Journal of Virological Methods 127 (2005) 33–39 Fluorometric cell-ELISA for quantify and heparin inhibiti arl s a,b, l de Sa que, Bo ebruar 2005 Abstract The purpo etectin toma cells in ) and a assay (cell-ELISA) technique; alkaline phosphatase was used as the antibody-marker enzyme and 4-methyl-umbelliferyl-phosphate as the fluorogenic substrate. The system was used for detecting up to 1:10,000 viral inoculums, followed by evaluating the effect of heparin on infection. Infected cultures were reliably differentiated from their respective negative controls in both assays allowing data to be analysed statistically. As reported in another study, heparin produces strong inhibition when the CVS-BHK viral strain is used for infection; it has thus been sugges time-consum for research © 2005 Else Keywords: R 1. Introdu Enzyme immunosor Porstmann tensively fo serological quiring sen also be app cedures in Classica antibody co body boun ∗ Correspon fax: +57 1 62 E-mail ad 0166-0934/$ doi:10.1016/j ted that it binds to the neural cell adhesion molecule and could be blocked by using this drug. This fluorometric method is less ing, has increased reproducibility and useful for quantitation of collected data and can therefore be considered as a useful tool . vier B.V. All rights reserved. abies virus; Human meduloblastoma cells; Heparin; Fluorometric method; Cell-ELISA; 4-Methyl-umbelliferyl-phosphate (MUP) ction immunoassay (EIA) and cell-enzyme linked bent assay (cell-ELISA) (Avrameas, 1992; and Kiessig, 1992) techniques have been used ex- r quantifying both antigens and antibodies. Such tests are useful when studying viral diseases re- sitive and specific quantitative methods. They can lied in basic research and routine diagnostic pro- clinical laboratories. l EIA/ELISA protocols involve using a secondary upled to an enzyme for detecting a primary anti- d to its antigen; however, many variations of this ding author. Tel.: +57 1 633 13 68x272 209; 5 20 30. dress:
[email protected] (J.E. Castellanos). method have been introduced. In addition to using enzymes, these protocols have involved using avidin or streptavidin for detecting secondary or primary biotinylated antibodies; these additional components improve sensitivity and versatil- ity. Immunoassay sensitivity depends on the enzyme marker and the substrate used. Several enzymes have been used suc- cessfully in ELISA tests, including peroxidase, alkaline phos- phatase, glucoamylase, urease and �-galactosidase. Alkaline phosphatase (AP) has been used most frequently, due to its high rate of enzyme turnover, sensitivity, reproducibility, low cost, excellent intrinsic stability, fast catalytic rate, easy con- jugation and resistance to inactivation by the usual labora- tory reagents. Alkaline phosphatase substrates are also non- toxic and relatively stable reagents (Hornbeck et al., 1992). The following represent the most used chromogenic sub- strates: p-nitrophenyl-phosphate (PNP), 5-bromo-4-chloro-3 indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT). 4-Methyl-umbelliferyl-phosphate (MUP) has been the flu- – see front matter © 2005 Elsevier B.V. All rights reserved. .jviromet.2005.03.002 Vero´nica Rinco´n a,b, Adriana Corredor a,b, M Jaime E. Castellano a Laboratorio de Neurociencias, Instituto Naciona b Instituto de Virologı´a, Universidad El Bos Received 18 November 2004; received in revised form 25 F Available online 7 April se of the present study was to implement a fluorometric method for d fected by two types of fixed rabies virus (CVS-MB and CVS-BHK ing rabies infection on e´n Martı´nez-Gutı´errez a,b, ∗ lud, Bogota´, Colombia gota´, Colombia y 2005; accepted 3 March 2005 g and quantifying viral antigens in human meduloblas- street virus using a cell-enzyme linked immunosorbent 34 V. Rinco´n et al. / Journal of Virological Methods 127 (2005) 33–39 orogenic substrate used most frequently for alkaline phos- phatase (Goers, 1993). Fluorogenic substrates can detect or measure even ex- tremely sm more sensi number of system for et al., 1992 lies in requ cost twice (Hornbeck The fact tion for det pH ranging MUP can activity. Th ELISA pro for determ enzyme (H and quantif 1992). Thi at room te hydrolysis produces a umbelifery measured b filters (Kyl This pa quantifying infected w nique. The cells at diff bies virus ( fluorometri the effect o that hepari from BHK virus harve indicated t virus infec expressing (1998) hyp Non-infect efficiently correlation direct micr immunocy method cou in both rese 2. Materia 2.1. Cell-l The hum in DMEM serum, antibiotics (100 U/ml penicillin and 100�g/ml strep- tomycin) and 0.25�g/ml anfotericin. Around 5000 cells per well were seeded in 96-well plates and 2000 cells in 24-well s (see t 37 ◦ Viral a e CV stitut abora ucing lls (C strain thal d ions (1 and 10 d in 2 1/10, 1 withd treet s and r rland Antibo ree an ic syst ad 72 12) a ast tw Neur n et a Fluoro 671, with tion a or 10 m eabili e pho c acid 10% b 0.14 M in bl hey w layer with 200 or n alka tor Lab enzy adde nd 50 ferent all quantities of antigen. They offer up to 1000 tivity than chromogenic substrates, so that the cells can be reduced five-fold, making this a useful screening assays (Labrousse et al., 1982; Huang ). The only disadvantage of fluorogenic system iring a fluorometer for reading the plates; this can as much as a conventional ELISA plate reader et al., 1992). that it is not necessary to raise the pH of the solu- ecting reaction product, as AP have an optimum from 9 to10, represents an additional advantage; thus be used continuously for monitoring their is substrate has been used in a wide variety of tocols (Avrameas, 1992; Hornbeck et al., 1992.) ining cell numbers based on an endogenous uschtscha et al., 1989; Parandoosh et al., 1998) ying PCR amplification products (Vlieger et al., s substrate can be kept in solution for months mperature without any significant spontaneous (Hornbeck et al., 1992). MUP dephosphorylation stable and highly fluorescent product, 4-methyl lferone (MU), having a 7.9 pKa and can be y using 360 nm excitation and 450 nm emission e et al., 1999). per reports the use of MUP for detecting and viral antigens in human meduloblastoma cells ith rabies virus by means of a cell-ELISA tech- system was used for detecting viral antigen in erent viral dilutions using two variants of fixed ra- CVS-BHK and CVS-MB) and a street virus. The c technique employed was used for evaluating f heparin (NCAM ligand) on infection, finding n inhibited infection by rabies virus harvested cells (CVS-BHK), contrary to that obtained with sted from adult mouse brain (CVS-MB). This hat heparin-NCAM binding could block rabies tion in the human TE671 meduloblastoma line NCAM, strongly supporting Thoulouze et al. othesis that NCAM is a receptor for rabies virus. ed negative control cultures were differentiated and reliably in the assay. There was also a good between data obtained by fluorometry and oscope count of cultures processed by indirect tochemistry, leading to the conclusion that this ld be used as an extremely useful screening tool arch and diagnosis in the field of virology. ls and methods ine an TE671 meduloblastoma cell-line was cultured medium supplemented with 10% fetal bovine plate tion a 2.2. Th the In tion l prod 21 ce ICR ing le dilut MB seede with were The s brain Dr. O 2.3. Th metr (Bior (PVE The l Viral (Lafo 2.4. TE fixed infec tine f perm kalin aceti with Tris; pared and t mono ature BA9 lutio (Vec MUP nally Tris a at dif below). Cultures were kept for 72 h before infec- C in 5% CO2. nd cell culture infection S-11 virus (challenge virus standard), donated by o Nacional de Salud’s anti-rabies vaccine produc- tory (PAHO/WHO reference virus), was used for two different viral variants: CVS adapted to BHK- VS-BHK) and CVS adapted to adult mouse brain (CVS-MB). They were titrated in vivo by calculat- ose50 (LD50) after intra-cerebrally injecting viral 05.3 DL50 for CVS-BHK, 106.7 DL50 for CVS- 8.1 DL50 for the street virus). TE671 cells were 4- and 96-well culture plates and infected for 1 h /100, 1/1000 and 1/10,000 viral dilutions, inocula rawn and cultures incubated for an additional 24 h. train rabies virus was isolated from a rabid dog’s eplicated in mouse brain (generously provided by o Torres, Colombian NIH). dies tibodies were employed for testing by the fluoro- em: polyclonal rabies virus nucleocapsid antibody 114C), monoclonal rabies glycoprotein antibody nd a rabies virus nucleoprotein antibody (PVA3). o reagents were a kind gift from Monique Lafon, o-immunology Unit at the Pasteur Institute, Paris l., 1983; Montan˜o-Hirose et al., 1995). metric technique plated in 96-well dishes (Corning 25870), were 4% paraformaldehyde for 30 min 24 h post- nd the monolayer was protected with 0.5% gela- in at 0 ◦C to prevent cell detachment. It was then sed with 0.1% Triton X-100 and endogenous al- sphatase blocked with levamisol (20 mM) in 5% for 1 h non-specific binding sites were saturated locking buffer (10% horse serum in TBS, 10 mM NaCl, pH 7.2). Primary antibodies were then pre- ocking buffer with 0.01%, Tween-20 was added ere then incubated for 1 h at 37 ◦C. After washing, s were then incubated for 30 min at room temper- secondary antibody (biotinylated anti-mouse IgG anti-rabbit IgG BA1000, Vector Labs). 1/250 di- line phosphatase conjugated streptavidin SA5100 s) was then added at room temperature for 30 min. me substrate (Molecular Probes, M-6491) was fi- d in developing buffer (100 mM NaCl, 100 mM mM MgCl2, pH 9.5); fluorescence was measured times on a PR-521 fluorometer (TecnoSuma, Ha- V. Rinco´n et al. / Journal of Virological Methods 127 (2005) 33–39 35 vana, Cuba) using 360 nm excitation and 450 nm emission filters. Pierce’s bicinchoninic acid (BCA) method was used for quantifying 1985; Wiec determined bovine seru were descr tal protein i done for ea cations of e by analysis block desig tiple mean using SIMS 2.5. Immu An indi to cultures by fluorom (10�g/ml) 353047). H pared in PB for 45 min. body (Bior chamber. C lated anti-r room temp SA5004 Ve perature an distilled wa 7.2). Cells 1 min, dehy concentrati Polyscienc to a triocul Image Soft complete 5 formation o way ANOV SIMSTAT 2.6. MTT a The cell for quantify ing the form tetrazolium dishes, wer (5, 10, 20 for 24 h. H was then ad removed an croplate rea optical den terpolating absorbance on a calibration curve having known cell numbers (1500–24,000 cells/well). ANOVA test of com- plete random block design and least significant difference were done. Heparin treatment and infection uman meduloblastoma cells, cultured in 96- and 24-well re plates, were pre-treated with heparin at different con- ations (5, 10, 20 and 40�g/ml) for 45 min at 37 ◦C. They inoculated for 1 h with the two fixed rabies virus vari- and the street virus and then evaluated by fluorometry mmunocytochemistry. The percentage of infection inhi- was calculated as being: 100× (infected cells without ent− infected cells with heparin treatment)/(infected without treatment). esults Relationship between substrate concentration and um de stand ne op hosph eptav conc hosph n, wh ved. Detec ometri ree a ficity medu . Curve squares e). total protein in each of the wells (Smith et al., helman et al., 1988). Protein concentration was by interpolating absorbance data on a known m albumin concentration calibration curve. Data ibed as the ratio of fluorescence units over the to- n each well. Three independent experiments were ch assay, each one being evaluated using four repli- ach condition (n= 12). All groups were compared of variance (ANOVA) using complete random n. Significant differences were analysed by mul- comparison test (Newman–Keuls and Tukey test) TAT software. nocytochemistry rect immunoperoxidase procedure was applied under to the same conditions as those tested etry. Culturing was done on round poly-l-lysine coated coverslips in 24-well culture plates (Falcon ydrogen peroxide (0.5%) in 50% methanol pre- S was used for quenching endogenous peroxidase A 1/500 dilution of anti-rabies polyclonal anti- ad) was then added for 45 min at 37 ◦C in a damp overslips were then incubated with a biotiny- abbit IgG antibody (Vector Labs) for 30 min at erature. Peroxidase-coupled streptavidin (1�g/�l, ctor Labs) was then added for 30 min at room tem- d developed with a 1:1 mix of H2O2 at 0.02% in ter and 0.1% diaminobenzidine in Tris–HCl (pH were counterstained with Mayer’s Haemalum for drated in 5-min ethanol baths at ever increasing ons and mounted with liquid resin (Poly-mount, es). A digital Sony DXC 107A camera connected ar microscope at 500× magnification and Scion ware were used for counting sufficient fields to 00 infected and uninfected cells. Angular trans- f infected cell proportions was examined by one- A and least significant differences test, using the software. ssay viability assay represents a colorimetric method ing viable cell mitochondrial activity by measur- ation of a dark blue formazan produced by MTT ring reduction. TE671 cells, seeded on 96-well e treated with heparin at different concentrations and 40�g/ml) for 45 min, washed and incubated undred microlitres of MTT solution (0.5 mg/ml) ded to each well for 3 h at 37 ◦C. The media were d formazan crystals dissolved in DMSO. A mi- der (ELISA Stat Fax) was then used for measuring sity at 570 nm. Cell number was determined by in- tests 2.7. H cultu centr were ants and i bition treatm cells 3. R 3.1. optim A termi line p of str MUP line p tratio obser 3.2. fluor Th speci man Fig. 1 tions ( enzym tection enzyme ard calibration curve was first drawn up to de- timal reference concentration for detecting alka- atase by MUP. The activity of two concentrations idin coupled to alkaline phosphatase at different entrations was measured. Fig. 1 shows that alka- atase was best detected at 3.6 mM MUP concen- ere the highest fluorescence emission peak was ting antibody signal specificity by c system ntibodies’ (PVE12, PVA3 and polyclonal) signal and quality was determined by fluorometry in hu- loblastoma cell cultures infected with CVS-MB showing different MUP and alkaline phosphatase concentra- represent 0.125�g/ml and triangles represent 0.250�g/ml of 36 V. Rinco´n et al. / Journal of Virological Methods 127 (2005) 33–39 Fig. 2. Antib (squares) and sponds to unin pendent expe (n= 12). strain; five ing from 1/ highest sig antibody di ies, where 3.3. Using in cells The fluo detecting v ties of infec of the thre significant can be seen the viral di greater the 6.3 units of wild-type r lation betw and infectio (Fig. 3). As quantity of in the lowe ages of inf 64.6, 33.9, 39.0 and 1 1/1000 and percentage strain. High ral strain d (p< 0.01). ral dilution ferences were found when infected and non-infected cells were compared (p< 0.05). These results were consistent with obtained by immunocytochemistry (Fig. 3, panel C); the ntages of infection obtained by immunocytochemistry odies evaluated by fluorometry: PVE12 (circles), PVA3 polyclonal anti-rabies nucleocapsid (triangles). Mock corre- fected culture. Values were obtained by averaging three inde- riments, having four replications for each condition±S.E.M. dilutions were thus tested for each antibody rang- 250 to 1/2000. Monoclonal PVE12 presented the nal to noise ratio in fluorometric detection in each lution tested, the reverse of the other two antibod- the signal fell with dilution (Fig. 2). a fluorometric system to detect viral antigen rometric detection system was highly sensitive for iral antigen present in cells at different multiplici- tion (1/10, 1/100, 1/1000 and 1/10,000 dilutions) e rabies virus strains studied, being statistically those perce respecting control having no virus (p< 0.05), as in Fig. 3. The antigen concentration agreed with lution (i.e. the greater the virus concentration, the emitted fluorescence (15.6 in 1/10 dilution cf. fluorescence in 1/10,000) except when high titre abies strain was used. There was also strong corre- een the data obtained by the fluorometric system n percentages obtained by immunocytochemistry pects of infected cells are shown in Fig. 4. A great viral antigen produced could be determined, even st quantity of viral dilution tested. The percent- ection obtained with the CVS-BHK strain were 10.5 and 2%, while percentages were 78.8, 75.3, 8.5% with CVS-MB variants using 1/10, 1/100, 1/10,000 viral dilutions, respectively, the highest s of infection being observed with the CVS-MB ly significant differences were found between vi- ilutions when different comparisons were made There were no significant differences between vi- s when a wild-type rabies virus was used, but dif- Fig. 3. Quantifying viral antigen produced in the human meduloblastoma line infected with CVS-BHK (panel A), CVS-MB (panel B) and street rabies (panel C) strains. The values are the mean of three independent experiments, having four replications for each condition±S.E.M. (n= 12) for evaluating the fluorometric technique (dark bars, left axis). The line on top of the bars in the panels mean of infec corresponds to immunocytochemical evaluation (right axis); ted cell percentages±S.E.M. (n= 6). V. Rinco´n et al. / Journal of Virological Methods 127 (2005) 33–39 37 Fig. 4. Rabies infected TE671 cells processed for peroxidase immunocytochemistry using an a infected with s infect the same way ctive ve were 94.7, and 1/10,00 ferences w dilutions (p 3.4. MTT a Exposin voke chang the cell num with hepar periments ( Fig. 5. Effect cells measure (5, 10, 20 an cell±S.E.M. ll via ded b Hepar re hen th rin on hepari the CVS-BHK strain. (B) Cells infected with the CVS-MB strain. (C) Cell . Note the dark staining in cellular cytoplasm, corresponding to immunorea 92.0, 92.3 and 89.5% using 1/10, 1/100, 1/1000 0 viral dilutions, respectively. No significant dif- ere observed in cell counts using different viral < 0.05). ssay g TE671 cells to heparin (5–40�g/ml) did not pro- es in MTT reduction. No significant differences in on ce exclu 3.5. cultu W hepa that ber were observed between culture cells treated in and untreated cultures in cell proliferation ex- n= 12), as seen in Fig. 5. The effects of heparin of heparin at different concentrations in uninfected TE671 d by MTT assay. Heparin exposition at different concentration d 40�g/ml) was during 45 min. Data represent the mean of (n= 12). virus, harv significant treatment ( with virus did not sho cells witho the 20�g/m data is pres Data ob inhibition o chemistry, was found chemistry. that rabies the virus to 4. Discuss Several tecting and described. nti-rabies polyclonal antibody, 24 h post-infection. (A) Cells ed with rabies street strain. (D) Uninfected cells processed in sicles for rabies virus (arrows). Scale bars, 40�m. bility, cellular adhesion or cytotoxicity had been y this assay. in effect on rabies virus infection in cell e system was implemented for testing the effect of rabies virus infection in cell culture, it was found n (NCAM ligand) inhibited infection by rabies ested from BHK (CVS-BHK) cells having highly differences regarding infection without heparin p< 0.05). This was contrary to the obtained respect harvested from mouse brain (CVS-MB), which w changes with respect to infection in the control ut treatment. The greatest inhibition occurred in l heparin concentration (46.0% inhibition); this ented in Fig. 6. tained by fluorometry agreed with percentages of btained by cell count when using immunocyto- even though greatest infection inhibition (51.2%) in the 40�g/ml concentration by immunocyto- These results support existing evidence suggesting virus could be using NCAM as receptor, allowing bind to and replicate in cells. ion modifications to the ELISA procedures for de- quantifying antigens and antibodies have been The choice of assay depends on the cell type and 38 V. Rinco´n et al. / Journal of Virological Methods 127 (2005) 33–39 Fig. 6. Inhibi toma cell-line infected with The data repr ing four replic (white bars) a replications p (dark bars). A untreated infe the problem methods, w ods, have b mended du at a specifi lows enzym reaction; th ing dose-de cannot be factors suc background It was al tecting alka sensitivity tibody mar cells. Pre-t activity is fundamental for diminishing background levels or eliminating false positives reactives; prior standardisation fferent inactivation times and levamisol concentration nece se fro perim nt con consta g high e TBS e were me ca tine pr rucial mples ge of al exp re pla of di being phata Ex reage kept teein Thre phas extre Gela was c all sa vanta sever cultu tion of rabies virus infection in human TE671 meduloblas- cultures pre-treated with different heparin concentrations and two rabies virus variants. (A) CVS-BHK and (B) CVS-MB. esent the mean of three independent experiments, each hav- as per condition±S.E.M. (n= 12) for the fluorometric system nd the mean of three independent experiments, each having two er each condition±S.E.M. (n= 6) for immunocytochemistry sterisks represent significant differences (p< 0.05) respecting cted control. of interest (Zheng et al., 2000). Fluorometric hich are more sensitive than colorimetric meth- een known for many years, being widely recom- e to their accurate detection of enzyme product c wavelength. Reading fluorescence flexibility al- e activity to be measured from the start of the is method is particularly convenient for compar- pendent assays having different signal levels. This done with a chromogenic set-up, due to limiting h as enzyme auto-reaction causing an increase in when samples are incubated for a long time. so found that MUP is an excellent substrate for de- line phosphatase activity in cell-ELISA, the high of this enzyme making it a good choice as an an- ker enzyme for this type of system in adherent reating cells for inactivating endogenous enzyme The stu gens presen quantified ent multipl virus strain highly sign controls wi Rabies v even thoug large varie Castellanos could use s target cells nicotinic re is a surface domains an to heparin, in mediatin domain has Ig domain conformati Cole and G Thoulou plays an im cellular su body or he susceptibil and murine and NIH 3 by rabies v tion of infe been incub antibodies It has be bachol) red fected by strain adap rabies viru ssary for efficiently inhibiting cell alkaline phos- m different sources. ental conditions (including incubation times, centration, temperature, washing time) must be nt in all experiments (as in all protocols), guaran- homogeneity amongst replicas and experiments. washes (PBS should not be used) during each seen to be sufficient and should be done with re to avoid losing or damaging cell monolayer. otection of cells after paraformaldehyde fixation for maintaining constant cell number. At the least, should also be tested in quadruplicate, taking ad- the system’s feasibility and ease of use, leading to erimental conditions being analysed on the same te, making this a very efficient technique. dy described above has shown that viral anti- t in human meduloblastoma cells were sensitively by the fluorometric technique employed at differ- icities of infection (viral dilutions) with the rabies s studied (CVS-BHK, CVS-MB and street rabies), ificant differences were found with respect to the thout infection (p< 0.05). irus has strong in vivo and in vitro neurotropism, h laboratory-adapted strains are able to infect a ty of cells in spite of their origin (Castan˜eda- et al., 2002). It has been proposed that rabies virus everal receptor molecules for effectively infecting , such as NCAM, p75NTR and the acetylcholine ceptor (Castellanos and Hurtado, 2001). NCAM glycoprotein consisting of five immunoglobulin d two fibronectin binding domains; it also binds a heparan sulphate functional analogue involved g their cell–cell interactions. The heparin binding been localised in the first (IgI) and second (IgII) of NCAM ectodomain, its interaction causing a onal change in the protein (Kiselyov et al., 1997; laser, 1986). ze et al., (1998) reported that NCAM expression portant role in rabies virus in vivo and in vitro sceptibility; specific blocking (anti-NCAM anti- paran sulphate) drastically reduces rabies virus ity. It has been found that both neuronal (human neuroblastoma) and fibroblastic (BHK-21, VERO T3) laboratory cell-lines susceptible to infection irus posses NCAM on their surface. The propor- cted cells diminished significantly when they have ated with anti-NCAM polyclonal and monoclonal and then become infected. en found that nicotinic agonists (nicotine and car- uce the percentage of adult sensory neurons in- CVS-MB variant of rabies virus but not by the ted to fibroblast cells, supporting the idea that this s variant uses the nicotinic receptor in its adsorp- V. Rinco´n et al. / Journal of Virological Methods 127 (2005) 33–39 39 tion process (Castan˜eda-Castellanos et al., 2002). The CVS virus could exist as a complex of quasi-species, each having genomic and intrinsic protein characteristics (Morimoto et al., 1998). The two fixed laboratory strains used in this study be- haved differently during the initial steps of infection. This could have indicated that these two viruses might have used different cell receptors for entry. They may have incorpo- rated host-specific sugar, lipids and proteins during each substrate v (Castan˜eda These re infections c suggests th preferred u fection, as is the first r viral antige fered by an cessing tim techniques inating obs and sensiti cellent tool allows relia Acknowled We wou Virology L Bernal and TecnoSum hanna Baq the chromo setting-up t Instituto N nologı´a (C tuto Nacion thank Dr. O virus and J this article. Reference Avrameas, S niques. J. Castan˜eda-Ca tial use of origin. J. Castellanos, J.E., Hurtado, H., 2001. Receptores para el virus de rabia. Biomedica 21, 389–401. Cole, G., Glaser, L., 1986. A heparin-binding domain from N-CAM is involved in neural cell-substratum adhesion. J. Cell. Biol. 102, 403– 412. Goers, J., 1993. Immunochemical Techniques. Laboratory Manual. Aca- demic Press, San Diego, pp. 69–78. Hornbeck, P., Winston, S., Fuller, S., 1992. Enzyme-linked immunoab- sorbent assays (ELISA). In: Ausubel, F., Brent, R., Kinhg, S., Moore, D., Seidman, J., Smith, J., Struhl, K. (Eds.), Short Protocols in olecular .1.6–11 , Z., O e ELIS fluoroge tscha, L r counti zyme a ov, V., e, A., hesion eraction heparin G., Su , Haug ted um lactosid usse, H ization orogen , M., W ies viru rol. 64, n˜o-Hiro ies vir 4. oto, K. chold, nesis. P oosh, Z measu v. Biol ann, T munol. , P.K., K no, M., al. Bio ouze, M fon, M ies vir r, A., M atama, tion pro chemil elman, inchon le for c , L., G lactosid 7. irus adaptation, thus modifying adsorption steps -Castellanos et al., 2002). sults provide evidence of NCAM participation in aused by fibroblast-adapted viral strain. The data at the mouse brain-adapted strain very possibly sing the acetylcholine nicotinic receptor for in- heparin did not induce blocking of infection. This eport in which MUP has been used for detecting ns in a cell-ELISA system. The advantages of- MUP fluorometric method include reduced pro- e respecting conventional immunocytochemical (since cell counting is no longer necessary, elim- erver error), increased reproducibility, specificity vity, allowing the technique to be used as an ex- for both research and diagnosis in virology, as it ble quantitative data to be obtained. gments ld like to thank the Instituto Nacional de Salud’s aboratory (Colombia), especially Maria del Pilar Gloria Rey for allowing us to read samples on their a Fluorometer PR-521. We would like to thank Jo- uero for her technical assistance in standardising genic cell-ELISA technique, a previous step to he fluorometric one. This work was supported by acional para el Fomento de la Ciencia y la Tec- olciencias, Project 2104-04-11724) and the Insti- al de Salud (Project 023/2002). We would like to rlando Torres for providing the street strain rabies ason Garry for patiently translating and revising s ., 1992. 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Meth. 234, 153– Fluorometric cell-ELISA for quantifying rabies infection and heparin inhibition Introduction Materials and methods Cell-line Viral and cell culture infection Antibodies Fluorometric technique Immunocytochemistry MTT assay Heparin treatment and infection Results Relationship between substrate concentration and optimum detection enzyme Detecting antibody signal specificity by fluorometric system Using a fluorometric system to detect viral antigen in cells MTT assay Heparin effect on rabies virus infection in cell culture Discussion Acknowledgments References