CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Nov. 1997, p. 671–675 1071-412X/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 4, No. 6
Development of an Enzyme-Linked Immunosorbent Assay for Measurement of Serum-Associated ALX40-4C P. J. PAYETTE,1 M. CORMIER,1 B. DABEK,2 P. YUNGBLUT,2 S. PRESSEAULT,2 S. CLIMIE,2 J. SAHAI,3 W. D. CAMERON,1,3 AND L. G. FILION1,3* Departments of Microbiology and Immunology1 and of Medicine,3 University of Ottawa, Faculty of Medicine, Ottawa, Ontario K1H 8M5, and Allelix Biopharmaceuticals, Mississauga, Ontario L4V 1V7,2 Canada Received 15 April 1997/Returned for modification 16 July 1997/Accepted 11 September 1997
ALX40-4C is an antiretrovirus agent that has been found to have some inhibitory properties against human immunodeficiency virus (HIV) replication in vitro. The compound was designed as a competitor of the HIV Tat protein for TAR binding. In addition to its anti-HIV properties, it has demonstrated the ability to inhibit in vitro replication of herpes simplex virus types 1 and 2 as well as human cytomegalovirus. Subsequently, in vivo pharmacokinetic evaluation of ALX40-4C necessitated the establishment of a detection system for the measurement of ALX40-4C in subject serum. For this purpose, an indirect-competition enzyme-linked immunosorbent assay with generated rabbit anti-ALX40-4C antiserum was developed. The original assay took 12 h to complete and required many manipulations. Herein, we describe alterations to the system that resulted in the overall reduction in assay time and manipulation. We demonstrate that our alterations do not affect the specificity or sensitivity of the assay compared to that of the original system. ALX40-4C levels in spiked serum samples as well as drug levels from patient samples were used to validate the assay. ALX40-4C (Allelix Biopharm, Mississauga, Ontario, Canada) is a novel antiretrovirus drug that inhibits the replication of human immunodeficiency virus type 1 (HIV-1) in vitro (8). The mechanism of action of ALX40-4C is not well-characterized; however, this compound can inhibit Tat-TAR binding and viral transactivation. A second mechanism of action that involves a cell surface event has also been identified but has not been well-characterized (8). ALX40-4C has also been shown to inhibit the replication of herpes simplex virus types 1 and 2 as well as human cytomegalovirus in vitro (10). To determine the levels of toxicity and metabolism of ALX40-4C, pharmacokinetic studies following administration of the compound to both dogs and rats were carried out. In both cases, the levels of ALX40-4C in serum were measured by an indirect-competition enzyme-linked immunosorbent assay (ELISA) employing a pooled rabbit anti-ALX40-4C antiserum (reference serum) and a goat anti-rabbit immunoglobulin (Ig) linked to alkaline phosphatase (unpublished data). The assay time was approximately 12 h, and the assay involved numerous technical manipulations. Our goal was to enhance the current assay by reducing its overall time and the number of manipulations. To achieve this goal, two aspects of the original assay were considered for alteration: the ELISA-based detection system was changed from an indirect process to a direct process by using an avidin-biotin complex (ABC) system, and the competition step that is performed in a separate plate was performed directly within the ELISA plate.
was linked to keyhole limpet hemocyanin (KLH) with the use of a step conjugation method employing glutaraldehyde (2a). The initial injection contained 250 mg of ALX40-4C linked to KLH (ALX40-4C–KLH) in 2 ml of Freund’s complete adjuvant (Gibco BRL, Chagrin Falls, Ohio) (1) at an antigen-toadjuvant ratio of 1:3. The injections were administered at one to three sites, either subcutaneously or intramuscularly. The animals were given three subsequent injections at 2-week intervals of 250 mg of ALX40-4C–KLH in 2 ml of Freund’s incomplete adjuvant (Gibco BRL) (7). The animals were bled 2 weeks following the last injection, and the antibody levels were assessed by a direct ELISA as described below. Isolation and biotinylation of rabbit IgG. The rabbit IgG was isolated from whole serum by affinity chromatography on a column of protein A-Sepharose (Pharmacia, Uppsala, Sweden) (3). The concentration of the dialyzed IgG antiALX40-4C sample (11.67 mg/ml) was determined by UV spectrophotometry. The purified rabbit IgG was biotinylated as directed by the manufacturer of the biotin labeling kit (Pierce, Rockford, Ill.). Seven biotinylated IgG fractions were tested for reactivity against ALX40-4C by the indirect ELISA as described below. This was done to ensure that the addition of biotin did not alter the antibody’s reactivity. Each sample was also assayed by the ABC system (4, 5, 12, 13) with streptavidin-horseradish peroxidase (HRP) (Jackson ImmunoResearch) as the detection method. Samples with similar binding patterns were pooled. The pooled sample of biotinylated rabbit anti-ALX40-4C IgG was retitrated, aliquoted, and stored at 280°C. Titration of anti-ALX40-4C antibodies in rabbit serum. A direct ELISA system was employed for the titration of anti-ALX40-4C antibodies in rabbit sera (6, 9). Nunc Maxisorp (96-well) microtiter plates (Gibco BRL) (11) were coated overnight at 4°C with 0.1 mg (2) of ALX40-4C per well in a carbonate buffer (pH 9.8). The ALX40-4C-coated plates were washed three times (0.005% Tween 20–phosphate-buffered saline [PBS]) (Sigma Chemical Co., St. Louis, Mo.), blocked by incubation overnight at 4°C with 200 ml of a 3.75% bovine serum albumin solution (Sigma), and washed three times (0.005% Tween 20–PBS) just prior to use. The rabbit sera were fourfold serially diluted in normal human serum (Gibco BRL) over eight wells, starting with a dilution of 1:100. The plates were incubated for 1 h at 37°C. The plates were washed, and goat anti-rabbit antibody conjugated to HRP (Jackson ImmunoResearch) at a dilution of 1: 20,000 in dilution buffer (PBS–1% Nonidet P-40–0.23% bovine serum albumin (BSA)) was added to all of the wells (except the blanks). The plates were incubated at 37°C for 1 h. The plates were washed three times (0.005% Tween 20–PBS), 100 ml of o-phenylenediamine dihydrochloride (OPD) substrate (Sigma Chemical Co.) was added to all wells, and the wells were incubated for 30 min at 37°C. The reaction was stopped by adding 50 ml of 1 M H2SO4 (BDH, Inc., Toronto, Ontario, Canada). The plates were read with a Bio-Tek EL310 Plate Reader (Bio-Tek Instruments Inc., Burlington, Vt.) at a wavelength of 490 nm. Test samples. Two types of samples were employed in this study to validate the assay. The first type consisted of spiked samples prepared by one of us (S.C.). These samples were analyzed in a blinded fashion. The second type consisted of samples from two clinical trials, and these were also tested in a blinded fashion, and included in one of the studies was a placebo.
MATERIALS AND METHODS Patients. All patients were from the Immunodeficiency Clinic at the Ottawa General Hospital. Informed written consent from each patient was obtained. Serum samples from patients were taken 1 h after infusion of ALX40-4C. Production of antibodies against ALX40-4C. Six New Zealand White rabbits (Charles River Laboratories) were hyperimmunized with ALX40-4C. ALX40-4C
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8M5. Phone: (613) 5625800, ext. 8308. Fax: (613) 562-5452. E-mail:
[email protected]. 671
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Measurement of levels of ALX40-4C in serum by an indirect ELISA. To quantitate the levels of ALX40-4C in serum, an indirect-competition ELISA (6, 9) was developed. This system involved use of two 96-well microtiter plates. The first plate (competition plate [NUNC tissue culture plate; Gibco BRL]) was blocked overnight at 4°C with 3.75% BSA in PBS and was used to incubate the competition step between ALX40-4C in patient serum and anti-ALX40-4C antiserum. The second plate (ELISA plate [NUNC Maxisorp ELISA plate; Gibco BRL]) (11) was coated overnight at 4°C with 0.1 mg (2) of ALX40-4C per well, washed three times (0.005% Tween 20–PBS), and blocked overnight at 4°C with 200 ml of a 3.75% BSA solution. This plate was used in the actual ELISA for the indirect quantitation of ALX40-4C in patient serum. The competition plates were washed three times (0.005% Tween 20–PBS) just prior to use. Normal human serum (100 ml/well) was added to each well of the competition plate and was used as a diluent for the standard curve and unknown samples. The standard curve consisted of a duplicate twofold stepwise dilution of 7.2 mg of ALX40-4C per ml in normal human serum across 11 wells. Test samples were plated in duplicate with three twofold stepwise dilutions for each unknown sample (see above). The dilutions ranged from 1:2 to 1:16 and resulted in data points that fell within the linear portion of the standard curve. All wells, with the exception of the blanks and negative controls, received 100 ml of rabbit anti-ALX40-4C antiserum (reference serum) at a dilution of 1/500 in dilution buffer. This resulted in a final rabbit anti-ALX40-4C antiserum dilution of 1/1,000 in a final volume of 200 ml. The plates were incubated for 2 h at 37°C. Two controls were included on each plate to assess the binding of the antiALX40-4C preparation. The first control consisted of normal human serum without ALX40-4C and rabbit anti-ALX40-4C antiserum. This provided us with the maximum binding of the anti-ALX40-4C preparation. The second control was a spiked normal human serum sample with a set amount of ALX40-4C and rabbit anti-ALX40-4C antiserum. This provided us with a control to determine if we could inhibit the anti-ALX40-4C and the bound ALX40-4C on the plate to the same extent. Four different negative controls were included on all plates and consisted of (i) normal human serum without rabbit anti-ALX40-4C antiserum, (ii) normal rabbit serum diluted in normal human serum, (iii) patient samples without rabbit anti-ALX40-4C antiserum, and (iv) normal human serum with dilution buffer. These four controls provided a clue to the nonspecific interactions in our assays. The ELISA itself was performed with the ELISA plates with the standards, controls, and test samples from the competition plates. The ELISA plates were washed three times (0.005% Tween 20–PBS). A 100-ml aliquot from each well of the competition plates was transferred into the ELISA plates and incubated for 1 h at 37°C. The plates were washed three times (0.005% Tween 20–PBS), and 100 ml of goat anti-rabbit IgG-alkaline phosphatase (AP), at a dilution of 1/1,000, was added to all of the wells except the blanks. The plates were incubated at 37°C for 1 h and washed three times (0.005% Tween 20–PBS), and 100 ml of pnitrophenylphosphate substrate was added to all wells, including the blanks. The plates were incubated for 30 min at 37°C, and the reaction was stopped by adding 100 ml of 1 M NaOH per well. The plates were read with a Bio-Tek EL310 plate reader at a wavelength of 450 nm. The resulting data were analyzed with Kineticalc V2.11 (Bio-Tek Instruments, Inc.). Direct-competition ELISA with the ABC system. A direct-competition ELISA with the ABC system (4, 5, 12, 13) was developed to simplify the measurement of ALX40-4C in patient serum. The use of a separate plate for the competition step was omitted, and the competition step was performed directly with the ELISA plate. Streptavidin linked to HRP was used as the detection system. Microtiter (96-well) plates (11) were coated overnight at 4°C with 0.1 mg (2) of ALX40-4C washed with 0.005% Tween 20 per well and blocked overnight at 4°C with 200 ml of a 3.75% BSA solution. The plates were washed, and 100 ml of normal human serum per ml was added as a diluent for the ALX40-4C standard curve and the unknown samples. The standard curve was generated by carrying a twofold stepwise dilution of a 7.2-mg/ml initial concentration of ALX40-4C in normal human serum across 11 wells in duplicate. Test samples, which were obtained as outlined above, were plated in duplicate with a total of three twofold stepwise dilutions for each unknown. The dilutions ranged from 1:2 to 1:16 and resulted in data points that fell within the linear portion of the standard curve. For some unknowns, dilutions ranged from 1:2 to 1:32 in order to maintain data points within the linear portion of the standard curve. All wells, except the blanks and negative controls, received 100 ml of biotinylated rabbit anti-ALX40-4C IgG at a dilution of 1/1,500 in dilution buffer, and the plates were incubated for 2 h at 37°C. The plates were washed three times (0.005% Tween 20–PBS), and 100 ml of streptavidin-HRP (30 ng/ml) was added to all of the wells except the blanks. The plates were incubated at 37°C for 30 min. The plates were washed, and 100 ml of OPD substrate was added to all of the wells, including the blanks. The plates were incubated at 37°C for 30 min. The reaction was stopped by the addition of 50 ml of 1 M H2SO4 per well. The plates were read with a Bio-Tek EL310 Plate Reader at a wavelength of 490 nm. The resulting data were analyzed with Kineticalc V2.11 (BIO-TEK Instruments Inc.).
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FIG. 1. Titration of anti-ALX40-4C antibodies. Six New Zealand White rabbits were immunized with ALX40-4C and bled. Anti-ALX40-4C antibodies were measured by the direct ELISA technique as described in Materials and Methods. Animals: 1, 3901; E, 3902; Œ, 3903; ƒ, 3921; h, 3922; }, 3924. F, reference serum; , normal rabbit serum.
RESULTS AND DISCUSSION Production of ALX40-4C antiserum was achieved with six New Zealand White rabbits that were immunized with ALX404C linked to KLH. The antibodies generated were to be used for the measurement of ALX40-4C levels in the sera of HIVinfected individuals undergoing therapy with ALX40-4C. The rabbits were bled, and their sera were screened for the presence of anti-ALX40-4C antibodies by the direct ELISA system described in Materials and Methods. The antibodies produced by each animal were assessed singly for titer and relative affinity, and the results are reported in Fig. 1. These data clearly demonstrate that all of the rabbits reacted similarly to ALX40-4C–KLH, except for rabbit 3902 and the reference rabbit anti-ALX40-4C antiserum used in the original assay, both of which had lower antibody titers. We tested the serum from each animal by the indirectcompetition ELISA, as described in Materials and Methods, to determine which animal would give the best and most-consistent standard curve. The serum dilutions to be used in the indirect assay were chosen from the titration curves. The selection of a proper dilution point was critical to ensure that a sensitive assay for measuring free drug would be developed (6). Dilution points that fell within the linear portion of the titration curve were chosen (6). The standard curve obtained with serum from animal 3922 was ideal, with a steep and linear slope over a broad range of ALX40-4C drug concentrations (Fig. 2) (6). The original assay that was used as the prototype for the method described in this paper was an indirect-competition ELISA as described in Materials and Methods. A secondary antibody was used to detect rabbit antibodies bound to ALX40-4C immobilized to the bottom of a 96-well plate. This assay required a total of 10 to 12 h to complete. We set out to improve the assay in the following two ways: with an ABC system, the ELISA-based detection system would be changed from an indirect process to a direct process, and the competition step that was performed in a separate plate would now be performed directly within the ELISA plate.
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FIG. 2. Standard curve for the measurement of ALX40-4C. The indirect ELISA system as described in Materials and Methods was used to generate the standard curves from serum sample dilutions from each animal as shown in Fig. 1. Animals: 1, 3901; E, 3903; Œ, 3921; ƒ, 3922; h, 3924.
The IgG fraction of animal 3922 was separated into seven samples, labeled with biotin, and tested via the direct ELISA as described in Materials and Methods. All preparations of the biotin-linked antibodies reacted similarly to ALX40-4C, except for sample no. 4. These results clearly demonstrate that the addition of biotin to the IgG fraction of animal 3922’s serum did not affect its reactivity to ALX40-4C (Fig. 3). A strong positive signal (i.e., optical density [OD] 5 1.6) and low background (OD , 0.05) could be observed. The titration curve was used to determine the dilution of the pooled anti-ALX40-4C IgG to be employed in the direct-competition ELISA. A dilution of 1:1,500 was agreed upon, since it gave a signal of more than 1.0 and fell within the linear portion of the titration curve (6). The steep slope of this curve predicts that any subtle changes in the chosen antibody dilution due to complex formation with free unbound ALX40-4C will result in a measurable change in OD. Second, a series of experiments were performed to determine if the competition of ALX40-4C-reactive IgG by free ALX40-4C could be measured directly on the ELISA plate coated with ALX40-4C. The competition step was originally performed with a separate plate that was not coated with ALX40-4C but that was blocked with 3.75% BSA. The assays revealed no detectable differences between the two methods of performing the competition step (data not shown); therefore, the competition step was carried out on the ELISA plate in all subsequent assays. The biotinylated IgG fraction from animal 3922 was used to generate a standard curve by the ABC method as described in Materials and Methods. The curve obtained with this antibody (i.e., biotin labeled) was compared to those obtained by the original indirect assay employing either the unprocessed antiALX40-4C antiserum from animal 3922 or the original rabbit
anti-ALX40-4C antiserum (reference serum) from the primary assay development. The results clearly demonstrated that the biotinylated IgG fraction produced an acceptable standard curve that varied little from that for the unprocessed serum from the same animal (Fig. 4). Standard curves obtained with the reference serum from the original assay displayed a slightly
FIG. 3. Titration of biotinylated anti-ALX40-4C. The IgG fraction from animal 3922 was isolated and aliquoted. Each aliquot was biotinylated separately and titrated by the direct ELISA. Samples 1 to 7 are represented by different symbols.
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FIG. 4. Comparison of standard curves to measure ALX40-4C with three different antibody preparations. Standard curves were generated with biotinylated anti-ALX40-4C (1/1,500) (animal 3922) (Œ), unfractionated serum from animal 3922 (1/1,000) (E), and reference serum (1/500) (1). Standard curves were generated, and the dilutions of serum were chosen so that the standard curves could be superimposable. The last two standard curves were generated by the indirect ELISA system, whereas the first standard curve was generated by the direct assay.
steeper slope and higher OD; however, the shape of the curve closely resembled that obtained with the two serum samples from animal 3922. The sensitivity and validity of the assay were assessed by measuring the levels of ALX40-4C in spiked samples of human serum that were prepared and processed in a double-blinded fashion. Samples from patients who received drugs in part of two phase-1 clinical trials were also tested. The spiked samples were assessed by each of three systems: (i) the indirect-competition ELISA with the original reference rabbit anti-ALX404C antiserum, (ii) the indirect-competition ELISA with the unprocessed anti-ALX40-4C antiserum from animal 3922, and (iii) the direct-competition ELISA with the biotinylated antiALX40-4C IgG fraction from animal 3922. The samples from
TABLE 1. Predicted values for unknown samples Predicted value by assay method Sample no.
1 2 3 4 5 6 7 8 9 10 11 a
Indirect assay
Direct assay
Reference % Vari- 3922 % Vari- 3922 bio- % Variserum ation serum ation tin label ation
0.158 1.014 3.292 0.137 7.039 —a — — — — —
—, offscale value.
58 15.9 5.9 174 0.55
0.042 0.641 3.328 0.036 7.402 0.023 0.036 0.182 0.314 1.361 0.032
58 27 5 29 6 17 414 17 28 22 128
0.05 0.784 3.74 0.036 9.35 0.018 — 0.159 0.353 1.542 —
50 10 7 29 34 35 28 19 12
Acutal (mg/ml)
0.1 0.875 3.5 0.05 7 0.027 0.007 0.22 0.438 1.75 0.014
FIG. 5. Levels of ALX40-4C in serum from patients infused with ALX40-4C. The patients were infused with ALX40-4C, and after 60 min following infusion a sample of blood was taken. The serum samples were obtained, labeled, and stored at 280°C until they were used in the assays. Groups of patients were infused with 0.07, 0.21, 0.42, 0.56, and 0.70 mg/kg of body weight. (A) Clinical trial in which a placebo group was also included in each group discussed above (with the results reported in a separate column). All samples were analyzed in a double-blinded fashion, and only after the trial was over were the identities of the samples made known. Points, mean levels of ALX40-4C in one patient; bars, means of all of the patients within a group. (A) Four patients per group, three of whom received ALX40-4C; (B) eight patients per group of whom each received the drug. Conc, concentration.
patients were assessed either by the indirect assay or by the direct-competition assay with biotinylated antibody. The results shown in Table 1 demonstrate that the directcompetition ELISA with the biotinylated anti-ALX40-4C IgG fraction from animal 3922 was as good as the indirect-competition ELISA carried out with the unprocessed serum from animal 3922 and was better than the indirect-competition ELISA with the original reference serum. We could detect ALX40-4C over a broader range of concentrations with serum from 3922 than with the original reference serum; the dynamic range of the assay allowed detection of ALX40-4C at concentrations ranging from 0.027 to 7 mg/ml. The capability of ALX40-4C detection with the antiserum from animal 3922 was maintained subsequent to development of the biotin-streptavidin system as assessed by comparison of percent variation values. Percent variation was calculated as follows: (predicted value 2 actual value/actual value) 3 100. The final assay configuration allowed samples to be analyzed in 6 h, with fewer manipulations. Both of these factors are important when working with samples from HIV-infected patients and reduce the chance of technical errors. The assays
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were used in two phase-1 trials to measure ALX40-4C from patient serum. Both trials used the same dose of ALX40-4C, and samples were drawn at the same time points. However, in one trial the indirect assay was used while in the second trial the direct assay was employed. The results show no significant difference in the levels of ALX40-4C in groups of patients receiving identical drug doses taken at the same time as measured by either assay. There is less variability with the direct assay than what was observed with the indirect assay (Fig. 5A and B). In conclusion, we have developed an assay that is sensitive and accurate for the determination of levels of ALX40-4C in serum samples of patients infected with HIV. This was achieved with the use of biotinylated antibodies and by performing all of the reactions on an ELISA plate. REFERENCES 1. Bomford, R. 1980. The comparative selectivity of adjuvants for humoral and cell-mediated immunity. Clin. Exp. Immunol. 39:426–434. 2. Cantarero, L. A., J. E. Butler, and J. W. Osborne. 1980. The absorptive characteristics of proteins for polystyrene and their significance in solidphase immunoassays. Anal. Biochem. 105:375–382. 2a.Coligan, J. E. 1997. Peptides, p. 9:0:1–9:8:15. In J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevack, and W. Strober (ed.), Current protocols in immunology. John Wiley & Sons, New York, N.Y.
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