Enzyme-Linked Immunosorbent Assay Using a Recombinant ... - NCBI

CLINICAL

AND

DIAGNOSTIC LABORATORY IMMUNOLOGY, Jan. 1994,

p.

Vol. 1, No. 1

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1071-412X/94/$04.00+0 Copyright ©D 1994, American Society for Microbiology

Enzyme-Linked Immunosorbent Assay Using a Recombinant Baculovirus-Expressed Bacillus anthracis Protective Antigen (PA): Measurement of Human Anti-PA Antibodies LAUREN C. IACONO-CONNORS,'* JEANNE NOVAK,2 CINDY ROSSI,3 JOSEPH MANGIAFICO,3 AND THOMAS KSIAZEK3t Virology Division, ' Bacteriology Division,2 and Applied Research Division,3 U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011 Received 15 April 1993/Accepted 15 July 1993

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera.

Bacillus anthracis is the etiologic agent of anthrax, a zoonotic disease of domestic livestock and wildlife. Human infection occurs through direct contact with infected animals, unprocessed animal products, or anthrax spores. Anthrax vaccines for humans and animals are currently available in the United States. The current veterinary vaccine is composed of the spores of an attenuated, toxigenic, nonencapsulated strain of B. anthracis originally described by Sterne (16). The primary immunogenic component of the current human vaccine in the United States, prepared by the Michigan Department of Public Health (MDPH), is B. anthracis protective antigen (PA). The vaccine consists of aluminum hydroxide-adsorbed supernatant from fermentor cultures of a toxigenic, nonencapsulated strain of B. anthracis, V770-NP1-R (15). PA appears to be the only essential component of an effective anthrax vaccine (3, 4, 6, 7). The detection and measurement of anti-PA antibodies in an immunized individual is the only method to date for evaluating the immunization schedule. Typically, an effective immunization schedule for humans requires a course of three biweekly vaccine doses followed by three additional doses given 6, 12, and 18 months later (1). Annual immunizations are administered to vaccinees to maintain anti-PA titers. One major component of recent anthrax vaccine research has been the development of efficient assays to measure anti-PA antibodies in vaccine recipients. The most recent assays developed are enzyme-linked immunosorbent assays (ELISAs) (9, 12, 17). In these assays, PA is immobilized on the

surface of 96-well microtiter plates, diluted serum samples are added to each well, and anti-PA antibody titers in serum are measured by adding a secondary antibody conjugated to a detector enzyme. The ELISA for detecting anti-PA antibodies in human serum, described by Johnson-Winegar (9), is relatively sensitive and rapid compared with the indirect hemagglutination assay (2). One drawback in both of these assays is that false-positive results can occur. A similar ELISA is still used today to monitor anti-PA antibody titers in humans and in laboratory animals (8, 13, 17). With this ELISA as modified by Little et al. (13), we also observed false-positive results in a routine serosurvey of anthrax vaccinees. A second drawback in these assays is the requirement for purified PA. Purifying PA from the Sterne strain of B. anthracis is labor intensive and requires biological laboratory containment. In addition, purified PA is not always readily available. The objectives of this study were to develop an ELISA for detecting human anti-PA antibodies that would utilize readily available and inexpensive antibody and antigen reagents and eliminate false-positive test results. In this study, we used PA produced in recombinant baculovirus-infected Spodoptera frugiperda (Sf-9) cells as a source of antigen to develop a more cost-effective ELISA. In addition, we developed an antigen capture ELISA which does not require purified PA and which eliminates false-positive results. In this assay, a monoclonal anti-PA antibody (12) fixed to 96-well microtiter plates is used to capture either the recombinant PA obtained from lysates of Sf-9 cells infected with a recombinant baculovirus containing the PA gene (5) or purified Sterne PA. We demonstrated that this capture ELISA can accurately detect anti-PA antibody in serum from vaccinated humans regardless of the source of PA. In addition, this antigen capture ELISA eliminated false-positive results. Thus, an antigen capture ELISA with recombinant baculovirusexpressed PA is accurate, safe, and inexpensive for detecting anti-PA antibodies in human serum.

* Corresponding author. Mailing address: Department of Virus Diseases, Division of Comm. Dis. and Immunol., Walter Reed Army Institute of Research, Washington, DC 20307-5100. Phone: (202) 576-3655. Fax: (202) 576-0442. t Present address: Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333.

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ELISA TO DETECT B. ANTHRACIS PA ANTIBODIES

Vot- l, 1994

TABLE 1. Ability of antibody to capture PA'

MATERIALS AND METHODS PA preparation. Purified B. anthracis Sterne PA was provided by John Ezzell (Bacteriology Division, U.S. Army Medical Research Institute of Infectious Diseases) and prepared by the method of Leppla (10). Recombinant PA was obtained from Sf-9 cells infected with a recombinant baculovirus carrying the PA gene (5). Baculovirus recombinant-infected Sf9 cells were infected with 10 PFU per cell and harvested at >72 h postinfection or when cell viability was approximately 50% as determined by a dye exclusion test. Infected cells were pelleted at 1,500 x g for 10 min, and the supernatant was stored at 4°C as the virus stock. Cell pellets were resuspended in phosphatebuffered saline (PBS) at 20% of the original culture volume and then subjected to one cycle of freezing at - 70°C and thawing at 37°C. The cell suspension was immediately sonicated on ice three times for 30 s each at maximum output in a cup horn sonicator. The crude cell lysate was clarified by centrifugation at 4,000 x g for 15 min, and the supernatant was stored at - 70°C and used as the source of recombinantbaculovirus-expressed PA in the assays described below. General assay procedures. ELISAs were performed as described previously (14). Ninety-six-well polyvinyl chloride (PVC) microtiter plates (Dynatech, Vienna, Va.) were coated with 100 [l of either antigen or antibody diluted in 0.01 M PBS (pH 7.4). After overnight incubation at 4°C, the plates were washed three times with PBS-Tw (PBS [pH 7.4], 0.1 % Tween 20). All subsequent antibody or antigen reagents added to the plates were diluted in PBS-Tw containing 5% skim milk (Difco, Detroit, Mich.). After the addition of each reagent, the plates were incubated at 37°C for h and then washed three times with PBS-Tw. Serum samples were serially diluted from 1:1)00 in fourfold dilutions and then added to the wells of PAor nonspecific antigen (uninfected Sf-9 cell lysate)-coated microtiter plates. Bound human immunoglobulin G (IgG) was measured by incubating microtiter plates with horseradish peroxidase-conjugated mouse antibody against human IgG (Accurate Chemical and Scientific Co., Westbury, N.Y.) for I h at 37°C. Plates were washed as described above and incubated at 37°C with 2,2'-azino-di(3-ethylbenzthiazoline sulfonate) (Kirkegaard and Perry, Gaithersburg, Md.). Plates were read spectrophotometrically at 410 nm (11). The optical densities (ODs) were adjusted by subtracting the OD at 410 nm (OD4,0) of the nonspecific-antigen-coated wells from that of the PA-coated wells for each ELISA format. Positive OD cutoff values were determined as follows. The antibody titers in serum samples from four unvaccinated individuals were determined in both ELISAs exactly as for the test sera. The mean of the adjusted OD was determined for all four samples, and the standard deviation was calculated. An OD value was considered positive if it was greater than the mean OD value for the samples from unvaccinated subjects plus 3 standard deviations. The titer is equal to the reciprocal of the last dilution that was above or equal to the OD cutoff value. A serum sample was considered positive if the titer was greater than or equal to 100. Preparation of PA-coated microtiter plates (direct ELISA). Purified Sterne PA was used to directly coat the wells of 96-well PVC microtiter plates. PA was diluted in 0.01 M PBS (pH 7.4), and 100 ng of PA was adsorbed to each well of a PVC microtiter plate as described above. The optimal concentration of PA was determined by checkerboard titration of the antigen adsorbed onto the plate. The plates were then processed as described above. Preparation of PA-immunocaptured microtiter plates (capture ELISA). Purified Sterne PA (250 ng) or baculovirusinfected Sf-9 cell lysate containing PA (75 ng of PA) was

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Baculovirus PA

antibody

OD

Sterne PA Dilution

OD

Dilution

2F9-1-1 14B7-1-1 14C7-1-1

0.32 0.75 0.32

1:500 1:8,000 1:2,000

0.32 0.92 0.40

1:500 1:8,000 1:2,000

Monoclonal

" Optimal dilutions and corresponding OD values are indicated for capture of PA by monoclonal antibodies (see Materials and Methods). Bound antigen was detected by human serum obtained from an individual who had recovered from cutaneous anthrax.

captured onto the surface of the wells of PVC microtiter plates by a bound anti-PA monoclonal antibody (14B7-1-1) contained in mouse ascitic fluid (12). The selection and optimal dilution of an anti-PA monoclonal antibody-containing ascitic fluid were determined empirically by screening 37 anti-PA ascitic fluids (12) for their ability to capture PA on microtiter plates. The ability of the bound monoclonal antibodies to present PA epitopes effectively was determined by using a convalescentphase serum obtained from an individual who had recovered from cutaneous anthrax (provided by John Ezzell). The optimal dilutions of the reagents, monoclonal antibody, and antigen used in these assays were determined by titration of the selected ascitic fluid, PA antigens, and the positive human serum (data not shown). Assays were then performed as described above. Serum bank. Sera used to evaluate the two ELISA formats were obtained from 32 individuals vaccinated with MDPH-PA at the U.S. Army Medical Research Institute of Infectious Diseases as volunteers in the Special Immunizations Program (1, 15). Human immunization schedules. The MDPH-PA vaccination schedule consisted of six vaccine doses scheduled for days 0, 14, 28, 182, 364, and 475. Blood samples were obtained from vaccine recipients before initiation of the vaccination schedule, at the time of each subsequent booster dose, and on day 850. The actual days of vaccination and blood sampling varied slightly among the vaccinees. Therefore, data are presented as the mean day for each vaccination dose or blood sampling. RESULTS Development of a capture ELISA for detection of human anti-PA antibodies. The original ELISA for measuring anti-PA antibodies in vaccinated individuals was developed by Johnson-Winegar (9). In this direct ELISA format, native PA is adsorbed directly to the surface of PVC microtiter plates. We developed a capture ELISA to measure human anti-PA antibodies. For our capture ELISA, an anti-PA monoclonal antibody is first fixed to the surface of a PVC microtiter plate, and then PA is added and captured by the plate-bound antibody. The advantage to the capture format is that either purified Sterne PA or a crude preparation of recombinant-baculovirusexpressed PA can be used as the source of antigen. We screened 37 anti-PA monoclonal antibodies (12) in the capture ELISA format to determine which were effective in capturing and presenting purified Sterne PA or crude recombinant PA to known-positive human serum samples (Table 1). Three monoclonal antibodies, 2F9-1-1, 14B7-1-1, and 14C71-1, captured PA and gave positive assay results with either PA source. 14B7-1-1 was used routinely in the capture ELISA. Comparative analysis of direct and capture ELISAs. We compared the direct ELISA with the capture ELISA for the ability to determine the anti-PA antibody titers in serum

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MEAN DAY FIG. 1. Measurement of human vaccinee antibody responses to B. anthracis PA by direct and capture ELISAs. Sera were tested in the direct ELISA with purified Sterne PA bound directly to the plates (0) or in the capture ELISA with either purified Sterne PA (0) or recombinant baculovirus-expressed PA (A) captured by a monoclonal antibody against PA. (A) Comparison of direct ELISA and capture ELISA with purified Sterne PA. (B) Comparison of capture ELISA with baculovirus-expressed PA and capture ELISA with Sterne PA. Vaccination occurred on Mean days 0, 14, 28, 182, 364, and 475. Serum samples were obtained on Mean days - 25, 28, 84, 182, 364, 475, and 850, as plotted. Error bars show

standard deviations.

samples from 32 anthrax vaccinees at various time points during the MDPH-PA vaccination schedule (Fig. 1). A control serum sample was collected from every vaccinee before the initial immunization. We evaluated two different sources of PA for use in the capture ELISA. Figure 1A shows a comparison of the direct ELISA with the capture ELISA with purified Sterne PA as the antigen source. Figure 1B shows a comparison of purified

Sterne PA with recombinant-baculovirus-expressed PA in the capture ELISA format. We found that none of the unvaccinated control sera were positive in the capture ELISA when we used the recombinant-baculovirus PA, but two samples scored positive (titer, 100) when Sterne PA was used in the capture ELISA format (data not shown). We also found that six samples from the prevaccination group scored positive when the direct ELISA was used (data not shown). In all cases in

ELISA TO DETECT B. ANTHRACIS PA ANTIBODIES

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TABLE 2. Comparison of direct ELISA and capture ELISA for false-positive resultsa Serum sample

Patient 1 5 January 31 March 20 April 31 May 19 December Patient recovered from cutaneous anthrax MDPH-PA vaccinee Known negative

Direct ELISA

Capture ELISA

OD

Titer

OD

Titer

1.15 1.41 1.20 1.27 1.37 1.90

800 800 1,600 800 800 1,600

0.03 0.01 0.00 0.00 0.05 0.65