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Bioconjugate Chem. 1999, 10, 496−501

Purified Protein Derivative (PPD) as an Immunogen Carrier Elicits High Antigen Specificity to Haptens Binodh S. De Silva,† Kamal L. Egodage,‡ and George S. Wilson* Department of Chemistry, Malott Hall, University of Kansas, Lawrence, Kansas 66045. Received June 23, 1998; Revised Manuscript Received February 12, 1999

The effectiveness of the carrier protein in eliciting antigen-specific antibodies was investigated. The effect of the carrier protein was independent of the conjugation chemistry involved. Keyhole limpet hemocyanin (KLH), purified protein derivative (PPD), and ovalbumin (OVA) were used as carrier proteins in the immunization of mice. Three antigens were studied: LY170881 (a small drug molecule), 4-[1′-cyanobenz(f)isoindolyl]butyric acid (CBI-butyric acid), and a seven residue peptide GPGRGPG (KLE1). The serum antibody response to the antigen or antigen:BSA conjugate was superior in the case where the PPD:antigen conjugates were used as the immunogen when compared to KLH and OVA. The specificity of the antibodies to the respective antigens vs cross-reactivity with the carrier protein was investigated. PPD-coupled antigen immunized mice generated a higher percentage of antigen-specific hybridomas compared to the other carrier proteins. These findings confirmed PPD as the best carrier molecule for the production of both polyclonal and monoclonal antibodies.

INTRODUCTION

With the advent of monoclonal antibodies (Ko¨hler and Milstein, 1975), many analytical and clinical applications have relied on the highly specific antigen antibody interaction made possible by this methodology. In theory, antibodies could be made to any synthetic molecule, protein, nucleic acid, carbohydrate, or lipoprotein. With the extensive understanding about processes involving the immune system, it has now become possible to characterize the physical requirements of an antigen which result in improved production of antibodies (Eshhar, 1985). Antigens in general can be classified into two major groups; those that are intrinsically immunogenic and those that have to be conjugated to a carrier to elicit an immune response. There is considerable interest in producing monoclonal antibodies to such small molecules including, for example, drugs, pesticides, and peptides. Typically, an antigen possesses an epitope that can be recognized by surface immunoglobulins on B-cells. However, this is not sufficient to elicit an antibody response. In addition to the B-cell epitope, the antigen has to possess a class II MHC T-cell receptor site that enables the cascade of events resulting in the production of antibodies to a particular antigen of interest. Haptens in classical immunology lack this T-cell epitope. Landsteiner (1936) showed that the T-cell epitope can be introduced by conjugating to a carrier protein, using what he called the “artificial conjugated antigens”. Since then, many approaches have attempted to improve the methodology for producing anti-hapten and anti-peptide antibodies. Some of the strategies used are incorporation of T-cell epitopes (Francis et al., 1987; Good et al., 1987), * To whom correspondence should be addressed. Phone: 785864-5152.Fax: 785-864-5156.E-mail: [email protected]. †Current address: Procter & Gamble Pharmaceuticals, Route 320, Woods Corners, Norwich, NY 13815. ‡ Current address: Monsanto Co., 700 Chesterfield Parkway North, St. Louis, MO 63198.

preparation of immunostimulating complexes (Morein et al., 1987), use of a variety of adjuvants and liposomes to enhance the longevity of the antigen of interest in the body (Kawamura and Berzofsky, 1986), and cross-linking of the antigen with antibodies against surface receptors on B-cells, T helper cells or other antigen presenting cells to target the antigen to the cells involved in the immune system (Snider et al., 1987; Hendrickson et al., 1994). Multiple antigen peptide (MAP) where both the B-cell and T-cell epitopes are incorporated into one peptide antigen and synthesized as a single molecule (Tam, 1989) has been successfully used to elicit immune response. This latter approach eliminates the conjugation of peptides to proteins and uses solid-phase peptide synthesis to prepare the immunogen. Even with the advancement of methodology for the preparation of monoclonal and polyclonal antibodies, the use of the hapten-protein conjugates or peptide-protein conjugates as immunogens still remains the most common method of choice. The simplicity of these techniques, which do not require sophisticated equipment, and the flexibility of choosing a conjugation method applicable to the analyte of interest (use of NH2, COOH, and SH groups for coupling) favors this mode of immunogen preparation. However, such preparations must strive to preserve the integrity of the antigen structure, since many applications involving these antibodies require the discrimination of analogues with very small conformational differences. There are number of important characteristics to consider when preparing these conjugates. For example, the specificity of the assay, the metabolites of interest, the characteristic site of the antigen that is important for the specificity of the assay, the carrier molecule, epitope density, the method of conjugation, and the chemistry of the linking group between the hapten and the carrier will determine the properties of the resulting antibody. An important unit of the conjugate is the carrier molecule which provides the T-cell epitope for the production of a successful immune response. Common car-

10.1021/bc9800724 CCC: $18.00 © 1999 American Chemical Society Published on Web 04/15/1999

Purified Protein Derivative as an Immunogen Carrier

rier proteins such as serum albumin (bovine, human) hemocyanin, ovalbumin, g-globulins of various species, thyroglobulin, tetanus toxoid, peptide antigens, and synthetic carriers such as poly-L-lysine have been used extensively (Plaue et al., 1990). The protein carrier chosen must come from a species different from the animal to be immunized (Walker et al., 1973). The proteins to be used as carriers must possess functional groups that can easily be substituted (such as amines or sulfhydryls). The molar ratio of the hapten:protein will greatly influence the immune response and this depends on the coupling method used. Generally a hapten:protein molar ratio of 10:1 is maintained in our laboratory (Tijssen, 1985). The hapten-protein conjugate once administered to an animal will elicit antibodies that have a specificity to the haptenic group as well as the carrier. While the carrierspecific antibodies could be removed by affinity purification using a column with immobilized carrier protein, antibodies that bind part of the hapten or peptide and the carrier protein will also be removed. More critical, however, is the impact of carrier-specific antibodies on the hybridoma screening process. In the preparation of monoclonal antibodies, the hybridoma supernatants are typically screened first against a hapten-protein conjugate, where the protein is different to that used in the immunization. These supernatants were subsequently screened against the carrier protein used in the immunogen to estimate the number of hybridomas specific to the carrier. If a positive response is obtained, then the well will likely be discarded even if it also may contain antigen-specific antibodies. Since carrier-specific antibodies can correspond to a significant fraction of the antibodies produced, their elimination will greatly simplify identification of the desired antibodies, thus resulting in significantly higher yields of specific antibody hybridoma cell lines. Keyhole limpet hemocyanin (KLH), ovalbumin (OVA), and bovine serum albumin (BSA) are typical carriers used in the preparation of hapten-protein conjugates. However, purified protein derivative (PPD), currently used in the measurement of T-cell reactivity in persons exposed to tuberculosis or vaccinated with Bacillus Calmette-Gue´rin (BCG), has been suggested as an ideal carrier candidate (Lachmann et al., 1986).Their studies indicate the advantages of PPD as a carrier molecule and as an adjuvant in vaccine preparations. The present study deals with the properties of PPD as a carrier for the preparation of anti-hapten antibodies. These studies were carried out using two haptens (small organic molecules) and a peptide. The antigens used were LY170881 (a small drug molecule), 4-[1′-cyanobenz(f)isoindolyl]butyric acid (CBI-butyric acid) (Figure 1) and a seven residue peptide, GPGRGPG (KLE1). EXPERIMENTAL SECTION

Materials and Methods. LY170881 and its conjugates with BSA, PPD, OVA, and KLH were provided by Lilly Laboratories (Indianapolis, IN). The seven residue peptide (GPGRGPG) (KLE1) was synthesized by Coast Scientific Products (La Jolla, CA). 4-[1′-Cyanobenz(f)isoindolyl]butyric acid (CBI-butyric acid) was synthesized at the University of Kansas (Orosz and Carlson, 1990). All buffer components used were of analytical grade and purchased from Fisher Scientific, Fairlawn, NJ. BSA, and KLH were purchased from Sigma Chemical Co. (St. Louis, MO). PPD was provided by Lederle Laboratories, NY.

Bioconjugate Chem., Vol. 10, No. 3, 1999 497

Figure 1. Structures of the antigens used. (A) 4-[1′-Cyanobenz(f)isoindolyl]butyric acid. (B) LY170881. Table 1. Properties and the Dosage of the Immunogens Used immunogen

linking group

dose/mouse (µg)

LY170881-PPD LY170881-KLH LY170881-OVA CBI-butyric acid-PPD CBI-butyric acid-KLH KLE1-PPD KLE1-KLH

anhydride hemisuccinate anhydride hemisuccinate anhydride hemisuccinate carbodiimide carbodiimide carbodiimide carbodiimide

30 30 30 30 10 25 25

Preparation of the Immunogens. KLH and PPD were used as the protein carriers in the conjugation of the peptide and CBI-butyric acid used for immunizations.The CBI-butyric acid and the KLE1 peptide were coupled through the -COOH group by first activating it with water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, Pierce, IL) according to the enhanced carbodiimide coupling method of Staros et al. (1986), using sulfo N-hydroxysuccinimide (Sulfo-NHS, Pierce, IL) as the enhancer. The molar ratio of KLH: antigen was 1:100. One milligram of antigen was dissolved in 1 mL of 0.01 M phosphate buffer (PB), pH 7.4, containing no azide. EDAC was added to a final concentration of 0.1 M. After stirring for a few minutes, sulfoNHS was added dropwise (1.25 times antigen concentration). Finally, 1 mg of KLH was added and the solution was made up to 2 mL. The reaction was allowed to proceed for 24 h at room temperature. The order of the addition of the reagents is critical to obtaining a successful conjugate. The PPD-antigen and the BSA-antigen conjugates were prepared in a similar manner using a molar ratio of protein:antigen of 1:10. All conjugates that contained the LY170881 antigen were prepared by the Lilly Laboratories using the mixed anhydride method (Bowsher et al., 1994). Immunization Schedule. Immunogens were administered to 4-6 week old female BALB/c mice. The immunogens used and their respective dosages are given in Table 1. One week prior to the first immunization with the respective PPD conjugates, the mice were sensitized with 50 µg of Bacillus Calmette-Gue´rin (BCG; RIBI Immunochemical Research Inc., MT) via the intramuscular (i.m.) route in 50 µL of sterile saline. The rest of the immunization protocol was as follows: the first immunization was intraperitoneal (i.p.) at four sites with 100 µL/site in complete Freund’s adjuvant (CFA; Gibco Laboratories, NY) at a 3:1 v/v (adjuvant:conjugate) ratio. The second immunization was intravenous (i.v.) in sterile saline at a ratio of 2:1 v/v (saline:conjugate) after 2 weeks. Biweekly i.p. immunizations (four sites with 100 µL/site)

498 Bioconjugate Chem., Vol. 10, No. 3, 1999

in incomplete Freund’s adjuvant (IFA; Gibco Laboratories, NY) were administered until a desired circulating antibody concentration (1:1000 dilution) specific to each immunogen was attained in the serum. A v/v ratio of 3:1 (IFA:conjugate) was maintained. The conjugates prepared with KLH and OVA as the carriers were employed in mouse immunization in a manner similar to that described above but without the BCG sensitization. Fusion. Four days after the final immunization, the spleen of the hyperimmunized mouse was fused with the myeloma cell line Sp2/0-Ag-14 (ATCC CRL 1581; American Type Culture Collection, Rockville, MD) according to Galfre´ and Milstein (1981). A spleen cell: myeloma cell ratio of 10:1 was used and poly(ethylene glycol), molecular weight 3350 (Sigma, St. Louis, MO), was used as the fusogen. Typically, the cell pellet of fused cells is diluted in a large volume (almost 40-50 mL) of growth medium to ensure a reasonable dilution and seeded on 10-12 plates on average, at 50 µL/well. After 2 weeks, the supernatants were screened for specific antibody production using enzyme linked immunosorbent assays (ELISA) as described below. Screening Procedures. All ELISA procedures were designed to screen for antibodies that reacted with the antigen of interest and the carrier used in the immunizations. Two general ELISA procedures are reported in this manuscript because the antigens used in this work were used for different analytical purposes. The ELISA methods have to be designed in such a way as to get the best antibodies that recognize the antigen of interest and not the carrier protein. Therefore, the following ELISA methods will be described in detail for each antigen. ELISA Method 1. This ELISA method will allow the selection of antibodies that would recognize the antigenprotein conjugate. The carrier protein used in the ELISA procedure is different from the one that was used in the immunization. This eliminates the selection of antibodies to the carrier protein used in the immunizations. The hybridoma clones that secrete antibodies recognizing the antigen and/or the linking group can be selected using this procedure. This ELISA method was used to screen against BSA-CBI butyric acid and BSA-LY170881 antigen conjugates. The ELISA method used was a modification of Bahr et al. (1980). The ELISA plates (Corning, NY) were coated with 100 µL of antigen-BSA conjugate or the respective carrier proteins (BSA/PPD/ KLH/ovalbumin) (10 µg/mL in 0.1 M carbonate buffer, pH 9.35) (plate coating buffer) overnight at 4 °C. The plates were washed four times with 0.01 M PBS containing 0.05% Tween 20 (Sigma, St. Louis, MO) (wash buffer) and patted dry on a stack of paper towels. Wash buffer containing 0.2% BSA (150 µL/well) (ELISA diluent buffer) was added and incubated for 1 h at 37 °C to block the remaining active sites on the plastic surface. The plates were washed four times with wash buffer and patted dry. Serum (1:100 dilution) or culture supernatant (50 µL/ well) was added and incubated for 1 h at 37°C. The plates were washed four times with wash buffer and patted dry. Horseradish peroxidase (HRP) labeled goat-anti-mouse (goat anti-Mo-HRP, Organon Teknika Corp., Durham, NC) immunoglobulin (100 µL, 1:10,000 dilution) was added and incubated at 37 °C for an additional hour. The plates were washed four times with Nanopure water (Barnstead, Nanopure II Sybron, Barnstead, MA) and patted dry. A total of 100 µL of HRP substrate (tetramethyl benzidine (TMB) and hydrogen peroxide, KP Laboratories, MD) was added, and after 20 min, the enzyme reaction was quenched with 1 M HCl. The optical density was read at 450 nm using a 96 well plate reader.

De Silva et al.

(Vmax, Molecular Devices, CA). The screening procedures for LY170881 used the wash buffer and the ELISA diluent buffer recommended by the Lilly Laboratories. In ELISA experiments, using supernatant, serum from an immunized mouse served as the positive control and serum from an unimmunized mouse was used as the negative control. ELISA Method 2. The primary purpose in preparing antibodies to the peptide, KLE1, was to select antibodies that recognized the native structure of the parent protein. Therefore, during the selection process, it was necessary to identify antibodies that recognized the component peptide only. The following ELISA method was used to fulfill this purpose: 100 µL of the KLE1 peptide antigen (20 µg/mL) was coated on ELISA plates in 0.1 M plate coating buffer and incubated overnight at 4 °C. The plates were washed four times with wash buffer and blocked with 100 µL of 0.2% gelatin in wash buffer to block the remaining active sites on the plates. The plates were incubated at 37 °C for 1 h. The plates were washed four times with wash buffer and patted dry. Serum (1:100 dilution) or culture supernatant (50 µL/well) was added and incubated for 1 h at 37 °C. The plates were washed four times with wash buffer and patted dry. A total of 100 µL of goat anti-Mo-HRP conjugate was added (1: 15000 dilution in wash buffer containing 0.2% gelatin) to each well and incubated at 37 °C for an additional hour. The plates were washed four times with Nanopure water and patted dry. A total of 100 µL of HRP substrate was added, and after 20 min, the enzyme reaction was quenched with 1 M HCl. The optical density was read at 450 nm using the 96 well plate reader. Data Analysis. The statistical analysis of the results was done using the Minitab statistical program (version 8.21, State College, PA). RESULTS

Serum Response to Antigens. The two ELISA procedures described above were specifically aimed at evaluating the antibody responses to suit the particular analytical method under development. Figures 2 and 3 show the antibody titers in serum for the respective antigens/antigen-BSA and carriers used. At least two mice were immunized with each antigen. The optical density data were statistically analyzed using the Minitab program, and the results for the different animals were pooled where the data showed no significant difference at a 95% confidence level as shown by a one way ANOVA. Figure 2 compares the antibody titers to the peptide, CBI-butyric acid-BSA conjugate, and their respective carrier proteins. When KLH was used as the carrier, the immune response to KLH was far superior to that for the antigen/antigen-BSA of interest. The immune response to PPD on the other hand was significantly lower compared to that for the respective antigens. These results agree with the fact that PPD is a T cell antigen and does not elicit a favorable immune response (Lachman et al., 1986). Figure 3 shows the serum titers to the LY170881-BSA conjugate and the respective carrier proteins used in the immunization procedures. The antibody responses to the BSA-LY170881 conjugate are comparable to that of the carriers in the case where PPD and OVA were used. The serum titer for PPD is around 51 200 and to OVA is 25 600. The KLH response is far superior to the BSALY170881 response. Fusion. A minimum of two fusions were performed from each category. The hybridoma supernatants were

Purified Protein Derivative as an Immunogen Carrier

Bioconjugate Chem., Vol. 10, No. 3, 1999 499

Figure 2. Serum dilution curves for the antigens and their respective carriers used in the immunizations. The solid symbols indicate the response to the antigen or the peptide, and the open symbols indicate the serum response to each carrier molecule. (a) KLE1PPD [(b, 1) antigen response for two mice, (O, 3) carrier for two mice], (b) KLE1-KLH [(b) average antigen response for two mice, (O) average carrier response for two mice], (c) CBI-butyric acid-PPD [(b, 9, 2, 1) antigen response for four mice, (O) average carrier response for four mice], (d) CBI-butyric acid-KLH [(b) average antigen response for two mice, (9) antigen response for one mouse, (3) average carrier response for two mice, (]) carrier response for one mouse].

tested for the production of antibodies against the BSAconjugated CBI butyric acid, BSA-conjugated LY170881, KLE1 peptide, and the respective carriers. The results are summarized in Table 2. The percent of specific wells were calculated as follows:

% specific wells ) (number of wells positive toward the antigen/carrier)/ (total number of growing wells) × 100 These results clearly indicate that PPD does elicit a superior antigen-specific immune response compared to that of KLH. The percentage of antigen-specific hybridoma wells (Table 2) is about four times higher for PPD than for KLH. The higher percentage of antigenspecific hybridomas makes available a larger number of cell lines from which to choose the appropriate monoclonal antibodies for the specific application. The percentage of specific wells to KLH and OVA was not calculated for the LY170881 antigen, since over 90% of the supernatants screened showed cross reactivity between the carrier and the antigen of interest. DISCUSSION

This study evaluates the effectiveness of different protein molecules as carriers used in the production of antibodies against haptens and small peptides irrespec-

tive of the conjugation process. The primary goal of this project was to enhance the production of anti-hapten/antipeptide antibodies in systems which are not intrinsically very immunogenic. The ELISA procedures described in the previous section served two purposes: the selection of antigen specific antibodies and the elimination of carrier-specific antibodies. These screening procedures were carefully designed in order to select antigen-specific antibodies which are useful in the analytical procedures that were envisioned for these antibodies. The serum and the fusion results clearly indicate a dramatically reduced carrier response of PPD over KLH and OVA. It has been reported that the efficacy of each carrier protein and the immune response it elicits are difficult to compare since they vary depending on the individual and the species (Pratt, 1978). These variations were taken into consideration during these experiments, and the conditions were kept as comparable as practically possible in designing the immunogens. Furthermore, the animals used were of similar age and belonged to the same species. The serum titer results we have obtained show great promise when preparing polyclonal antibodies (Figure 2 and Figure 3) and were measured in the serum after the immunization. The low/no titers shown for PPD response is a clear indication of the exceptional effect of PPD in eliciting a specific antibody response to the antigen of

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De Silva et al. Table 2. Fusion Results

immunogen

% antigen specificity (a)

KLE-PPD KLE-KLH CBI-butyric acid-PPD CBI-butyric acid-KLH LY170881-PPD

20 7 30 29 4.1

a

Figure 3. Serum dilution curves for LY170881-BSA conjugate and the carrier proteins used in the immunization. (a) LY170881-PPD [(b) average antigen response for two mice, (O) average carrier response for two mice], (b) LY170881-KLH [(b) average antigen response for two mice, (O) average carrier response for two mice], (c) LY170881-OVA [(b) average antigen response for two mice, (1) antigen response for one mouse, (O) average carrier response for all three mice].

interest without itself eliciting and immune response. When both KLH and OVA were used, the majority of the time the serum antibody response to the carrier obscured that of the antigen of interest. This poses a great difficulty when purifying the serum to obtain polyclonal antibodies, which are antigen specific, due to the high concentration of antibodies to carrier molecules. The strong carrier response has the effect of reducing the antigen-specific antibody concentration in the IgG fraction.

% carrier specificity (b) PPD

KLH

[(a)/(a + b)] × 100

13 na 7.5 na 2

naa 40 na 95 na

61 15 80 23 68

na, not applicable.

The major advantage of producing antigen-specific antibodies lies in the preparation of monoclonal antibodies. When the specificity toward the antigen is greater than that of the carrier, the selection of optimal antibodies for a particular assay becomes trivial. The high percentage of antigen-specific hybridomas (Table 2) in PPD immunized mice confirm the reduction of carrierspecific hybridomas by using PPD as a carrier molecule. In the case of KLE1 peptide, when PPD was used as a carrier molecule 61% of the wells screened for carrier and antigen were antigen specific as opposed to 15% for KLH. The 4-fold increase in the antigen-specific hybridomas will increase the probability of selecting the optimal cell line for the subsequent analytical procedure. The serum antibody response may or may not be a true predictor of the number of specific hybridoma cells that are produced during a fusion. However, the number of hybridoma wells that cross-reacted with KLH in both the peptide and the CBI-butyric acid was greater than those for PPD (Table 2). The specificity to the antigen is almost 12 times greater when PPD was used as the carrier in CBI-butyric acid. It could be argued that the antigen was not exposed to the surface (epitopic suppression) in KLH due to the large molecular weight of this protein. However, this should be not the case since the molar ratios in each situation were comparable. These results strongly support the fact that PPD is indeed a T-cell antigen. The KLE1 peptide specificity results are also very encouraging. The fusion results were much superior when the PPD conjugates were used as the immunogen when compared to KLH (Table 2). The results show no advantage to using KLH in obtaining peptide-specific antibodies. Peptides typically have low antigenicity, particularly if they are naturally occurring. To increase the antigenicity, the most common approach has been to use KLH whose immunogenicity KLH masks the production of peptide-specific antibodies, a major disadvantage when preparing peptide-specific antibodies. The role of PPD therefore is to act as a T-cell activation leaving the B-cells to respond to the peptide (Lachmann et al., 1986). PPD has not been extensively used in the preparation of protein-hapten conjugates. The principal practical use of PPD for the last half century has been as an in vivo diagnostic test for previous encounters with Mycobacterium tuberculosis (Lachmann, 1988). However, its potential in the “heterogenization” of tumor cells and the results of these studies in the production of antibodies against tumor antigens has been very encouraging (Lachmann et al., 1981; Sia et al., 1984). PPD has being used by Lachmann et al. (1986) in the preparation of peptide conjugates that were used in the production of anti-peptide antibodies. Antibodies against PPD cannot be precipitated in the Farr assay (Lachmann, 1988), but there has been an ELISA method developed to detect PPD-specific antibodies in the detection of tuberculosis in children (Barrera et al., 1989). Our results do show a

Purified Protein Derivative as an Immunogen Carrier

weak antibody response to PPD compared to the other carriers used. On the basis of the Farr assay, Lachmann (1988) defines PPD as a poor B-cell antigen. However, the antibodies that are present in our experiments could be against the impurities present in the protein preparations since it is not clear that the PPD used in most of the clinical testing is of 100% purity (Lachmann et al., 1986). KLH, BSA, and OVA are excellent carrier proteins commonly used in the preparation of hapten-protein conjugates. We have clearly described the exceptional ability of PPD as a carrier in the production of hapten-specific antibodies. Although this unique characteristic of PPD as a carrier protein is not completely understood, it is assumed that the denatured structure of PPD could be responsible for the activation of T-cell receptors. It is conceivable that as the T-cells recognize processed antigenic epitopes, the heat-denatured PPD could be a potential target for their activation. The activity of PPD is enhanced only in animals previously exposed to Mycobacterium tuberculosis or immunized with BCG. These animals show a delayed-type hypersensitivity (DTH) when challenged with PPD. This, on the other hand, may cause a retention of the antigen for a prolonged period of time at the site of the injection, thus giving rise to an adjuvant effect. This has invoked an interest in using PPD in vaccine preparations. Thus PPD has demonstrated its usefulness as a carrier molecule in the preparation of hapten-protein/peptide-protein conjugates and its potential in vaccine preparation as an adjuvant. ACKNOWLEDGMENT

This work was supported in part by the Center for BioAnalytical Research at University of Kansas, the Procter and Gamble Company, and Eli Lilly Laboratories. We thank Lederle Laboratories for the generous donation of the PPD. LITERATURE CITED (1) Bahr, G. M., Rook, G. A. W., Moreno, E., Lydyard, P. M., Modabber, F. Z., and Stanford, J. L. (1980) Use of the ELISA to screen for anti-thymocyte and anti-b2-microglobulin antibodies in leprosy and SLE. Immunology 41, 865-873. (2) Barrera, L., Miceli, I., Ritacco, V., Torrea, G., Broglia, B., Botta, R., Maldonado, C. P., Ferrero, N., Pinasco, A., Cutillo, I., Cornejo, M., Prokopio, E., and De Kantor, I. (1989) Detection of circulating antibodies to purified protein derivative by enzyme-linked immunosorbent assay: its potential for the rapid diagnosis of tuberculosis. Pediatr. Infect. Dis. J. 8, 763-767. (3) Bowsher, R. R., Compton, J. A., Kirkwood, J. A., Place, G. D., Jones, C. D., Mabry, T. E., Hyslop, D. L., Hatcher, B. L., and De Sante, K. A. (1994) Sensitive and specific radioimmunoassay for Fialuridine: Initial assessment of pharmacokinetics after single oral doses of healthy volunteers. Antimicrobial Agents Chemother. 38 (9), 2134-2142. (4) Eshhar, Z. (1985) Monoclonal antibody strategy and techniques. in Hybridoma technology in the Biosciences and Medicine (T. A. Springer, Ed.) pp 3-41, Plenum Press, New York. (5) Francis, M. J., Hastings, G., Syred, A. D., McGinn, B., Brown, F., and Rowlands, D. J. (1987) Nonresponsiveness to a foot and mouth disease virus peptide overcome by of foreign helper T-cell determinants. Nature 330, 168-170.

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