were positive in the immunofiltration test with a sensitivity of 93%. All other sera, with titers ranging from R8 to R128 proved to be positive when tested using the immunofiltration test with a sensitivity of 100%. All these RPR / TP-PA positive sera were found to be treponemal spot-positive when tested by the immunofiltration test. Number of RPR Positive Samples RPR Non-Trep Spot % 100% 100% % 93% % 100% 5 100% 0 R1 R2 R4 R8 R16 R32 R64 R128 RPR Titer Figure 4.9 Sensitivity of the immunofiltration test nontreponemal spot by RPR titer. In addition, 104 serum samples from patients with known stages of syphilis were selected from a CDC clinical serum panel. With the exception of sera obtained from two cases of untreated latent disease, and one case of treated latent, there was complete concordance between the results obtained with rapid dual test and the comparator tests (Table 4.11). 142

24 Table Documented cases of syphilis tested with the rapid dual test and the comparator RPR and TPPA tests. Number of serum samples reactive Syphilis category Total Immunofiltration test Comparator Non-Trep Spot Trep Spot RPR TP-PA Primary untreated Primary treated Secondary untreated Secondary treated Latent untreated Latent treated Total A further 23 sera were selected that were known to be RPR positive but negative by the TP- PA. These sera have been classified as biological false positives (BFPs). Of these, 16 were found to be nontreponemal spot-positive in the rapid dual test, but showed no reactivity in the treponemal spot. Forty-nine serum samples obtained from patients with diseases other than syphilis (DOS) were also tested. One sample, from a patient with pinta (a non-venereal treponematosis) was, as expected, found to be reactive in both the dual test as well as the comparator RPR and TP-PA tests. One serum from a case of malaria was found to be reactive in the RPR test, but negative in the nontreponemal spot in the rapid dual test. In addition, four further serum samples from malaria cases were positive in the nontreponemal spot in the rapid dual test but negative in the RPR test. Since the same samples were negative in both the TP-PA and the treponemal spot test in the dual test, none of these patients would have been selected for treatment for syphilis. Similarly, among the 22 serum samples obtained from patients with leprosy, six were positive in the nontreponemal spot test in the dual test, but negative in the RPR test, but all treponemal tests (both TP-PAs and treponemal spot tests) proved negative (Table 4.12). 143

25 Table Serum samples obtained from patients with diseases other than syphilis (DOS) tested with the rapid dual test and the comparator RPR and TP-PA tests. Dual rapid test spot Comparator Non-Trep Trep. RPR TPPA Category of sample Total R NR R NR R NR R NR Malaria Leprosy Mumps Rubeola Mononucleosis Toxoplasma HSV CMV Pinta Total R = reactive, NR = non-reactive. Evaluation of the immunofiltration test using both the IgG/IgM gold conjugate and the protein A/G conjugate gave comparable results when tested against the laboratory based RPR nontreponemal and TP-PA treponemal tests. A positive screen and confirm test was characterized by the appearance of three red/magenta dots; a negative result was viewed as the absence of dots where the treponemal and nontreponemal antigens had been applied. The presence of a control red/magenta colored spot was indicative of the validity of the test. A visible treponemal antigen spot [1] and control spot [C] and no detectable cardiolipin antigen spot [2] was interpreted as probably an early primary or an old or previously treated case of syphilis. A visible cardiolipin [2], and control spot [C] with no visible treponemal spot [1] was interpreted as a false positive nontreponemal reaction. A completely negative reaction with no history of syphilis or other disease was demonstrated by the appearance of only a red/magenta control spot [C]. The results are shown in Fig

26 Figure 4.10 Reactivity pattern of the dual treponemal and nontreponemal immunofiltration test for syphilis with positive and negative sera. These results indicate that the dual treponemal and nontreponemal immunofiltration assay could be used as a screen and confirmatory test for the serological diagnosis of syphilis. If this test is applied in point-of-care settings, the test results can be obtained within two to ten minutes and patients can be counseled regarding treatment and the need to notify partners. This test would be more valuable in remote or resource poor settings where there is a need to provide counseling and treatment at the initial consultation, while the patient waits for the result. The application of this test would therefore increase the effectiveness of syphilis control efforts. 145

27 4.8 Immunochromatographic lateral flow test Determination of protein content and ph range for the goat (Fab) anti-human IgM(µ) serum and protein A conjugate A different approach was taken to detect both IgG and IgM when using the immunochromatographic lateral flow test. Protein A was used to detct IgG, while goat antihuman IgM (µ) was used to detect attachment of human immunoglobulins to the test lines in the assay. The protein content and ph range of the goat anti-human IgM (µ) and protein A conjugate used in the immunochromatographic lateral flow test was performed according to procedure (Tables 4.13 & 4.14) Table 4.13 Determination of protein content and ph range for goat (Fab) Anti human IgM (µ) Column 1 Column 2 Column 3 Column 4 Column 5 Goat anti human IgM ph 6.0 ph 7.0 ph 8.0 ph 9.0 ph 10.0 A* R R R R R 10.0µg B R R R R R 5.0µg C R R R R R 2.5µg D R R R R R 1.25µg E ± R R R R 0.625µg F B ± R ± ± 0.312µg G B B B B B 0.156µg H B B B B B 0.078µg R = Complete coating ± = Partial coating B = No coating *Goat Anti-Human IgM (1mg/mL) (or 10µg) 146

28 A concentration of µg/100 µl or 3.12 µg/ml at ph 8.0 was suitable for conjugation to colloidal gold for use in the immunochromatographic test. Table 4.14 Determination of protein content and ph range for protein A. Column 1 Column 2 Column 3 Column 4 Column 5 ph 6.0 ph 7.0 ph 8.0 ph 9.0 ph 10.0 A* R R R R R 10.0µg B R R R R R 5.0µg C R R R R R 2.5µg D R R R R R 1.25µg E ± R R R R 0.625µg F B ± R R R 0.312µg G B B R ± ± 0.156µg H B B ± B B 0.078µg R = Complete coating ± = Partial coating B = No coating *Protein A (1 mg/ml) (or 10µg) A concentration of µg per 100 µl or 1.56 µg/ml at ph 8.0 was suitable for conjugation to colloidal gold Evaluation of the immunochromatographic lateral flow test A total of 1601 serum samples obtained the Georgia Department of Community Health laboratory were included in the evaluation, together with 105 sera from cases with known stages of syphilis, 179 sera from diseases other than syphilis and 14 sera previously classified as biological false positives (RPR+ve/TPPA-ve). Of the 1601Georgia serum samples tested, 834 were reactive in both RPR and TP-PA tests, while 173 were reactive in the TP-PA test alone. The remaining 594 sera were non-reactive in both treponemal and non-treponemal reference tests. A comparison of the results obtained for the non-treponemal line in the dual POC test with those of the RPR yielded an overall sensitivity of 88.6% and a specificity of 98.6%. The 834 serum samples reactive in both the RPR test and TP-PA were tested 147

29 quantitatively using the RPR test and the distribution of RPR titers and rates of reactivity with the nontreponemal line of the dual POC test are shown in (Figure 4.11). The sensitivity of the nontreponemal line increased dramatically to 98.4% with a specificity of 98.6% when the RPR titers of sera were 1:2. The sensitivity and specificity of the treponemal line, when compared to the TP-PA test were 96.5% and 95.5% respectively (Table 4.15). Table 4.15 Comparison of the performance of the dual immunochromatographic lateral flow test with the RPR and the TP-PA tests. Description RPR TP-PA Pos Neg Total Pos Neg Total Immunochromatographic Test Pos Neg Total The performance of the nontreponemal line of the immunochromatographic test compared to the laboratory based RPR was calculated according to the following formula where TP = True Positive, FN = False Negative, TN=True Negative and FP=False Positive: Sensitivity = TP x 100 = 739 x 100 = 88.6 % TP + FN Of the 95 samples found nonreactive with dual POC test, 85 had an RPR titer of 1:1. With an RPR titer of 1:2 the sensitivity was 98.4% Specificity = TN x 100 = 756 x 100 = 98.6 % TN + FP The performance of the treponemal line of the immunochromatographic test compared to the laboratory based TPPA was similarly calculated: 148

30 Sensitivity = TP x 100 = 972 x 100 = 96.5 % TP +FN Specificity = TN x 100 = 567 x 100 = 95.5 % TN + FP Fig Sensitivity of the nontreponemal line of the immunochromatographic test by RPR titer. The results of testing 105 sera from patients with known stages of syphilis by the dual POC and comparator tests are shown in Table Complete concordance between the dual rapid test and the comparator tests was obtained in all cases of primary and untreated secondary disease. The dual POC test detected nontreponemal antibody in all cases of untreated disease, but was unable to detect this antibody in one case of treated secondary disease and six cases of treated latent disease where the RPR reactivity could only be detected in undiluted serum or when diluted 1:2. The dual POC test was also unable to detect treponemal antibody in three cases of treated and one case of untreated latent disease. One hundred and seventy nine serum samples obtained from patients with diseases other than syphilis were also tested using the RPR, TP-PA and dual POC test. One sample, from a 149

31 patient with pinta (a non-venereal treponematosis), was found to be reactive in both lines of the dual DPP-POC test as well as the comparator RPR and TP-PA tests. One serum from a patient with malaria was reactive in the RPR test, but non-reactive with the nontreponemal line in the dual POC test. However, four sera from malaria patients were reactive with the nontreponemal line in the dual POC test but non-reactive in the RPR test. Since these same samples also proved non-reactive with the treponemal line in the POC test and in the TP-PA, these patients would not have received treatment for syphilis having been classified as biological false positives. Similarly, six of 22 sera from patients with leprosy were found to be reactive with the nontreponemal line of the dual POC test, but non-reactive in the RPR test. All these sera were non-reactive in all confirmatory treponemal tests (Table 4.17). Among 14 RPR-positive, TP-PA-negative (classified as biological false-positive) sera, seven were found to be reactive with the nontreponemal line of the dual POC test. All were nonreactive with the treponemal line. Table 4.16 Documented cases of syphilis tested with the dual immunochromatographic test and with the RPR and the TP-PA tests. Number of serum samples reactive Syphilis category Total Immunochromatographic test Comparator Non-Trep a Trep b RPR TP-PA Primary untreated 7 7(100*) 7(100) 7 7 Primary treated 14 9(100) 13(100) 9 13 Secondary untreated 6 6(100) 6(100) 6 6 Secondary treated 28 22(84.7) 28(100) Latent untreated 5 3(100) 4(80) 3 5 Latent treated 45 35(85.4) 44(97.8) Total * Percent. a Non-Trep, Nontreponemal Line. b Treponemal Line 150

32 Table 4.17 Serum samples of diseases other than syphilis (DOS) tested with the dual POC test and the comparator rapid plasma reagin (RPR) and Treponema pallidum passive particle agglutination (TP-PA) tests. 151

33 4.9 Development of a nontreponemal ELISA test Evaluation of oxidized cardiolipin attached to a protein carrier in the ELISA test RPR titers have long been recognized as the key serological markers for the diagnosis of active syphilis and are used to monitor the efficacy of drug therapy. Currently these nontreponemal tests are visually read which can lead to subjective assessment and person to person variation in reading of end-point titers. Unfortunately, native cardiolipin cannot be used as an antigen in ELISA tests owing to its fatty chemical structure which results in inconsistent adherence to the plastic of microtiter plate wells. However, oxidized cardiolipin, the product of the procedure described in can be used as antigenis an objective quantitative test designed to reduce the human error in interpreting the results. The oxidized cardiolipin was attached to a protein carrier for direct coupling to ELISA plates. Four proteins were selected for the covalent attachment; BSA, KLH, MAPS and IgY to the activated carboxyl group of oxidized cardiolipin. The purpose of the test was to determine the usefulness of the protein carrier in allowing the cardiolipin antigen to react with the syphilitic antisera. A 1:50 dilution of reference reactive and nonreactive sera was used for the comparison. The ELISA plate contains 10 µg/ml concentration of the antigens or 1 µg per well (Table 4.18). The test was performed in triplicate. Three times the negative OD was considered as the difference between positive and negative reactions. Table 4.18 Reactivity of Cardiolipin antigen attached to various proteins carriers. Antigen Serum I.D No. Dil OD 1 OD 2 OD 3 Ave. 3xneg MAPS Reactive WS : Nonreactive PT009 1: BSA Reactive WS : Nonreactive PT009 1: KLH Reactive WS : Nonreactive PT009 1: IgY Reactive WS : Nonreactive PT009 1:

34 The results of the test indicated that the protein carriers BSA, MAPS and IgY were similar in reactivity and any of them could be used in the ELISA test. However, oxidized cardiolipin attached to KLH gave high nonspecific reaction and could not be used Evaluation of oxidized Cardiolipin attached to BSA It was of interest to determine the optimal concentration range of the oxidized cardiolipin attached to BSA with various concentrations of the antigen. For this purpose a reference reactive sera was selected and was diluted 1:50 to 1:1600 (Table 4.19). Table 4.19 Evaluation of oxidized cardiolipin attached to BSA tested with a reference serum with a RPR titer of 1:128. Antigen concentration Serum ID. Di l 1000 µg/ml 500 µg/ml 250 µg/ml 100 µg/ml 50 µg/ml 25 µg/ml WS : : : : : : Bl k Antigen concentration Serum ID. Di l 10 µg/ml 5 µg/ml 2.5 µg/ml 1.25 µg/ml 0.62 µg/ml Neg cont WS : : : : : : Bl k The reference reactive sera had a RPR titer of 1:128. An antigen concentration of 25 µg/ml to 2.5 µg/ml was regarded as suitable for the performance of the test. Reference reactive sera with lower RPR titer gave similar results with lower OD values. It was then established that by selecting the BSA carrier protein with an antigen concentration ~10 µg/ ml is suitable for development of an ELISA nontreponemal test. 153

35 Table 4.20 Evaluation of oxidized cardiolipin attached to BSA tested with a reference serum with a RPR titer of 1:32. Antigen concentration Serum ID. Dil 500 µg/ml 250 µg/ml 125 µg/ml 50 µg/ml 25 µg/ml 12.5 µg/ml WS : : : : : : Blk Antigen concentration Serum ID. Dil 5 µg/ml 2.5 µg/ml 1.25 µg/ml 0.62 µg/ml 0.31 µg/ml Neg cont WS : : : : : : Blk With reference reactive antiserum with a RPR titer of 1:32 an antigen concentration range of 12.5 µg/ml to 5 µg/ml was adequate to demonstrate the suitability of the test. Five reference sera with RPR titers of 1:128 and 1:512 were tested by using a 10 µg/ml concentration of oxidized cardiolipin attached to BSA following procedure The serum dilutions were at 1:20, 1:40, 1:80 and 1:160 (Figure 4.12). A negative control serum was also diluted at the same concentrations. With these particular sets of serum samples the 1:20 dilution of the test serums showed a greater differentiation between the negative control sera and the positive serum samples. 154

36 Figure 4.12 Correlation of RPR serum titer and optical density with 5 reactive and one nonreactive control sera Figure 4.13 Correlation of a serum with an RPR titer of 1:128 at dilutions of 1:20, 1:40, 1:80 and 1:160 and the corresponding OD value. 155

37 The oxidized cardiolipin attached to BSA protein carrier was evaluated with various concentration of antigen ranging from 100 µg/ml to 0.31 µg/ml and the reference reactive and nonreactive serums were diluted from 1:10 to 1:160. The cut off value was established as three times the negative OD value. At serum dilutions of 1:10, 1:20 and 1:40 the concentrations of the cardiolipin BSA antigen were found to be optimal at 7.5 µg/ml, 10 µg/ ml and 50 µg/ml respectively (Table 4.21). The RPR titer of the reference serum was 1:64. Table 4.21 Evaluation of oxidized cardiolipin attached to BSA with various concentration of antigen. Antigen concentration Serum ID. Dil 100 µg/ml 50 µg/ml 25 µg/ml 12.5 µg/ml 10 µg/ml 7.5 µg/ml Reactive WS : Nonreactive TP 010 1: x Neg Reactive WS : Nonreactive TP 010 1: x Neg Reactive WS : Nonreactive TP 010 1: x Neg Reactive WS : Nonreactive TP 010 1: x Neg Reactive WS : Nonreactive TP 010 1: x Neg Antigen concentration Serum ID. Dil 5 µg/ml 2.5 µg/ml 1.25 µg/ml 0.62 µg/ml 0.31 µg/ml Blank Reactive WS : Nonreactive TP 010 1: x Neg Reactive WS : Nonreactive TP 010 1: x Neg Reactive WS : Nonreactive TP 010 1: x Neg Reactive WS : Nonreactive TP 010 1: x Neg Reactive WS : Nonreactive TP 010 1: x Neg Reactive serum WS 2001 RPR titer 1:64 156

38 The pattern of reactivity of the oxidized cardiolipin attached to BSA carrier showed a range related to the RPR titer of the serum at various dilutions and the concentration of the antigen attached to the ELISA plate. This system while workable was improved by the direct attachment of the oxidized cardiolipin antigen to aminated ELISA plates. The well to well reproducibility of the test was done by repeating the samples 10 times with 7 reference sera with RPR titers of 1:1(R1), 1:2 (R2), 1:4 (R4), 1:8 (R8), 1:16 (R16), 1:32 (R32) and 1:128 (R128) and one negative control (Figure 4.14). There was a distinct difference from the OD value of the nonreactive control and the minimal reactive 1:1(R1) sera. With serum sample of RPR titer of 1:1 (R1) and 1:16 (R16) there was clear linearity between the OD values of each of the corresponding repeat wells. However, with samples with RPR titers 1:32 (R32) and 1:128 (R128) there was an excess antibody concentration resulting in a partial nonlinearity. This phenomenon can be solved by dilution the serum samples 1:10 and projecting the OD value.(figure 4.14) Cardiolipin antigen 10 µg/ml Figure 4.14 Well-to-well reproducibility of the test by using seven reactive and one nonreactive control sera. 157

39 The correlation between the RPR titer and the OD value of the ELISA test was also demonstrated by infecting a rabbit with a live culture of T. pallidum and monitoring the antibody response for a period of one year past infection. Samples were drawn at intervals during the course of the untreated infection. Untreated syphilis follows a declined in nontreponemal cardiolipin antibody that expand for several years. However, in the rabbit model the decline of antibody content to the cardiolipin antigen is more pronounce and the time period is reduced. Serum samples were collect at 16, 26, 46, 56, 59, 66, 247 and 495 days and were stored frozen at -20ºC during the collection period. For this study we obtained these samples courtesy of the Centers of Disease Control and Prevention Atlanta Georgia USA. The samples were thaw and tested quantitatively by the RPR test and according to procedure for the ELISA test. Because of the nature of the sample the goat antihuman IgG HRP conjugate was substituted by goat anti-rabbit IgG HRP. The test results demonstrated an accurate correlation between the RPR titer and the OD values (Figure 4.15). Figure Correlation between the RPR titer and ELISA OD of sera obtained from a syphilis infected rabbit Evaluation of attachment of oxidized cardiolipin to aminated ELISA plates The direct attachment of oxidized cardiolipin to ELISA plate was made possible by using Nunc immobilizer amino plates. The carboxyl group of the oxidized cardiolipin was activated with EDC/NHS and covalently attached to the amine group of the Nunc plate. 158

40 This procedure avoided the necessity of attaching the cardiolipin to a protein carrier thus simplifying the procedure and ensuring a more reproducible system. With this configuration the head group of cardiolipin was oriented upward exposing the head group to the antiserum. The first step of the evaluation was to determine the cardiolipin antigen coating concentration attached to the aminated Nunc plate (Table 4.22). The coating concentrations ranged from 25 µg/ ml to 200 µg/ml. The test was performed in duplicate for each concentration and the cut off was determined as three times the negative value. In order to measure and compare the various antigen concentrations the goat anti-human HRP conjugate was made constant at 1:5000 dilution. Acceptable concentrations ranged from 75 µg/ml to 200 µg/ml. However, by comparing the ratio of positive reactive test to the corresponding cut off value the 150 µg/ml was better and thus selected for consequent studies. Table Determination of cardiolipin antigen coating concentration attached to the aminlated Nunc plate. Nonreactive Serum Cardioilipin antigen concentration µg/ml No. BM13840 Dilution 25 µg/ml 25 µg/ml Avg 50 µg/ml 50 µg/ml Avg 75 µg/ml 75 µg/ml Avg 1: : : Blank Reactive Serum Cardioilipin antigen concentration µg/ml No Dilution 25 µg/ml 25 µg/ml Avg 50 µg/ml 50 µg/ml Avg 75 µg/ml 75 µg/ml Avg 1: : : Blank Cut off Dilution 3 x Neg 1: x Neg 1: x Neg 1: Nonreactive Serum Cardiolipin antigen coating concentration µg/ml No. BM13840 Dilution 100 µg/ml100 µg/ml Avg 150 µg/ml150 µg/ml Avg 200 µg/ml200 µg/ml Avg 1: : : Blank Reactive Serum Cardiolipin antigen coating concentration µg/ml No Dilution 100 µg/ml100 µg/ml Avg 150 µg/ml150 µg/ml Avg 200 µg/ml200 µg/ml Avg 1: : : Blank Cut off Dilution 3 x Neg 1: x Neg 1: x Neg 1:

41 4.9.4 Determination of the anti-human IgG and IgM conjugate ratio The ratio of goat anti-human IgG and IgM conjugate was determined by making cocktails of IgG and IgM (three volume of IgG to 0.5 volumes of IgM, four volumes of IgG to 0.5 volume of IgM, and four volumes of IgG to one volume of IgM). The mixture was diluted 1:7500. The serum samples were diluted 1:100 for this test. The ELISA plates were coated with 100 µl of cardiolipin antigen at a 150 µg/ml concentration or 15 µg per well. Eight reactive and 18 nonreactive samples were selected for the evaluation of the conjugate. Seven of the samples had a low RPR titer ranging from 1:1 (R1) to 1:4 (R4). This was done with the purpose of severely challenging the system to determine the best conjugate combination. At a cut off value of 0.199, the sensitivity of the conjugate was 72%, and the sensitivity of the conjugate was 80%. However, the sensitivity and specificity of the conjugate was 100% (Table 4.23). Table Determination of the ratio of goat anti-human IgG and IgM conjugate. Anti-huma IgG + Anti-human IgM conjugate ratio IgG + IgM IgG + IgM IgG + IgM Serum No. Reactive Abs Abs AVEG Abs Abs. AVEG Abs Abs AVEG Blank R R R R R R R Serum No. Nonreactive Abs Abs AVEG Abs Abs. AVEG Abs Abs AVEG Blank Cardiolipin antigen concentration 150 µg/ml. Serum dilution 1:100. Conjugate dilution 1:7500. Cut off value

42 4.9.5 Determination of the anti-human IgG and IgM conjugate dilutions. The anti-human IgG + anti-human IgM ratio of was selected for the determination of best conjugate dilution. ELISA plates were coated with 100 µl of cardiolipin antigen at a 150 µg/ml concentration or 15 µg per well. The conjugate was diluted 1:5000, 1:7500 and 1:10,000. One nonreactive and one reactive serum were diluted 1:25, 1:50 and 1:100 (Table 4.24). Table 4.24 Determination of dilution of goat anti-human IgG and IgM conjugate. Anti-human IgG + Anti-human IgM conjugate Dil 1:5000 Dil 1:7500 Dil 1:10000 Nonreactive Dilution Abs Abs Aveg Abs Abs Aveg Abs Abs Aveg Sample No. 1: : : Blank Reactive Abs Abs Aveg Abs Abs Aveg Abs Abs Aveg Sample No. 1: R8 1: : Blank x Neg 1: x Neg 1: x Neg 1: Comparison of the conjugate dilutions to the serum dilutions, the ratio of the average of the reactive serum divided by the 3 x negative was calculated as follows: Conjugate Dil 1:7500 Serum Dil. Abs Ave. Nonreactive serum 1: : : Ave x 3 neg value = Reactive serum 1: : : Ave

43 It was determined that the anti-human IgG + Anti-human IgM conjugate at a ratio of with a dilution of 1:7500 and a cardiolipin antigen concentration of 150 µg/ml was optimal for the performance of the test Evaluation of oxidized cardiolipin BSA and KLH complex attached to nitrocellulose membrane for the Immuno-Dot test procedure In the Immuno-Dot test, if human antibodies to cardiolipin are present in the serum sample, they will bind to the cardiolipin protein complex attached to the nitrocellulose membrane. The addition of anti-human serum conjugated to AP will give a purple to magenta color dot if the human serum is attached to the antigen bound to the nitrocellulose membrane. A nitrocellulose membrane was spotted with 500, 250, 125, 62.5 and 31.2 µg/ml of cardiolipin BSA protein carrier. The anti-human serum conjugated to AP was diluted 1:2000 and the reactive and nonreactive serum samples were diluted serially 1:0, 1:20, 1:40, 1:80 and 1:160 (Figure 4.16). Figure Evaluation of oxidized cardiolipin BSA on Immuno-Dot test. 162

44 The cardiolipin BSA antigen spotted with a 125 µg/ml concentration was visible with a 1:80 dilution of the reactive serum. At equivalent concentration with the nonreactive sera there was no visible reaction. A nitrocellulose membrane was spotted with 500, 250, 125, 62.5 and 31.2 µg/ml of cardiolipin KLH protein carrier complex (Figure 4.17). The anti-human serum conjugated to AP was diluted 1:2,000 and the reactive and nonreactive serum samples were diluted serially 1:0, 1:20, 1:40, 1:80, 1:160 and 1:320. The evidence of reactivity was determined by the visual appearance of red magenta dots for the reactive sera and no visible dots for the nonreactive sera. Figure Evaluation of oxidized cardiolipin KLH on Immuno-Dot test. 163