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Densitometric analysis of Western blot assays for feline immunodeficiency virus antibodies
Veterinary Immunology and Immunopathology 79 (2001) 261±271
Densitometric analysis of Western blot assays for feline immunode®ciency virus antibodies M. Calandrellaa, D. Matteuccib, P. Mazzettib, A. Polia,* a
Department of Animal Pathology, University of Pisa, Viale delle Piagge 2, I-56124 Pisa, Italy b Department of Biomedicine and Retrovirus Center, University of Pisa, Via San Zeno 37, I-56127 Pisa, Italy Received 29 June 2000; received in revised form 8 March 2001; accepted 16 March 2001
Abstract Western blot (WB) strips for antibodies directed to feline immunode®ciency virus (FIV) were analysed using re¯ectance densitometry by a semiautomatic densitometer. This method was used to quantify the antibody responses to different FIV proteins in both vaccinated and naturally or experimentally-infected cats. In order to increase reproducibility, reagents and protocols were accurately standardised and internal controls were added. In a ®rst format, an internal control band consisting of feline IgG was added to each blot to minimise the effect of band intensity variation. In a second format, antibody concentrations were calculated from the ratio of the dens ities produced by test sera and by positive and negative standard sera. The sera under scrutiny were also examined by standard enzyme-linked immunosorbent assay (ELISA) and the results obtained compared with those of the corresponding WB. A statistically signi®cant positive correlation was found between the results obtained with the two methods, and this was especially evident when ELISA titres were compared to corrected WB values (P 0:001). Densitometric analysis of WB assays allowed to quantify the antibodies against FIV proteins and might be useful to investigate possible humoral immune correlates of protection in FIV vaccination studies and antibody production in the early phase of infection. The quantitation of antibodies to Gag and Env FIV antigens might be used to obtain further informations on the course of FIV disease, as previously demonstrated in human immunode®ciency virus-1 (HIV-1) infections. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Western blot assay; Quantitative analysis; FIV; Antibody response
Abbreviations: BSA, bovine serum albumin; CV, coef®cient of variability; DR, density of re¯ectance; ELISA, enzyme-linked immunosorbent assay; FIV, feline immunode®ciency virus; HIV, human immunode®ciency virus; Ig, immunoglobulin; PCR, polymerase chain reaction; PBS, phosphate-buffered solution; RI, relative intensity; RIPA, radioimmunoprecipitation assay; SPF, speci®c pathogen free; WB, Western blot * Corresponding author. Tel.: 39-50-575970; fax: 39-50-540644. E-mail address: [email protected] (A. Poli). 0165-2427/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 2 6 5 - 3
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1. Introduction Feline immunode®ciency virus (FIV) is a lentivirus that induces persistent infections of domestic cats characterised by progressive deterioration of immune functions, neuropathological changes, and opportunistic infections similar to those associated with human immunode®ciency virus-1 (HIV-1) infection of humans. This virus also shares several biological and biochemical properties with HIV and it may help develop a rationale for the development of effective vaccines and therapies against AIDS infection (Bendinelli et al., 1995). Besides its importance as an animal model, FIV is a major pathogen of cats and therefore, important in veterinary practice. Genetic analysis of FIV has shown that the structural genes gag, env and pol are similar the ones of other lentiviruses and several proteins encoded by these genes have been identi®ed (Talbott et al., 1989; Morikawa et al., 1991). Serological studies have shown that, following infection, cats develop antibodies to both the nucleocapsid proteins and the envelope glycoproteins, but that qualitative and quantitative differences in the immune response occur between individual cats (Hosie and Jarrett, 1990). The humoral immune response of FIV-infected or immunised cats has been extensively investigated to identify the effectors responsible for protection and to de®ne possible correlates of vaccine-induced protection (Mazzetti et al., 1999). Antibodies to individual FIV proteins can be detected by Western blot (WB) and radioimmunoprecipitation assay (RIPA) using native FIV proteins (Steinman et al., 1990; Hosie and Jarrett, 1990) and enzyme-linked immunosorbent assays (ELISAs) using recombinant FIV Gag and Env proteins (Mermer et al., 1992; Rimmelzwaan et al., 1994; Calzolari et al., 1995). The development of these sensitive and speci®c ELISAs based on recombinant proteins allows the investigation of the production of antibodies to individual FIV antigens, but such studies are very timeand labour-demanding. WB and RIPA provide highly speci®c results, but they are usually not reliable for quantitating antibody production. The need for a more objective WB assay to better de®ne the immune status against FIV antigens prompted us to develop a quantitative WB assay. This report describes two WB assays that enabled us to determine the antibody response against single FIV antigens by using a computer assisted densitometer for image analysis. The results obtained using these quantitative WB assays were also compared with the results obtained by a standard ELISA. 2. Materials and methods 2.1. Cat sera The study was conducted on sera from 10 FIV-seropositive and 15 FIV-seronegative ®eld cats, and from 10 noninfected and 12 FIV-immunised speci®c pathogen free (SPF) cats. All cats were monitored serologically at selected time intervals for 28 months. The immunised cats were injected with a previously described vaccine (Matteucci et al., 1997), consisting of MBM cells acutely infected with a stock of the PISA M2 strain of FIV inactivated with paraformaldehyde. The cats were immunised subcutaneously at weeks 0, 3 and 6, and three boosters were administered 4, 10 and 16 months later,
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respectively. Of the 15 seronegative ®eld cats 2 seroconverted during the study period after they became infected, as demonstrated by virus isolation and PCR studies. Both ®eld and SPF cats were vaccinated against feline panleukemia virus, herpesvirus, and calicivirus (Feligen CR/P1, Virbac, Milan, Italy). The FIV-noninfected SPF control cats were housed in isolation units (Techniplast, Gazzada, Italy) at the Retrovirus Center of the University of Pisa. 2.2. Preparation of antigen for Western blotting The WB assays were performed with home-made strips, prepared with whole FIV antigen. Cell-free FIV-Petaluma antigen was produced from the supernatant of persistently infected FL4 cells and puri®ed by sucrose gradient puri®cation, followed by sonication and treatment with Triton X-100 (Yamamoto et al., 1991). 2.3. Western blotting procedures WB assays were performed as described previously (Poli et al., 1995) on Trans-Blot transfer medium (Bio-Rad Lab., Hercules, CA, USA) strips prepared using Mini-Protean II system (Bio-Rad). Brie¯y, FIV proteins were separated by preparative electrophoresis of detergent-treated, heat inactivated (1008C for 5 min) antigen preparation on sodium dodecyl sulfate-polyacrylamide (10%) gels in discontinuous buffer system and electrophoretically transferred to nitrocellulose sheets by Mini Trans-Blot Electrophoretic Transfer cell (Bio-Rad). The membrane was cut longitudinally into 2 mm wide strips. Each strip was treated with blocking buffer (5% dried milk in NaCl 150 mM, Tris±Cl 100 mM pH 7.5) for 2 h, and WB assays were conducted. Sera, diluted in PBS±Tween 20 (0.05%) containing 1% skim milk, were incubated with strips for 2 h at room temperature. After two washes with 0.05% PBS±Tween 20 for 10 min each, the reactivity of antibodies with antigen was detected by adding a rabbit horseradish peroxidase-conjugated anti-cat immunoglobulin (Ig)G (heavy- and light-chain), speci®c IgG (Nordic, Tilburg, The Netherlands) for 1 h. The strips were washed again and incubated in the dark with the enzyme substrate for peroxidase consisting of a solution of 10 ml methanol containing 6 mg of 4-chloro-1-naphthol (Sigma, St. Louis, MO, USA) added immediately before use to a solution of 45 ml of Tris pH 6.8 containing 6 ml of 10% hydrogen peroxide. Membranes were washed in distilled water and air dried. Analysis of the results was accomplished by capturing the strip images, locating bands using pre-stained molecular weight markers (Bio-Rad), measuring the re¯ectance density (DR) of anti-p15, -p25, -p31, gp40 and -gp95 antigen bands, and calculating the relative intensity (RI) by a video densitometer (Gel Blot-Pro Ultra Violet Prod., Cambridge, UK). To increase reproducibility of measurements and score band intensity two different assays with internal controls were developed and performed every time the sera were tested. In a ®rst format, WB A, puri®ed feline IgG (Jackson ImmunoResearch Lab., West Grove, PA, USA) were included in each strip and used as internal control to account for inter-assay variability. A serial dilution on a scale 1:2 was made from a concentrated solution of feline IgG (250 mg/1 ml PBS). From each dilution, 10 ml were withdrawn and transferred on the nitrocellulose sheets by using vacuum manyfold (BioRad). The RI of
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the single antigen bands was hence quantitated by comparing the DR of each band with the DR of the internal IgG control on each strip after substraction of the RI detected using negative control cat serum. In the second format, WB B, the reproducibility of measurements was increased by using the ratio of antibody concentrations as determined by including positive and negative controls in each experiment (Lacheretz et al., 1995). The DR was calculated with the formula: DR of test sample DR of negative control/DR of positive control DR of negative control. Because some FIV-negative cat sera can show weak bands against FIV antigen, sera from 10 SPF FIV-noninfected cats were used to determine cut-off values for the RI of each antigen band. The cut-off values were quanti®ed by adding three times the standard deviation to the means of the readings obtained with the negative control sera. 2.4. Standard ELISA for anti-FIV antibodies The antibody response to FIV was also determined by a standard ELISA, using for coating FIV-Petaluma virus grown in FL4 cells as described (Matteucci et al., 1997). ELISA plates (Probind, Falcon, Italy) were coated overnight with 0.5 mg per well gradient puri®ed, SDS-disrupted virus in 100 ml carbonate buffer (pH 9.6). After four washes with PBS±Tween 20, the plates were post-coated with 150 ml of PBS containing 1% bovine serum albumin (BSA) (PBS±BSA) for 1 h. Cat sera, serially diluted in 100 ml PBS containing 1% skim milk, 5% fetal calf serum, and 0.05% Tween 20 (dilution buffer), were added to the plate and incubated 1 h. Bound IgG antibodies were revealed by a horseradish peroxidase-conjugated mouse mAb anti-cat IgG (Sigma, St. Louis, MO, USA) diluted in 100 ml PBS±Tween 20±BSA. All steps were performed at room temperature. The enzyme reaction was carried out with 100 ml tetramethylbenzidine solution (0.4 mg/ml; KPL, Gaithersburg, MD, USA) and stopped with 100 ml 0.1N H2SO4; the absorbance was measured at 450 nm. The antibody titre was expressed as the reciprocal of the highest dilution of serum that gave optical density readings higher than the average values obtained with 20 control FIV-negative serum samples plus three times the standard deviation. 2.5. Statistical analysis To assess reproducibility, intra-assay (one serum with a strong reactivity for all FIV proteins in eight replicates) and inter-assay (three serum samples examined eight times) analysis were performed. Mean RI of each band and corresponding coef®cient of variability (CV) were calculated. The correlation between data obtained using the standard ELISA and the different WB assays was analysed by regression ANOVA and the Pearson correlation coef®cient using SPSS 8.0 (SPSS, Bologna, Italy). 3. Results To determine optimal WB conditions, different amounts of antigen and different dilutions of serum samples and of the conjugate were tested to obtain the highest
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Fig. 1. Quantitative Western blot assays and development of speci®c antibodies against individual FIV proteins in a cat immunised with an inactivated vaccine and boosted at 3, 6, 16, 40 and 66 weeks post-vaccination (arrow heads). Antibody production against single FIV proteins was quanti®ed: (A) comparing the relative intensity (RI) of each band with the RI of the internal feline IgG standard (WB A); (B) calculating the ratio of the RIs produced by test sera and by positive and negative standard sera (WB B).
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signal-to-noise ratios for positive and negative sera, respectively (results not shown). The optimal amount of antigen for preparing the strips was found to be 5 ng per strip. Optimal signal-to-noise ratios were found with sera diluted 1:100 and conjugate diluted 1:200. In the examination of the different dilutions of feline IgG used as internal control, a linear relationship between DR of bands and feline IgG dilution was observed (r 0:869; P 0:05). The optimal amount of feline IgG for quantifying the single bands of anti-FIV antibodies was found to be 0.04 mg per strip. Representative examples of the two quantitative WB assays are shown in Fig. 1. Serum samples of an immunised cat were examined with both WB formats. WB A used feline IgG as internal control (Fig. 1A), WB B was developed together with positive and negative control sera to determine ratio of antibody concentrations (Fig. 1B). With both formats, the highest intensities were detected against the gag products p25 and p15. Other FIV proteins consistently detected were, although with a lower RI, the env products gp95 and gp40 and the integrase p31. Cut-off values of RI obtained for each band corresponding to FIV antigens are presented in Table 1. Using WB A, the cut-off values for each FIV protein were lower than those observed with WB B. Both WB assays were also used to determine the seroconversion pro®le in cats which acquired infection during the follow-up period. Fig. 2 shows the course of seroconversion for p15, p25, p31, gp40, and gp95 determined using WB A. In the pre-infection samples, the RI of bands of the above proteins were constantly lower than the cut-off determined by using the sera from 10 SPF FIV-negative control cats. In the ®rst subject (Gassata; Fig. 2A), RI of antibodies to p15, p25 and p31 rose faster than RI of antibodies against gp40 and gp95. In the second cat (Umberto; Fig. 2B), the antibodies against all the FIV antigens increased concomitantly. Table 2 shows the relationships between the RI obtained with the two WB assays for the different bands corresponding to individual FIV protein and the results of a standard ELISA. A strong correlation was found between the results obtained with the two different WB assays for all the FIV antigens. The measure of antibody production against individual FIV antigens measured obtained with both WB formats was also statistically correlated with the antibody titres against whole FIV determined using a standard ELISA.
Table 1 Cut-off valuesa of relative intensity obtained for each antigen band using the two Western blot formats developedb FIV protein
WB A
WB B
p15 p25 p31 gp40 gp95
0.06 0.06 0.05 0.07 0.05
0.20 0.25 0.31 0.40 0.50
a
Mean of negative control sera plus three standard deviations. WB A: Western blot A with internal feline IgG standard; WB B in which the relative intensity (RI) of bands was calculated using the ratio of the RIs produced by test sera and by positive and negative standard sera. b
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Fig. 2. Seroconversion to FIV antigens after natural FIV infection. The levels of antibodies to single FIV antigens were quanti®ed using a quantitative Western blot assay with internal feline IgG standard (WB A). The ®gure shows the course of seroconversion of two cats (Gassata; A and Umberto; B).
To further evaluate the reproducibility of quantitation of FIV antibodies, the RI obtained using both strips from the same blot or antigen bound to different blots were compared in intra- and inter-assay tests. The results are presented in Table 3. Intra-assay CV ranged from 2.4% for gp40 and 95 to 3.4% for p31, while inter-assay CV ranged from 2.3% for p25 to 7.8% for p31. Table 2 Correlation between the results obtained with the two Western blot (WB) formats and between the relative intensities obtained with the two WB and anti-FIV titre determined with a standard ELISA FIV protein
p15 p25 p31 gp40 gp95
WB A vs. WB B
WB A vs. ELISA
WB B vs. ELISA
r
P
r
P
r
P
0.901 0.856 0.924 0.969 0.874
<0.00001 <0.00001 <0.00001 <0.00001 <0.00001
0.510 0.464 0.759 0.714 0.734
0.001 0.001 <0.0001 <0.0001 <0.0001
0.639 0.778 0.707 0.738 0.685
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
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Table 3 Intra- and inter-assay reproducibility of Western blot tests developed FIV protein
p15 p25 p31 gp40 gp95 Feline IgG
Intra-assaya
Inter-assayb
Mean relative intensityc
CVc (%)
Mean relative intensityc
CVc (%)
60 169.6 60.9 58.3 40.8 123.9
2.7 2.7 3.4 2.4 2.4 3.2
80.7 170.8 65 65.4 44.4 127.0
5.1 2.3 7.8 5.2 5.1 4.0
a Intra-assay reproducibility was determined using one serum with a strong reactivity for all FIV proteins in eight replicate. b Inter-assay reproducibility was calculated with three serum samples examined eight times. c Mean relative intensities of each band and corresponding coef®cients of variability (CV) were calculated.
4. Discussion Two quantitative WB assays, which were read using a video camera based densitometer connected to a personal computer for quantitative image analysis, are described. To evaluate the performances of the two assays, the humoral response to individual FIV proteins was studied in infected cats and in a longitudinal study of cats immunised with a ®xed cell vaccine. Using both formats, it was possible to determine the course of antibody production against the various FIV antigens. Of the two WB assays developed, WB A with the internal feline IgG standard, showed cut-off values for single FIV antigens markedly lower than WB B, when sera from SPF FIV-noninfected controls were used. In the WB A assay, the signals given by bands for anti-FIV antibodies using FIV-negative and -positive sera differ widely. Taking into account that automated quanti®cation of band intensities gives objective results, the presence of a low cut-off allowed to avoid the misclassi®cation of bands. A statistically signi®cant concordance was found when the results obtained with the two different WB assays were compared: the Pearson correlation coef®cients estimated for each pair of variables ranged from 0.969 for gp40 to 0.856 for p24 (P < 0:0001). The highest correlation was found for the RI of the bands p15, p31 and gp40. The RIs obtained with both WB assays for the individual FIV proteins were also correlated with total anti-FIV antibody titres as determined by a standard ELISA against whole virus. This demonstrated that the quanti®cation of antibodies against single FIV antigens by quantitative WB assays is comparable with the titres obtained using a standard ELISA. With regard to reproducibility of the results, both intra- and inter-assay precision tests gave acceptable results. It must be noted that the inter-assay CV of the RIs in the WB was lower than 10% for all the bands. In intra-assay tests, the CV was always below 5% for each band. Of the two different formats of quantitative WB developed, WB A, with internal feline IgG standard, was found to be preferable due to good correlation with the results obtained
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with a standard ELISA and to low cut-off values. Correct interpretations of band intensities is important because previous studies demonstrated that sera from ®eld and SPF cats can produce clearly recognisable single bands in WB although these subjects are not infected with FIV (Calzolari et al., 1995). This might be due to the fact that FIV sequences are also present in other pathogens of cats as was observed between HIV and pathogens such as Plasmodium falciparum and human T-cell lymphotropic virus-1 and -2 (McLaughlin et al., 1987). Gradient puri®ed retroviruses also contain components of the cell in which the virus is propagated or components of the fetal calf serum used for substrate cell growth (Pedersen and Barlough, 1991). Vaccination of cats with viral vaccines produced in feline cells is also likely to induce immune response to such antigens. Therefore, some cats vaccinated with viral antigens grown in cell culture may show false positive results with FIV viral antigens. The results of our study demonstrated that the quantitative WB assay with internal feline IgG standard (WB A) proved convenient for characterisation of speci®c antibody production against single FIV antigens. Mazzetti et al. (1999) previously described a quantitative WB to determine anti-FIV antibodies, in which strips were exposed to cat sera serially diluted until end point reactivity was reached. WB A has the advantage that speci®c anti-FIV antibody can be quanti®ed using a single dilution of test sera, thus saving time and labour. Accurate analysis of humoral responses in the FIV cat model is of particular importance in attempts to identify in vitro predictors of protective immunity in vaccination experiments and to characterise the pattern of antibody production after seroconversion in experimentally and naturally FIV-infected cats. In this longitudinal study of vaccinated cats, the quantitative WB assay developed proved easy to perform and offered signi®cant advantages over other methods, such as standard ELISA methods, which would require that the individual FIV antigens are ®rst obtained in puri®ed form to measure speci®c antibodies against single FIV antigens. Quanti®cation of antibody production in cats after seroconversion con®rmed that these subjects develop detectable antibodies to both FIV Gag and Env proteins, but that qualitative and quantitative differences in the immune response occur in individual cats (Rimmelzwaan et al., 1994). A further ®eld study on a large number of subjects following seroconversion could allow to determine the speci®city of the antibodies developed in the early phase of FIV infection. This would clarify whether the development of antibodies to Gag p25 protein occurs before (Hosie and Jarrett, 1990) or after antibodies to Env gp40 protein (Calzolari et al., 1995). Furthermore, the use of a computer assisted video camera offered the advantage of a permanent storage of image data, thus allowing comparison between samples analysed at different times, while membranes stained with chromogenic substrates can lose their colour differentiation overtime. Although the technical equipment employed in this study is still mainly a research tool, its potential for a clinical application could be explored. WB is the standard con®rmatory test for detecting antibodies to FIV and HIV-1. The pattern of antibody response may vary both in cats and humans. In HIV-infected patients, anti-p24 antibodies received considerable attention as a potential clinical marker early in the epidemic (Lange and Goudsmit, 1987; Schmidt et al., 1987). Subsequent studies in which antibody to speci®c
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HIV antigen were quantitated showed a strong relationship between baseline values of p24 antibodies and subsequent disease progression, demonstrating that low levels of these antibodies herald disease aggravation (Hogervorst et al., 1995). Further investigation demonstrated that the ratio of p24 and gp41 antibodies was highly predictive of the risk of developing AIDS (Strathdee et al., 1995). The use of an internally-controlled WB assay for measuring anti-FIV antibodies could be applied to study whether the changes in production of antibodies to Gag or Env proteins might give further information on the course of FIV infection. Acknowledgements This work was supported by grants from Ministero della SanitaÁ, Istituto Superiore di SanitaÁ, ``Progetto per l'AIDS'' and by the Ministero della UniversitaÁ e Ricerca Tecnologica, Rome, Italy. References Bendinelli, M., Pistello, M., Lombardi, S., Poli, A., Garzelli, C., Matteucci, D., Ceccherini-Nelli, L., Malvaldi, G., Tozzini, F., 1995. Feline immunode®ciency virus: an interesting model for AIDS studies and an important cat pathogen. Clin. Microbiol. Rev. 8, 87±112. Calzolari, M., Young, E., Cox, D., Davis, D., Lutz, H., 1995. Serological diagnosis of feline immunode®ciency virus infection using recombinant transmembrane glycoprotein. Vet. Immunol. Immunopathol. 46, 83±92. Hogervorst, E., Jurriaans, S., de Wolf, F., van Wijk, A., Wiersma, A., Valk, M., Roos, M., van Gemen, G.B., Coutinho, R., Miedema, F., 1995. Predictors for non- and slow-progression in human immunode®ciency virus (HIV) type 1 infection: low viral RNA copy numbers in serum and maintenance of high HIV-1 p24speci®c but not V3-speci®c antibody levels. J. Infect. Dis. 171, 811±821. Hosie, M.J., Jarrett, O., 1990. Serological responses of cats to feline immunode®ciency virus. AIDS 4, 215±220. Lacheretz, A., Berthon, A.F., Vialard, J., Mahl, P., 1995. EÂtude de la reÂponse humorale dans le syndrome immuno-de®citaire feÂlin par ELISA et Western blot. Revue MeÂd. VeÂt. 146, 847±854. Lange, J., Goudsmit, J., 1987. Decline of antibody reactivity to HIV core protein secondary to increased production of HIV antigen [letter]. Lancet 1, 448. McLaughlin, G.L., Benedik, M.J., Campbell, G.H., 1987. Repeated immunogenic amino acid sequences of Plasmodium species share sequence homologies with proteins from humans and human viruses. Am. J. Trop. Med. Hyg. 37, 258±262. Matteucci, D., Pistello, M., Mazzetti, P., Giannecchini, S., Del Mauro, D., Lonetti, I., Zaccaro, L., Pollera, C., Specter, S., Bendinelli, M., 1997. Studies of AIDS vaccination using an ex vivo feline immunode®ciency virus model: protection conferred by a ®xed-cell vaccine against cell-free and cell-associated challenge differs in duration and is not easily boosted. J. Virol. 71, 8368±8376. Mazzetti, P., Giannecchini, S., Del Mauro, D., Matteucci, D., Portincasa, P., Merico, A., Chezzi, C., Bendinelli, M., 1999. AIDS vaccination studies using an ex vivo feline immunode®ciency virus model: detailed analysis of the humoral immune response to a protective vaccine. J Virol. 73, 1±10. Mermer, B., Hillman, P., Harris, R., Krogmann, T., Tonelli, Q., Palin, W., Andersen, P., 1992. A recombinantbased feline immunode®ciency virus antibody enzyme-linked immunosorbent assay. Vet. Immunol. Immunopathol. 35, 133±141. Morikawa, S., Lutz, H., Aubert, A., Bishop, D.H.L., 1991. Identi®cation of conserved and variable regions in the envelope glycoprotein sequences of two feline immunode®ciency viruses isolated in ZuÈrich, Switzerland. Virus Res. 21, 53±63.
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Pedersen, N.C., Barlough, J.E., 1991. Clinical overview of feline immunode®ciency virus. J. Am. Vet. Med. Assoc. 199, 1298±1305. Poli, A., Falcone, M.L., Bigalli, L., Massi, C., Hofmann-Lehmann, R., Lombardi, S., Bendinelli, M., Lutz, H., 1995. Circulating immune complexes and analysis of renal immune deposits in feline immunode®ciency virus-infected cats. Clin. Exp. Immunol. 101, 254±258. Rimmelzwaan, G.F., Siebelink, K.H., Bross, H., Drost, G.A., Weijer, K., van Herwijnen, R., Osterhaus, A.D., 1994. Gag- and Env-speci®c serum antibodies in cats after natural and experimental infection with feline immunode®ciency virus. Vet. Microbiol. 39, 153±165. Schmidt, G., Amiraian, K., Frey, H., Stevens, R.W., Berns, D.S., 1987. Densitometric analysis of Western blot (immunoblot) assays for human immunode®ciency virus antibodies and correlation with clinical status. J. Clin. Microbiol. 25, 1993±1998. Steinman, R., Dombrowski, J., O'Connor, T., Montelaro, R.C., Tonelli, Q., Lawrence, K., Seymour, C., Goodness, J., Pedersen, N.C., Andersen, P.R., 1990. Biochemical and immunological characterisation of the major structural proteins of feline immunode®ciency virus. J. Gen. Virol. 71, 701±706. Strathdee, S.A., Franck, J.W., McLaughlin, J., Leblanc, M., Major, C., O'Shaughnessy, M.V., Read, S.E., 1995. Quantitative measures of human immunode®ciency virus-speci®c antibodies predict progression to AIDS. J. Infect. Dis. 172, 1375±1379. Talbott, R.L., Sparger, E.E., Lovelace, K.M., Fitch, W.M., Pedersen, N.C., Luciwm, P.A., Elder, J.H., 1989. Nucleotide sequence and genomic organisation of feline immunode®ciency virus. Proc. Natl. Acad. Sci. U.S.A. 86, 5743±5747. Yamamoto, J.K., Ackley, C.D., Zochlinski, H., Louie, H., Pembroke, E., Torten, M., Hansen, H., Munn, R., Okuda, T., 1991. Development of IL-2-independent feline lymphoid cell lines chronically infected with feline immunode®ciency virus: importance for diagnostic reagents and vaccines. Intervirology 32, 361±375.
Densitometric analysis of Western blot assays for feline immunode®ciency virus antibodies M. Calandrellaa, D. Matteuccib, P. Mazzettib, A. Polia,* a
Department of Animal Pathology, University of Pisa, Viale delle Piagge 2, I-56124 Pisa, Italy b Department of Biomedicine and Retrovirus Center, University of Pisa, Via San Zeno 37, I-56127 Pisa, Italy Received 29 June 2000; received in revised form 8 March 2001; accepted 16 March 2001
Abstract Western blot (WB) strips for antibodies directed to feline immunode®ciency virus (FIV) were analysed using re¯ectance densitometry by a semiautomatic densitometer. This method was used to quantify the antibody responses to different FIV proteins in both vaccinated and naturally or experimentally-infected cats. In order to increase reproducibility, reagents and protocols were accurately standardised and internal controls were added. In a ®rst format, an internal control band consisting of feline IgG was added to each blot to minimise the effect of band intensity variation. In a second format, antibody concentrations were calculated from the ratio of the dens ities produced by test sera and by positive and negative standard sera. The sera under scrutiny were also examined by standard enzyme-linked immunosorbent assay (ELISA) and the results obtained compared with those of the corresponding WB. A statistically signi®cant positive correlation was found between the results obtained with the two methods, and this was especially evident when ELISA titres were compared to corrected WB values (P 0:001). Densitometric analysis of WB assays allowed to quantify the antibodies against FIV proteins and might be useful to investigate possible humoral immune correlates of protection in FIV vaccination studies and antibody production in the early phase of infection. The quantitation of antibodies to Gag and Env FIV antigens might be used to obtain further informations on the course of FIV disease, as previously demonstrated in human immunode®ciency virus-1 (HIV-1) infections. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Western blot assay; Quantitative analysis; FIV; Antibody response
Abbreviations: BSA, bovine serum albumin; CV, coef®cient of variability; DR, density of re¯ectance; ELISA, enzyme-linked immunosorbent assay; FIV, feline immunode®ciency virus; HIV, human immunode®ciency virus; Ig, immunoglobulin; PCR, polymerase chain reaction; PBS, phosphate-buffered solution; RI, relative intensity; RIPA, radioimmunoprecipitation assay; SPF, speci®c pathogen free; WB, Western blot * Corresponding author. Tel.: 39-50-575970; fax: 39-50-540644. E-mail address: [email protected] (A. Poli). 0165-2427/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 2 6 5 - 3
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1. Introduction Feline immunode®ciency virus (FIV) is a lentivirus that induces persistent infections of domestic cats characterised by progressive deterioration of immune functions, neuropathological changes, and opportunistic infections similar to those associated with human immunode®ciency virus-1 (HIV-1) infection of humans. This virus also shares several biological and biochemical properties with HIV and it may help develop a rationale for the development of effective vaccines and therapies against AIDS infection (Bendinelli et al., 1995). Besides its importance as an animal model, FIV is a major pathogen of cats and therefore, important in veterinary practice. Genetic analysis of FIV has shown that the structural genes gag, env and pol are similar the ones of other lentiviruses and several proteins encoded by these genes have been identi®ed (Talbott et al., 1989; Morikawa et al., 1991). Serological studies have shown that, following infection, cats develop antibodies to both the nucleocapsid proteins and the envelope glycoproteins, but that qualitative and quantitative differences in the immune response occur between individual cats (Hosie and Jarrett, 1990). The humoral immune response of FIV-infected or immunised cats has been extensively investigated to identify the effectors responsible for protection and to de®ne possible correlates of vaccine-induced protection (Mazzetti et al., 1999). Antibodies to individual FIV proteins can be detected by Western blot (WB) and radioimmunoprecipitation assay (RIPA) using native FIV proteins (Steinman et al., 1990; Hosie and Jarrett, 1990) and enzyme-linked immunosorbent assays (ELISAs) using recombinant FIV Gag and Env proteins (Mermer et al., 1992; Rimmelzwaan et al., 1994; Calzolari et al., 1995). The development of these sensitive and speci®c ELISAs based on recombinant proteins allows the investigation of the production of antibodies to individual FIV antigens, but such studies are very timeand labour-demanding. WB and RIPA provide highly speci®c results, but they are usually not reliable for quantitating antibody production. The need for a more objective WB assay to better de®ne the immune status against FIV antigens prompted us to develop a quantitative WB assay. This report describes two WB assays that enabled us to determine the antibody response against single FIV antigens by using a computer assisted densitometer for image analysis. The results obtained using these quantitative WB assays were also compared with the results obtained by a standard ELISA. 2. Materials and methods 2.1. Cat sera The study was conducted on sera from 10 FIV-seropositive and 15 FIV-seronegative ®eld cats, and from 10 noninfected and 12 FIV-immunised speci®c pathogen free (SPF) cats. All cats were monitored serologically at selected time intervals for 28 months. The immunised cats were injected with a previously described vaccine (Matteucci et al., 1997), consisting of MBM cells acutely infected with a stock of the PISA M2 strain of FIV inactivated with paraformaldehyde. The cats were immunised subcutaneously at weeks 0, 3 and 6, and three boosters were administered 4, 10 and 16 months later,
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respectively. Of the 15 seronegative ®eld cats 2 seroconverted during the study period after they became infected, as demonstrated by virus isolation and PCR studies. Both ®eld and SPF cats were vaccinated against feline panleukemia virus, herpesvirus, and calicivirus (Feligen CR/P1, Virbac, Milan, Italy). The FIV-noninfected SPF control cats were housed in isolation units (Techniplast, Gazzada, Italy) at the Retrovirus Center of the University of Pisa. 2.2. Preparation of antigen for Western blotting The WB assays were performed with home-made strips, prepared with whole FIV antigen. Cell-free FIV-Petaluma antigen was produced from the supernatant of persistently infected FL4 cells and puri®ed by sucrose gradient puri®cation, followed by sonication and treatment with Triton X-100 (Yamamoto et al., 1991). 2.3. Western blotting procedures WB assays were performed as described previously (Poli et al., 1995) on Trans-Blot transfer medium (Bio-Rad Lab., Hercules, CA, USA) strips prepared using Mini-Protean II system (Bio-Rad). Brie¯y, FIV proteins were separated by preparative electrophoresis of detergent-treated, heat inactivated (1008C for 5 min) antigen preparation on sodium dodecyl sulfate-polyacrylamide (10%) gels in discontinuous buffer system and electrophoretically transferred to nitrocellulose sheets by Mini Trans-Blot Electrophoretic Transfer cell (Bio-Rad). The membrane was cut longitudinally into 2 mm wide strips. Each strip was treated with blocking buffer (5% dried milk in NaCl 150 mM, Tris±Cl 100 mM pH 7.5) for 2 h, and WB assays were conducted. Sera, diluted in PBS±Tween 20 (0.05%) containing 1% skim milk, were incubated with strips for 2 h at room temperature. After two washes with 0.05% PBS±Tween 20 for 10 min each, the reactivity of antibodies with antigen was detected by adding a rabbit horseradish peroxidase-conjugated anti-cat immunoglobulin (Ig)G (heavy- and light-chain), speci®c IgG (Nordic, Tilburg, The Netherlands) for 1 h. The strips were washed again and incubated in the dark with the enzyme substrate for peroxidase consisting of a solution of 10 ml methanol containing 6 mg of 4-chloro-1-naphthol (Sigma, St. Louis, MO, USA) added immediately before use to a solution of 45 ml of Tris pH 6.8 containing 6 ml of 10% hydrogen peroxide. Membranes were washed in distilled water and air dried. Analysis of the results was accomplished by capturing the strip images, locating bands using pre-stained molecular weight markers (Bio-Rad), measuring the re¯ectance density (DR) of anti-p15, -p25, -p31, gp40 and -gp95 antigen bands, and calculating the relative intensity (RI) by a video densitometer (Gel Blot-Pro Ultra Violet Prod., Cambridge, UK). To increase reproducibility of measurements and score band intensity two different assays with internal controls were developed and performed every time the sera were tested. In a ®rst format, WB A, puri®ed feline IgG (Jackson ImmunoResearch Lab., West Grove, PA, USA) were included in each strip and used as internal control to account for inter-assay variability. A serial dilution on a scale 1:2 was made from a concentrated solution of feline IgG (250 mg/1 ml PBS). From each dilution, 10 ml were withdrawn and transferred on the nitrocellulose sheets by using vacuum manyfold (BioRad). The RI of
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the single antigen bands was hence quantitated by comparing the DR of each band with the DR of the internal IgG control on each strip after substraction of the RI detected using negative control cat serum. In the second format, WB B, the reproducibility of measurements was increased by using the ratio of antibody concentrations as determined by including positive and negative controls in each experiment (Lacheretz et al., 1995). The DR was calculated with the formula: DR of test sample DR of negative control/DR of positive control DR of negative control. Because some FIV-negative cat sera can show weak bands against FIV antigen, sera from 10 SPF FIV-noninfected cats were used to determine cut-off values for the RI of each antigen band. The cut-off values were quanti®ed by adding three times the standard deviation to the means of the readings obtained with the negative control sera. 2.4. Standard ELISA for anti-FIV antibodies The antibody response to FIV was also determined by a standard ELISA, using for coating FIV-Petaluma virus grown in FL4 cells as described (Matteucci et al., 1997). ELISA plates (Probind, Falcon, Italy) were coated overnight with 0.5 mg per well gradient puri®ed, SDS-disrupted virus in 100 ml carbonate buffer (pH 9.6). After four washes with PBS±Tween 20, the plates were post-coated with 150 ml of PBS containing 1% bovine serum albumin (BSA) (PBS±BSA) for 1 h. Cat sera, serially diluted in 100 ml PBS containing 1% skim milk, 5% fetal calf serum, and 0.05% Tween 20 (dilution buffer), were added to the plate and incubated 1 h. Bound IgG antibodies were revealed by a horseradish peroxidase-conjugated mouse mAb anti-cat IgG (Sigma, St. Louis, MO, USA) diluted in 100 ml PBS±Tween 20±BSA. All steps were performed at room temperature. The enzyme reaction was carried out with 100 ml tetramethylbenzidine solution (0.4 mg/ml; KPL, Gaithersburg, MD, USA) and stopped with 100 ml 0.1N H2SO4; the absorbance was measured at 450 nm. The antibody titre was expressed as the reciprocal of the highest dilution of serum that gave optical density readings higher than the average values obtained with 20 control FIV-negative serum samples plus three times the standard deviation. 2.5. Statistical analysis To assess reproducibility, intra-assay (one serum with a strong reactivity for all FIV proteins in eight replicates) and inter-assay (three serum samples examined eight times) analysis were performed. Mean RI of each band and corresponding coef®cient of variability (CV) were calculated. The correlation between data obtained using the standard ELISA and the different WB assays was analysed by regression ANOVA and the Pearson correlation coef®cient using SPSS 8.0 (SPSS, Bologna, Italy). 3. Results To determine optimal WB conditions, different amounts of antigen and different dilutions of serum samples and of the conjugate were tested to obtain the highest
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Fig. 1. Quantitative Western blot assays and development of speci®c antibodies against individual FIV proteins in a cat immunised with an inactivated vaccine and boosted at 3, 6, 16, 40 and 66 weeks post-vaccination (arrow heads). Antibody production against single FIV proteins was quanti®ed: (A) comparing the relative intensity (RI) of each band with the RI of the internal feline IgG standard (WB A); (B) calculating the ratio of the RIs produced by test sera and by positive and negative standard sera (WB B).
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signal-to-noise ratios for positive and negative sera, respectively (results not shown). The optimal amount of antigen for preparing the strips was found to be 5 ng per strip. Optimal signal-to-noise ratios were found with sera diluted 1:100 and conjugate diluted 1:200. In the examination of the different dilutions of feline IgG used as internal control, a linear relationship between DR of bands and feline IgG dilution was observed (r 0:869; P 0:05). The optimal amount of feline IgG for quantifying the single bands of anti-FIV antibodies was found to be 0.04 mg per strip. Representative examples of the two quantitative WB assays are shown in Fig. 1. Serum samples of an immunised cat were examined with both WB formats. WB A used feline IgG as internal control (Fig. 1A), WB B was developed together with positive and negative control sera to determine ratio of antibody concentrations (Fig. 1B). With both formats, the highest intensities were detected against the gag products p25 and p15. Other FIV proteins consistently detected were, although with a lower RI, the env products gp95 and gp40 and the integrase p31. Cut-off values of RI obtained for each band corresponding to FIV antigens are presented in Table 1. Using WB A, the cut-off values for each FIV protein were lower than those observed with WB B. Both WB assays were also used to determine the seroconversion pro®le in cats which acquired infection during the follow-up period. Fig. 2 shows the course of seroconversion for p15, p25, p31, gp40, and gp95 determined using WB A. In the pre-infection samples, the RI of bands of the above proteins were constantly lower than the cut-off determined by using the sera from 10 SPF FIV-negative control cats. In the ®rst subject (Gassata; Fig. 2A), RI of antibodies to p15, p25 and p31 rose faster than RI of antibodies against gp40 and gp95. In the second cat (Umberto; Fig. 2B), the antibodies against all the FIV antigens increased concomitantly. Table 2 shows the relationships between the RI obtained with the two WB assays for the different bands corresponding to individual FIV protein and the results of a standard ELISA. A strong correlation was found between the results obtained with the two different WB assays for all the FIV antigens. The measure of antibody production against individual FIV antigens measured obtained with both WB formats was also statistically correlated with the antibody titres against whole FIV determined using a standard ELISA.
Table 1 Cut-off valuesa of relative intensity obtained for each antigen band using the two Western blot formats developedb FIV protein
WB A
WB B
p15 p25 p31 gp40 gp95
0.06 0.06 0.05 0.07 0.05
0.20 0.25 0.31 0.40 0.50
a
Mean of negative control sera plus three standard deviations. WB A: Western blot A with internal feline IgG standard; WB B in which the relative intensity (RI) of bands was calculated using the ratio of the RIs produced by test sera and by positive and negative standard sera. b
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Fig. 2. Seroconversion to FIV antigens after natural FIV infection. The levels of antibodies to single FIV antigens were quanti®ed using a quantitative Western blot assay with internal feline IgG standard (WB A). The ®gure shows the course of seroconversion of two cats (Gassata; A and Umberto; B).
To further evaluate the reproducibility of quantitation of FIV antibodies, the RI obtained using both strips from the same blot or antigen bound to different blots were compared in intra- and inter-assay tests. The results are presented in Table 3. Intra-assay CV ranged from 2.4% for gp40 and 95 to 3.4% for p31, while inter-assay CV ranged from 2.3% for p25 to 7.8% for p31. Table 2 Correlation between the results obtained with the two Western blot (WB) formats and between the relative intensities obtained with the two WB and anti-FIV titre determined with a standard ELISA FIV protein
p15 p25 p31 gp40 gp95
WB A vs. WB B
WB A vs. ELISA
WB B vs. ELISA
r
P
r
P
r
P
0.901 0.856 0.924 0.969 0.874
<0.00001 <0.00001 <0.00001 <0.00001 <0.00001
0.510 0.464 0.759 0.714 0.734
0.001 0.001 <0.0001 <0.0001 <0.0001
0.639 0.778 0.707 0.738 0.685
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
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Table 3 Intra- and inter-assay reproducibility of Western blot tests developed FIV protein
p15 p25 p31 gp40 gp95 Feline IgG
Intra-assaya
Inter-assayb
Mean relative intensityc
CVc (%)
Mean relative intensityc
CVc (%)
60 169.6 60.9 58.3 40.8 123.9
2.7 2.7 3.4 2.4 2.4 3.2
80.7 170.8 65 65.4 44.4 127.0
5.1 2.3 7.8 5.2 5.1 4.0
a Intra-assay reproducibility was determined using one serum with a strong reactivity for all FIV proteins in eight replicate. b Inter-assay reproducibility was calculated with three serum samples examined eight times. c Mean relative intensities of each band and corresponding coef®cients of variability (CV) were calculated.
4. Discussion Two quantitative WB assays, which were read using a video camera based densitometer connected to a personal computer for quantitative image analysis, are described. To evaluate the performances of the two assays, the humoral response to individual FIV proteins was studied in infected cats and in a longitudinal study of cats immunised with a ®xed cell vaccine. Using both formats, it was possible to determine the course of antibody production against the various FIV antigens. Of the two WB assays developed, WB A with the internal feline IgG standard, showed cut-off values for single FIV antigens markedly lower than WB B, when sera from SPF FIV-noninfected controls were used. In the WB A assay, the signals given by bands for anti-FIV antibodies using FIV-negative and -positive sera differ widely. Taking into account that automated quanti®cation of band intensities gives objective results, the presence of a low cut-off allowed to avoid the misclassi®cation of bands. A statistically signi®cant concordance was found when the results obtained with the two different WB assays were compared: the Pearson correlation coef®cients estimated for each pair of variables ranged from 0.969 for gp40 to 0.856 for p24 (P < 0:0001). The highest correlation was found for the RI of the bands p15, p31 and gp40. The RIs obtained with both WB assays for the individual FIV proteins were also correlated with total anti-FIV antibody titres as determined by a standard ELISA against whole virus. This demonstrated that the quanti®cation of antibodies against single FIV antigens by quantitative WB assays is comparable with the titres obtained using a standard ELISA. With regard to reproducibility of the results, both intra- and inter-assay precision tests gave acceptable results. It must be noted that the inter-assay CV of the RIs in the WB was lower than 10% for all the bands. In intra-assay tests, the CV was always below 5% for each band. Of the two different formats of quantitative WB developed, WB A, with internal feline IgG standard, was found to be preferable due to good correlation with the results obtained
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with a standard ELISA and to low cut-off values. Correct interpretations of band intensities is important because previous studies demonstrated that sera from ®eld and SPF cats can produce clearly recognisable single bands in WB although these subjects are not infected with FIV (Calzolari et al., 1995). This might be due to the fact that FIV sequences are also present in other pathogens of cats as was observed between HIV and pathogens such as Plasmodium falciparum and human T-cell lymphotropic virus-1 and -2 (McLaughlin et al., 1987). Gradient puri®ed retroviruses also contain components of the cell in which the virus is propagated or components of the fetal calf serum used for substrate cell growth (Pedersen and Barlough, 1991). Vaccination of cats with viral vaccines produced in feline cells is also likely to induce immune response to such antigens. Therefore, some cats vaccinated with viral antigens grown in cell culture may show false positive results with FIV viral antigens. The results of our study demonstrated that the quantitative WB assay with internal feline IgG standard (WB A) proved convenient for characterisation of speci®c antibody production against single FIV antigens. Mazzetti et al. (1999) previously described a quantitative WB to determine anti-FIV antibodies, in which strips were exposed to cat sera serially diluted until end point reactivity was reached. WB A has the advantage that speci®c anti-FIV antibody can be quanti®ed using a single dilution of test sera, thus saving time and labour. Accurate analysis of humoral responses in the FIV cat model is of particular importance in attempts to identify in vitro predictors of protective immunity in vaccination experiments and to characterise the pattern of antibody production after seroconversion in experimentally and naturally FIV-infected cats. In this longitudinal study of vaccinated cats, the quantitative WB assay developed proved easy to perform and offered signi®cant advantages over other methods, such as standard ELISA methods, which would require that the individual FIV antigens are ®rst obtained in puri®ed form to measure speci®c antibodies against single FIV antigens. Quanti®cation of antibody production in cats after seroconversion con®rmed that these subjects develop detectable antibodies to both FIV Gag and Env proteins, but that qualitative and quantitative differences in the immune response occur in individual cats (Rimmelzwaan et al., 1994). A further ®eld study on a large number of subjects following seroconversion could allow to determine the speci®city of the antibodies developed in the early phase of FIV infection. This would clarify whether the development of antibodies to Gag p25 protein occurs before (Hosie and Jarrett, 1990) or after antibodies to Env gp40 protein (Calzolari et al., 1995). Furthermore, the use of a computer assisted video camera offered the advantage of a permanent storage of image data, thus allowing comparison between samples analysed at different times, while membranes stained with chromogenic substrates can lose their colour differentiation overtime. Although the technical equipment employed in this study is still mainly a research tool, its potential for a clinical application could be explored. WB is the standard con®rmatory test for detecting antibodies to FIV and HIV-1. The pattern of antibody response may vary both in cats and humans. In HIV-infected patients, anti-p24 antibodies received considerable attention as a potential clinical marker early in the epidemic (Lange and Goudsmit, 1987; Schmidt et al., 1987). Subsequent studies in which antibody to speci®c
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HIV antigen were quantitated showed a strong relationship between baseline values of p24 antibodies and subsequent disease progression, demonstrating that low levels of these antibodies herald disease aggravation (Hogervorst et al., 1995). Further investigation demonstrated that the ratio of p24 and gp41 antibodies was highly predictive of the risk of developing AIDS (Strathdee et al., 1995). The use of an internally-controlled WB assay for measuring anti-FIV antibodies could be applied to study whether the changes in production of antibodies to Gag or Env proteins might give further information on the course of FIV infection. Acknowledgements This work was supported by grants from Ministero della SanitaÁ, Istituto Superiore di SanitaÁ, ``Progetto per l'AIDS'' and by the Ministero della UniversitaÁ e Ricerca Tecnologica, Rome, Italy. References Bendinelli, M., Pistello, M., Lombardi, S., Poli, A., Garzelli, C., Matteucci, D., Ceccherini-Nelli, L., Malvaldi, G., Tozzini, F., 1995. Feline immunode®ciency virus: an interesting model for AIDS studies and an important cat pathogen. Clin. Microbiol. Rev. 8, 87±112. Calzolari, M., Young, E., Cox, D., Davis, D., Lutz, H., 1995. Serological diagnosis of feline immunode®ciency virus infection using recombinant transmembrane glycoprotein. Vet. Immunol. Immunopathol. 46, 83±92. Hogervorst, E., Jurriaans, S., de Wolf, F., van Wijk, A., Wiersma, A., Valk, M., Roos, M., van Gemen, G.B., Coutinho, R., Miedema, F., 1995. Predictors for non- and slow-progression in human immunode®ciency virus (HIV) type 1 infection: low viral RNA copy numbers in serum and maintenance of high HIV-1 p24speci®c but not V3-speci®c antibody levels. J. Infect. Dis. 171, 811±821. Hosie, M.J., Jarrett, O., 1990. Serological responses of cats to feline immunode®ciency virus. AIDS 4, 215±220. Lacheretz, A., Berthon, A.F., Vialard, J., Mahl, P., 1995. EÂtude de la reÂponse humorale dans le syndrome immuno-de®citaire feÂlin par ELISA et Western blot. Revue MeÂd. VeÂt. 146, 847±854. Lange, J., Goudsmit, J., 1987. Decline of antibody reactivity to HIV core protein secondary to increased production of HIV antigen [letter]. Lancet 1, 448. McLaughlin, G.L., Benedik, M.J., Campbell, G.H., 1987. Repeated immunogenic amino acid sequences of Plasmodium species share sequence homologies with proteins from humans and human viruses. Am. J. Trop. Med. Hyg. 37, 258±262. Matteucci, D., Pistello, M., Mazzetti, P., Giannecchini, S., Del Mauro, D., Lonetti, I., Zaccaro, L., Pollera, C., Specter, S., Bendinelli, M., 1997. Studies of AIDS vaccination using an ex vivo feline immunode®ciency virus model: protection conferred by a ®xed-cell vaccine against cell-free and cell-associated challenge differs in duration and is not easily boosted. J. Virol. 71, 8368±8376. Mazzetti, P., Giannecchini, S., Del Mauro, D., Matteucci, D., Portincasa, P., Merico, A., Chezzi, C., Bendinelli, M., 1999. AIDS vaccination studies using an ex vivo feline immunode®ciency virus model: detailed analysis of the humoral immune response to a protective vaccine. J Virol. 73, 1±10. Mermer, B., Hillman, P., Harris, R., Krogmann, T., Tonelli, Q., Palin, W., Andersen, P., 1992. A recombinantbased feline immunode®ciency virus antibody enzyme-linked immunosorbent assay. Vet. Immunol. Immunopathol. 35, 133±141. Morikawa, S., Lutz, H., Aubert, A., Bishop, D.H.L., 1991. Identi®cation of conserved and variable regions in the envelope glycoprotein sequences of two feline immunode®ciency viruses isolated in ZuÈrich, Switzerland. Virus Res. 21, 53±63.
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