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Microwave irradiation-accelerated in situ hybridization technique for HIV detection
Journal of Virological Methods, 35 (1991) 4!%58 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/91/SO3.50 ADONIS 0166093491005556
49
VIRMET 01233
Microwave irradiation-accelerated in situ hybridization technique for HIV detection Aldar S. Bourinbaiar, Vanaja R. Zacharopoulos The Population Council. Center for Biomedical Research,
(Accepted
and David M. Phillips New York, NY, U.S.A.
13 June 1991)
Summary
High frequency irradiation generated in a common household microwave oven was used to establish an in situ hybridization technique for rapid detection of HIV sequences in infected cells. A biotin-labeled DNA probe was subsequently detected either by an alkaline phosphatase-based calorimetric reaction or by fluorescence. When compared to standard hybridization procedures with radioactive or nonradioactive probes, microwave energymediated hybridization results in equal sensitivity and diminished background. The main advantage of this method, however, is the drastic reduction in time, allowing completion of the whole procedure, from sample preparation to hybrid signal visualization, within one hour. In addition to HIV detection, the approach described can be applied for the diagnosis of other viral infections and may stimulate the development of nucleic acid hybridization techniques based on microwave irradiation. HIV;
In
situ
hybridization; Microwave; Viral diagnosis; Biotin; DNA probe
Introduction
During the prolonged latent stage characteristic of human immunodeliciency virus (HIV) infection, the number of cells carrying proviral transcripts appears to be higher than the number of cells actively expressing viral protein (Folks et al., 1986; Fauci, 1988). Thus, it has proven difficult to identify viral presence Correspondence to: Aldar S. Bourinbaiar. c/o Prof. Jean-Marie HBpital Laenntc, 42 rue de Scvres, 75007 Paris, France.
Andrieu,
Hematologie/Oncologie,
solely by immunological methods (Daugharty et al., 1990). In situ hybridization offers the advantage of being a direct detection technique which provides information on the type of infected tissue as well as on the localization of viral genome or viral transcripts within the infected cell. Several reports have described the detection of HIV in infected cells by using an isotope-labeled probe (Harper et al., 1986; Chayt et al., 1986; Stoler et al., 1986) and recently three laboratories reported the use of biotin- or alkaline-phosphatase-labeled DNA probes (Singer et al., 1989; Unger et al., 1989; Spadoro et al., 1990). Nevertheless, the in situ hybridization method of HIV detection has not become as popular as immunological detection techniques such as ELISA or Western blotting. This is partly because in situ hybridization is considered to be a technique which is too laborious and time-consuming to be handled routinely in clinical or research virology laboratories. High energy irradiation generated by magnetron-powered ovens has been exploited extensively to reduce the lengthy staining and fixation procedures in immunohistochemistry (for review see Boon and Kok, 1989). Here we describe a simple and rapid in situ hybridization process using an HIV-specific biotinylated DNA probe and a common household microwave oven.
Materials and Methods Cells HIV-l strain HTLV-IIIB-infected lymphocytic cells MOLT-4 (Minowada et al., 1972; kindly provided by Jun Minowada, Fujisaki Cell Center, Okayama, Japan), monocytic cells U l/HIV-l (courtesy of AIDS Research and Reference Reagent Program, Rockville, MD; contributor: Thomas Folks) and a latently infected epithelial cell line 1407/YH5 (established as described previously by Bourinbaiar and Phillips, 1991) were used to develop the technique. These cell lines exist in paired, virus-infected and uninfected (MOLT-4, U937, Intestine 407) forms. Cells were grown in RPM1 1640 medium supplemented with 10% fetal calf serum (Whittaker, Walkersville, MD) in a humidified incubator containing 5% CO1 at 37°C. Suspension-grown cells were washed twice in RPM1 1640 and readjusted to an appropriate concentration in a mild hypotonic solution consisting of one part RPM1 1640/10% FCS and two parts of distilled water. Mixtures of infected and uninfected cells were also prepared. About 25 ~1 of cell suspension were spread in each well (8-mm diameter) of teflon coated multiwell glass slides (Carlson Scientific Inc., Peotone, IL). The total number of cells in a well ranged from 5-10 x lo4 cells. Air-dried slides were fixed in Carnoy’s II solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for 10 min at room temperature and then postfixed in absolute acetone for another 10 min. Such a double fixation procedure provides the necessary pertneabilization of the cell membrane for easier access of the viral probe. Monolayers of epithelial 1407/YH5 cells were
grown on glass Lab-Tek chamber-slides (Nunc Inc., Naperville, IL) and fixed for hybridization in the same way as the suspension-grown cells. HIV probes
A biotinylated HIV-specific 9-kb DNA probe and an 35S-labeled RNA probe were purchased from Oncor (Gaithersburg, MD) and diluted to 100 ng/ ml and lo8 dpm/ml respectively in a hybridization mixture consisting of 50% deionized formamide (Oncor), 10% dextran sulfate (Oncor) and 2 x SSC at pH 7.4. In situ hybridization procedures
Detection of HIV-specific signal by autoradiography was carried out as described by Harper and colleagues (1986) with modifications according to Keller and Manak (1990). In situ hybridization with the biotinylated probe involving overnight incubation at 42°C was performed as described by Singer et al. (1989) with modifications according to Spadoro et al. (1990). In situ hybridization using microwave irradiation was performed as follows: 2-5 ,ul (usually 5 ~1 of biotinylated probe is largely sufficient for an 8-mm diameter well) of hybridization mixture was applied dropwise and gently spread over the fixed cells by placing individual sheets of autoclavable plastic cut out from a biohazard bag. The slide was placed in a 600-W microwave oven (Sharp Carousel II, Japan) equipped with a carousel. The slide was positioned about 6 cm from the center of the carousel. Following initial trials the optimal position of the slide was marked. A 150-ml beaker tilled with tap water was placed in the center of the carousel as a water load. The slide was exposed to microwave irradiation for 30 s preselected on the ‘Defrost’ program. As incubation time is crucial for this operation, the duration of irradiation will presumably need to be optimized individually for different models of microwave ovens. The slide was removed from the oven and rinsed with 2 x SSC, pH 7.4, from a plastic squeeze bottle. Care was exercised not to direct the jet of buffer onto the cells. The washing procedure removes the hybridization mixture as well as the plastic coverslips. Detection of biotinylated probes
Hybridization product was detected using either avidin-conjugated alkaline phosphatase or an FITC-conjugated anti-biotin antibody. For enzymatic detection of hybrid signal the In Situ Hybridization Detection System for Biotinylated Probes (Dakopatts, Carpinteria, CA) was used. Reagents were diluted according to the manufacturer’s instructions but all incubation steps were carried out in the microwave oven. Biotinylated hybrids were detected by incubating first with appropriately diluted streptavidin and then with biotin-
52
alkaline phosphatase conjugate followed by BCIP/NBT substrate. For every reaction the slide was placed in a humidified chamber made of a plastic box and irradiated for 15 s on the ‘High’ setting. Each step was followed by washing with 0.9% saline from a plastic squeeze bottle. Incubation with substrate solution was at 37°C for 5-15 min. Slides were washed in tap water and mounted in 50% buffered glycerol. For fluorescent detection of the signal the Detek I-f Signal Generating System (Enzo Inc., New York, NY) was used. This kit is designed for the detection of biotinylated DNA probes and was used according to the manufacturer’s instruction. The target cells were first incubated with rabbitanti-biotin IgG followed by goat-anti-rabbit FITC conjugate. Slides were washed with saline and mounted in 50% glycerol/PBS containing 25 mg/ml of 1,4-diazobicyclo[2.2.2]octane (Sigma, St.Louis, MO) as an antifade reagent (Johnson et al., 1982).
Results Fixed smears with high-producing MOLT-4/HTLV-IIIB lymphocytes or Ul promonocytes containing only two copies of proviral genome (Spadoro et al., 1990) as well as monolayers of latently-infected virus-carrying 1407/YH5 epithelial cells were examined by three different in situ hybridization methods using either a biotinylated HIV DNA probe or an 35S-labeled HIV RNA probe. Figs. 1 and 2 show the results of conventional hybridization methods with an overnight incubation of the probe with target sequences followed by detection of the hybrids by autoradiography and by the enzymatic method, respectively. These results are compared with results from a hybridization procedure using brief microwave irradiation for 30 s (Figs. 3-6). The slides were examined under a microscope at low magnification. Positive cells displayed dark-blue deposits located in the cytoplasm of HIV carrying cells. For negative controls we used uninfected MOLT-4 lymphocytes, U937 monocytes, and Intestine 407 epithelial cells. These cells were run on the same slide with virus-carrying cells along with mixtures of infected and uninfected cells. Both non-isotopic methods gave comparable results. The intensity of the signal was generally the
Figs. l-6. Comparative in situ analysis of HIV-l sequences in virus-carrying cells by three different hybridization methods. Fig. 1 shows the distribution of the positive signal in MOLT-4/HTLV-IIIB lymphocytes by autoradiography; Fig. 2 shows HIV-positive cells after the fixed slides were exposed overnight to biotin-labeled DNA probe at 42°C and the hybridization label was revealed by alkaline phosphatase detection system; Fig. 3 displays the virus-specific signal in MOLT-4/HTLV-IIIB as detected by microwave hybridization procedure followed by accelerated immunoenzyme staining in the microwave oven; Fig. 4 shows the results of the similar microwave irradiation-accelerated hybridization followed by fluorescence detection; Fig. 5 demonstrates differential staining pattern when the mixture of infected and uninfected MOLT-4 lymphocytes (1: 100) has been subjected to in situ analysis; Fig. 6 reveals cytoplasmic distribution of HIV-l transcripts in CDCnegative epithelial 1407/YH5 cells derived from fetal small intestine.
53
54
same or stronger in microwaved slides; however, on some occasions overnight incubation resulted in a more uniform pattern of signal distribution throughout the well. This could be due to the irregularity of microwave distribution within the oven chamber which makes the positioning of the slide on the carousel critical. This problem can be particularly important for an oven without a carousel. The intensity of the signal usually correlated with the activity of viral production. Chronically infected Ul monocytic cells with the lowest number of proviral HIV were the least stained (data not shown). The MOLT-4/HTLVIIIB with higher post-transcriptional activity (supernatant from these cells contains at least 1000 times more viral gag p24 than medium from Ul) stained significantly stronger. We also investigated a CD4-negative epithelial cell line, 1407/YH5, which usually releases very low amounts of viral particles as judged by p24 ELISA or by reverse transcriptase (RT) activity (~24 was below 10 pg/ ml - the value of the cut-off threshold for this particular antigen detection kit is 7.8 pg/ml (Cellular Products Inc., Buffalo, NY) and RT activity was at the limit of detectability). However, despite low levels of viral production these cells exhibited a strong positive signal in the cytoplasm, indicating the presence of HIV mRNA. This is in agreement with the fact that the infectious capacity of these cells by cell contact is equivalent to that of MOLT-4/HTLV-IIIB (unpublished). In negative-control wells and in wells with the probe omitted there were no indications of reactivity by all three methods. Incubation of the DNA probe with positive cells for 30 s at 42°C did not produce any sign of hybridization. The specificity of the viral probe has also been confirmed by mixing known proportions of infected and uninfected cells. The frequency of positive cells detected either by an isotopic probe or a biotinylated probe was identical (Fig. 5). The fixation procedure appears to destroy the endogenous alkaline phosphatase activity as there was no discernible reaction when the BCIP/NBT substrate was incubated with unhybridized intestinal cells alone. It is noteworthy that the resolution of non-isotopic probes at the level of individual cells is significantly better than that obtained by autoradiography. In the latter method, due to the thickness of the photographic emulsion, the black grains corresponding to the hybridized 35S-labeled probe do not coincide with exact localization of virus within the cell (Fig. 1). Initially, following the hybridization step the slide was dipped in a Coplin jar filled with 50% formamide in 2 x SSC and irradiated for 24 s on the ‘High’ setting. Under these conditions the temperature of the stringency solution reaches about 52°C as determined using the temperature probe provided with the microwave oven. This stringency washing step was thought to prevent background staining. However, it was noted later that the background was often less (or absent) in irradiated slides than in overnight incubated slides, even if the slides had not been processed through stringent washing. Lower background staining was probably due to the ‘shaking off of non-specific binding by microwave irradiation and because of that the disassociation of non-covalent binding was no longer required.
55
Discussion
The in situ hybridization technique is a potentially powerful diagnostic tool which can be used alone or in conjunction with immunological tests for viral infection. However, in order to pick up a reliable positive signal by autoradiography, classical in situ hybridization with a radiolabeled probe requires from several days to several weeks of X-ray-film exposure. In addition, the signal resolution in terms of cellular localization of the positive signal is not as good when compared to hybridization results derived from enzymatic or fluorescence detection. The introduction of biotin-labeled nonradioactive probes (Langer et al., 1981) has decreased the duration of the hybridization procedure dramatically by eliminating the lengthy step of signal recovery (Brigati et al., 1983). In addition, the use of biotinylated DNA probes has eliminated inconveniences such as the potential hazards of cumulative exposure to radioactivity and the problems of radioactive waste disposal. Still, the average amount of time spent on the entire in situ HIV technique requires two working days (Singer et al., 1990). The major part of this time is usually attributed to the several hours, or more commonly, the overnight incubation period necessary for satisfactory covalent binding of the labeled probe with the target viral sequences. Such a time-lag is a major drawback for wider applications of the in situ techniques, especially for diagnostic applications. Two recently published protocols (Coates et al., 1987; Boon and Kok, 1989) describe the use of microwaves for rapid detection of hybridized biotin-labeled DNA probes. However, to our knowledge, there are no reported attempts to reduce the duration of the hybridization process itself. Two standard, isotopic and nonisotopic, in situ hybridization techniques for the detection of HIV transcripts were compared to a newly developed hybridization technique based on the use of a microwave oven. Although all three techniques were equally sensitive there is a dramatic difference when one compares the gain in time. Instead of overnight incubation the microwave-based procedure requires only 30 seconds. In addition to the use of microwave irradiation there were several steps in the protocol where modifications were made. During preliminary trials it became clear that successful hybridization required an optimal fixation procedure which would not only preserve the cellular morphology but would also allow easy access of the bulky 9-kb probe to the viral sequences located within the host cell. The most commonly used fixative - formaldehyde - does not provide adequate permeabilization of the cell membrane and also creates a problem of nonspecific binding due to the cross-linking nature of the alkylation. A number of cumbersome postfixation steps is usually required to eliminate these shortcomings. However, membrane detergents, proteases, and acetic anhydride used for these aims have an adverse effect on cell morphology. These inconveniences prompted us to search for a simpler fixation procedure. After several trials a procedure similar to the one used for chromosome hybridization (Pinkel, personal communication; Pinkel et al., 1986) has been tested and
56
adopted as very simple and adequate for routine use. It is also relevant that suspending cells in a mild hypotonic solution before fixation resulted in an expanded or ‘swollen’ cytoplasm and contributed to better resolution of the intracellular signal. It also became apparent that even in the absence of stringency conditions there was no significant background in microwaved slides. It is possible that 3 billion/s oscillations generated by an average 600-W magnetron effectively eliminates non-covalent binding associated with background staining (Boon and Kok, 1989). Thus, the hybridization reaction can be followed immediately by biotin detection steps. All these technical tips would have little relevance if the application perspectives of the technique are limited. It was originally believed that HIVinfected cells are found at very low frequency (1 in 10 OO&lOO 000 cells) in peripheral blood (Harper et al., 1986). Later it was demonstrated (Harnish et al., 1987) that the occurrence of virus-infected cells in seropositive infants detected by flow cytometry ranges from 4 to 13%. These numbers were about the same, independent of the stage of AIDS. In other studies (Ho et al., 1989; Spear et al., 1990) it was estimated that in symptomatic and asymptomatic patients at least 1 in 400-1000 circulating mononuclear cells harbored HIV-l. These and a recent study using confocal microscopy (Lewis et al., 1990) suggest that the frequency of HIV-carrying cells in the blood of infected individuals is much higher than originally estimated. Therefore, it is possible that this in situ HIV detection in addition to research applications can also be used routinely for viral diagnosis. In conclusion, using microwave irradiation we were able to develop a simple and rapid in situ hybridization method. In addition to being rapid and sensitive this technique provides an extremely good localization of the signal and low background. The total amount of time required for detection of viral sequences in infected cells is less than one hour, as opposed to two days or more by standard protocols. It is hoped that the advantages of this approach will be appreciated and that they will stimulate widespread use of the microwave oven for hybridization purposes.
Acknowledgements The fun of experimenting with microwaves was inspired by Dr. Ariyasu who brought a microwave oven into the lab to melt agarose solutions. It was due to this interest that we subsequently discovered the unique ‘Surrealistic Microwave Cookbook’ by Boon and Kok and really appreciated their goodhumored way of exposing elaborate technical details. Initial steps in the development of this technique were supported by Hayashibara International Cancer Research Program. Further financing was provided primarily by grants from CONRAD No. CSA- 88-037, The Rockefeller Foundation No. GAHS 8938, No. GAPS 8921 and National Institutes of Health No. HD 27433.
57
References Boon, M.E. and Kok, L.P. (1989) Microwave Cookbook of Pathology, The Art of Microscopic Visualization. 2”d edit., Coulomb Press, Leiden. Bourinbaiar, A.S. and Phillips, D.M. (1991) Transmission of human immunodeliciency virus from monocytes to epithelia. J. Acquir. Immune. Del%. Syndr. 4, 5663. Brigati, D.J., Myerson, D., Leary, J.J., Spalholz, B., Travis, S.Z., Fong, C.K.Y., Hsiung, G.D. and Ward, D.C. (1983) Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology 126, 32-50. Chayt, K.J., Harper, M.E., Marselle, L.M., Lewin, E.B., Rose, R.M., Oleske, J.M., Epstein, L.G., Wong-Staal, F. and Gallo, R.C. (1986) Detection of HTLV-III RNA in lungs of patients with AIDS and pulmonary involvement. J. Am. Med. Assoc. 256, 23562359. Coates, P.J., Hall, P.A., Butler. M.G. and D’Ardenne, A.J. (1987) Rapid technique of DNA-DNA in situ hybridization on formalin fixed tissue sections using microwave irradiation. J. Clin. Pathol. 40, 865-869. Daugharty, H., Long, E.G., Swisher, B.L., Warfield, D.T. and Feorino P.M. (1990) Comparative study with in situ hybridization and immunocytochemistry in detection of HIV-l in formalinfixed paraffin-embedded cell cultures. J. Clin. Lab. Analys. 4, 283-288. Fauci, A.S. (1988) The human immunodeficiency virus: Infectivity and mechanisms of pathogenesis. Science 239, 617622. Folks, T., Powell, D.M., Lightfoote, M.M., Benn, S., Martin, M. and Fauci, A.S. (1986) Induction of HTLV-III/LAV from a non virus producing T cell line: implications for latency. Science 231, 600-602. Harnish, D.G., Hammerberg, O., Walker, I.R. and Rosenthal, K.L. (1987) Early detection of HIV infection in a newborn. New. Engl. J. Med. 316, 272-273. Harper, M.E., Marselle, L.M., Gallo, R.C. and Wong-Staal, F. (1986) Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc. Natl. Acad. Sci. USA 83, 772-776. Ho, D.D., Moudgil, T. and Alam, M. Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. New. Engl. J. Med. 321, 1621-1625. Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D. and Holborow, E.J. (1982) Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. J. Immunol. Methods 55, 231-242. Keller, G.H. and Manak, M.M. (1990) DNA Probes. Stockton Press, New York. Langer, P.A., Waldrop, A.A. and Ward D.C. (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633-6637. Lewis, D.E., Minshall, M., Wray, N.P., Paddock, S.W., Smith, L.C. and Crane, M.M. (1990) Confocal microscopic detection of human immunodeticiency virus RNA-producing cells. J. Infect. Dis. 162, 1373-1378. Minowada, J., Ohnuma, T. and Moore, G.E. (1972) Rosette-forming human lymphoid cell line. I. Establishment and evidence for origin of thymus-derived lymphocytes. J. Natl. Cancer. Inst. 49, 891-895. Pinkel, D., Straume, T. and Gray, J.W. (1986) Cytogenetic analysis using quantitative, highsensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83, 29342938. Singer, R.H., Byron, K.S., Lawrence, J.B. and Sullivan, J.L. (1989) Detection of HIV-l-infected cells from patients using nonisotopic in situ hybridization. Blood 74, 22952301. Singer, R.H., Byron, K., Lawrence, J.B., Sullivan, J.L., Yin, S. and Lederman (1990) In situ hybridization to detect HIV: elements of a nonradioactive test. In: A. Aldovini and B.D. Walker (Eds), Techniques in HIV Research. Stockton Press, New York, pp. 30-39. Spadoro, J.P., Payne, H., Lee, Y. and Rosenstraus, M.J. (1990) Single copies of HIV proviral DNA detected by fluorescent in situ hybridization. Biotechniques 9, 186195. Spear, G.T., Ou, C.-Y., Kessler, H.A., Moore, J.L., Schochetman,. G. and Landay, A.L. (1990) Analysis of lymphocytes, monocytes, and neutrophils from human immunodeficiency virus (HIV)-infected persons for HIV DNA. J. Infect. Dis. 162, 1239-1244.
58 Stoler, M.H., Eskin, T.A., Berm, S., Angerer, R.C. and Angerer, L.M. (1986) Human T-cell lymphotropic virus type III infection of the central nervous system. A preliminary in situ analysis. J. Am. Med. Assoc. 256, 236&2364. Unger, E.R., Chandler, F.W., Chenggis, M.L., Ou, C.-Y., Warfield, D.T. and Feorino P.M. (1989) Demonstration of human immunodeticiency virus by calorimetric in situ hybridization: a rapid technique for formalin-fixed paraffin-embedded material. Mod. Pathol. 2, 200-204.
49
VIRMET 01233
Microwave irradiation-accelerated in situ hybridization technique for HIV detection Aldar S. Bourinbaiar, Vanaja R. Zacharopoulos The Population Council. Center for Biomedical Research,
(Accepted
and David M. Phillips New York, NY, U.S.A.
13 June 1991)
Summary
High frequency irradiation generated in a common household microwave oven was used to establish an in situ hybridization technique for rapid detection of HIV sequences in infected cells. A biotin-labeled DNA probe was subsequently detected either by an alkaline phosphatase-based calorimetric reaction or by fluorescence. When compared to standard hybridization procedures with radioactive or nonradioactive probes, microwave energymediated hybridization results in equal sensitivity and diminished background. The main advantage of this method, however, is the drastic reduction in time, allowing completion of the whole procedure, from sample preparation to hybrid signal visualization, within one hour. In addition to HIV detection, the approach described can be applied for the diagnosis of other viral infections and may stimulate the development of nucleic acid hybridization techniques based on microwave irradiation. HIV;
In
situ
hybridization; Microwave; Viral diagnosis; Biotin; DNA probe
Introduction
During the prolonged latent stage characteristic of human immunodeliciency virus (HIV) infection, the number of cells carrying proviral transcripts appears to be higher than the number of cells actively expressing viral protein (Folks et al., 1986; Fauci, 1988). Thus, it has proven difficult to identify viral presence Correspondence to: Aldar S. Bourinbaiar. c/o Prof. Jean-Marie HBpital Laenntc, 42 rue de Scvres, 75007 Paris, France.
Andrieu,
Hematologie/Oncologie,
solely by immunological methods (Daugharty et al., 1990). In situ hybridization offers the advantage of being a direct detection technique which provides information on the type of infected tissue as well as on the localization of viral genome or viral transcripts within the infected cell. Several reports have described the detection of HIV in infected cells by using an isotope-labeled probe (Harper et al., 1986; Chayt et al., 1986; Stoler et al., 1986) and recently three laboratories reported the use of biotin- or alkaline-phosphatase-labeled DNA probes (Singer et al., 1989; Unger et al., 1989; Spadoro et al., 1990). Nevertheless, the in situ hybridization method of HIV detection has not become as popular as immunological detection techniques such as ELISA or Western blotting. This is partly because in situ hybridization is considered to be a technique which is too laborious and time-consuming to be handled routinely in clinical or research virology laboratories. High energy irradiation generated by magnetron-powered ovens has been exploited extensively to reduce the lengthy staining and fixation procedures in immunohistochemistry (for review see Boon and Kok, 1989). Here we describe a simple and rapid in situ hybridization process using an HIV-specific biotinylated DNA probe and a common household microwave oven.
Materials and Methods Cells HIV-l strain HTLV-IIIB-infected lymphocytic cells MOLT-4 (Minowada et al., 1972; kindly provided by Jun Minowada, Fujisaki Cell Center, Okayama, Japan), monocytic cells U l/HIV-l (courtesy of AIDS Research and Reference Reagent Program, Rockville, MD; contributor: Thomas Folks) and a latently infected epithelial cell line 1407/YH5 (established as described previously by Bourinbaiar and Phillips, 1991) were used to develop the technique. These cell lines exist in paired, virus-infected and uninfected (MOLT-4, U937, Intestine 407) forms. Cells were grown in RPM1 1640 medium supplemented with 10% fetal calf serum (Whittaker, Walkersville, MD) in a humidified incubator containing 5% CO1 at 37°C. Suspension-grown cells were washed twice in RPM1 1640 and readjusted to an appropriate concentration in a mild hypotonic solution consisting of one part RPM1 1640/10% FCS and two parts of distilled water. Mixtures of infected and uninfected cells were also prepared. About 25 ~1 of cell suspension were spread in each well (8-mm diameter) of teflon coated multiwell glass slides (Carlson Scientific Inc., Peotone, IL). The total number of cells in a well ranged from 5-10 x lo4 cells. Air-dried slides were fixed in Carnoy’s II solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for 10 min at room temperature and then postfixed in absolute acetone for another 10 min. Such a double fixation procedure provides the necessary pertneabilization of the cell membrane for easier access of the viral probe. Monolayers of epithelial 1407/YH5 cells were
grown on glass Lab-Tek chamber-slides (Nunc Inc., Naperville, IL) and fixed for hybridization in the same way as the suspension-grown cells. HIV probes
A biotinylated HIV-specific 9-kb DNA probe and an 35S-labeled RNA probe were purchased from Oncor (Gaithersburg, MD) and diluted to 100 ng/ ml and lo8 dpm/ml respectively in a hybridization mixture consisting of 50% deionized formamide (Oncor), 10% dextran sulfate (Oncor) and 2 x SSC at pH 7.4. In situ hybridization procedures
Detection of HIV-specific signal by autoradiography was carried out as described by Harper and colleagues (1986) with modifications according to Keller and Manak (1990). In situ hybridization with the biotinylated probe involving overnight incubation at 42°C was performed as described by Singer et al. (1989) with modifications according to Spadoro et al. (1990). In situ hybridization using microwave irradiation was performed as follows: 2-5 ,ul (usually 5 ~1 of biotinylated probe is largely sufficient for an 8-mm diameter well) of hybridization mixture was applied dropwise and gently spread over the fixed cells by placing individual sheets of autoclavable plastic cut out from a biohazard bag. The slide was placed in a 600-W microwave oven (Sharp Carousel II, Japan) equipped with a carousel. The slide was positioned about 6 cm from the center of the carousel. Following initial trials the optimal position of the slide was marked. A 150-ml beaker tilled with tap water was placed in the center of the carousel as a water load. The slide was exposed to microwave irradiation for 30 s preselected on the ‘Defrost’ program. As incubation time is crucial for this operation, the duration of irradiation will presumably need to be optimized individually for different models of microwave ovens. The slide was removed from the oven and rinsed with 2 x SSC, pH 7.4, from a plastic squeeze bottle. Care was exercised not to direct the jet of buffer onto the cells. The washing procedure removes the hybridization mixture as well as the plastic coverslips. Detection of biotinylated probes
Hybridization product was detected using either avidin-conjugated alkaline phosphatase or an FITC-conjugated anti-biotin antibody. For enzymatic detection of hybrid signal the In Situ Hybridization Detection System for Biotinylated Probes (Dakopatts, Carpinteria, CA) was used. Reagents were diluted according to the manufacturer’s instructions but all incubation steps were carried out in the microwave oven. Biotinylated hybrids were detected by incubating first with appropriately diluted streptavidin and then with biotin-
52
alkaline phosphatase conjugate followed by BCIP/NBT substrate. For every reaction the slide was placed in a humidified chamber made of a plastic box and irradiated for 15 s on the ‘High’ setting. Each step was followed by washing with 0.9% saline from a plastic squeeze bottle. Incubation with substrate solution was at 37°C for 5-15 min. Slides were washed in tap water and mounted in 50% buffered glycerol. For fluorescent detection of the signal the Detek I-f Signal Generating System (Enzo Inc., New York, NY) was used. This kit is designed for the detection of biotinylated DNA probes and was used according to the manufacturer’s instruction. The target cells were first incubated with rabbitanti-biotin IgG followed by goat-anti-rabbit FITC conjugate. Slides were washed with saline and mounted in 50% glycerol/PBS containing 25 mg/ml of 1,4-diazobicyclo[2.2.2]octane (Sigma, St.Louis, MO) as an antifade reagent (Johnson et al., 1982).
Results Fixed smears with high-producing MOLT-4/HTLV-IIIB lymphocytes or Ul promonocytes containing only two copies of proviral genome (Spadoro et al., 1990) as well as monolayers of latently-infected virus-carrying 1407/YH5 epithelial cells were examined by three different in situ hybridization methods using either a biotinylated HIV DNA probe or an 35S-labeled HIV RNA probe. Figs. 1 and 2 show the results of conventional hybridization methods with an overnight incubation of the probe with target sequences followed by detection of the hybrids by autoradiography and by the enzymatic method, respectively. These results are compared with results from a hybridization procedure using brief microwave irradiation for 30 s (Figs. 3-6). The slides were examined under a microscope at low magnification. Positive cells displayed dark-blue deposits located in the cytoplasm of HIV carrying cells. For negative controls we used uninfected MOLT-4 lymphocytes, U937 monocytes, and Intestine 407 epithelial cells. These cells were run on the same slide with virus-carrying cells along with mixtures of infected and uninfected cells. Both non-isotopic methods gave comparable results. The intensity of the signal was generally the
Figs. l-6. Comparative in situ analysis of HIV-l sequences in virus-carrying cells by three different hybridization methods. Fig. 1 shows the distribution of the positive signal in MOLT-4/HTLV-IIIB lymphocytes by autoradiography; Fig. 2 shows HIV-positive cells after the fixed slides were exposed overnight to biotin-labeled DNA probe at 42°C and the hybridization label was revealed by alkaline phosphatase detection system; Fig. 3 displays the virus-specific signal in MOLT-4/HTLV-IIIB as detected by microwave hybridization procedure followed by accelerated immunoenzyme staining in the microwave oven; Fig. 4 shows the results of the similar microwave irradiation-accelerated hybridization followed by fluorescence detection; Fig. 5 demonstrates differential staining pattern when the mixture of infected and uninfected MOLT-4 lymphocytes (1: 100) has been subjected to in situ analysis; Fig. 6 reveals cytoplasmic distribution of HIV-l transcripts in CDCnegative epithelial 1407/YH5 cells derived from fetal small intestine.
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same or stronger in microwaved slides; however, on some occasions overnight incubation resulted in a more uniform pattern of signal distribution throughout the well. This could be due to the irregularity of microwave distribution within the oven chamber which makes the positioning of the slide on the carousel critical. This problem can be particularly important for an oven without a carousel. The intensity of the signal usually correlated with the activity of viral production. Chronically infected Ul monocytic cells with the lowest number of proviral HIV were the least stained (data not shown). The MOLT-4/HTLVIIIB with higher post-transcriptional activity (supernatant from these cells contains at least 1000 times more viral gag p24 than medium from Ul) stained significantly stronger. We also investigated a CD4-negative epithelial cell line, 1407/YH5, which usually releases very low amounts of viral particles as judged by p24 ELISA or by reverse transcriptase (RT) activity (~24 was below 10 pg/ ml - the value of the cut-off threshold for this particular antigen detection kit is 7.8 pg/ml (Cellular Products Inc., Buffalo, NY) and RT activity was at the limit of detectability). However, despite low levels of viral production these cells exhibited a strong positive signal in the cytoplasm, indicating the presence of HIV mRNA. This is in agreement with the fact that the infectious capacity of these cells by cell contact is equivalent to that of MOLT-4/HTLV-IIIB (unpublished). In negative-control wells and in wells with the probe omitted there were no indications of reactivity by all three methods. Incubation of the DNA probe with positive cells for 30 s at 42°C did not produce any sign of hybridization. The specificity of the viral probe has also been confirmed by mixing known proportions of infected and uninfected cells. The frequency of positive cells detected either by an isotopic probe or a biotinylated probe was identical (Fig. 5). The fixation procedure appears to destroy the endogenous alkaline phosphatase activity as there was no discernible reaction when the BCIP/NBT substrate was incubated with unhybridized intestinal cells alone. It is noteworthy that the resolution of non-isotopic probes at the level of individual cells is significantly better than that obtained by autoradiography. In the latter method, due to the thickness of the photographic emulsion, the black grains corresponding to the hybridized 35S-labeled probe do not coincide with exact localization of virus within the cell (Fig. 1). Initially, following the hybridization step the slide was dipped in a Coplin jar filled with 50% formamide in 2 x SSC and irradiated for 24 s on the ‘High’ setting. Under these conditions the temperature of the stringency solution reaches about 52°C as determined using the temperature probe provided with the microwave oven. This stringency washing step was thought to prevent background staining. However, it was noted later that the background was often less (or absent) in irradiated slides than in overnight incubated slides, even if the slides had not been processed through stringent washing. Lower background staining was probably due to the ‘shaking off of non-specific binding by microwave irradiation and because of that the disassociation of non-covalent binding was no longer required.
55
Discussion
The in situ hybridization technique is a potentially powerful diagnostic tool which can be used alone or in conjunction with immunological tests for viral infection. However, in order to pick up a reliable positive signal by autoradiography, classical in situ hybridization with a radiolabeled probe requires from several days to several weeks of X-ray-film exposure. In addition, the signal resolution in terms of cellular localization of the positive signal is not as good when compared to hybridization results derived from enzymatic or fluorescence detection. The introduction of biotin-labeled nonradioactive probes (Langer et al., 1981) has decreased the duration of the hybridization procedure dramatically by eliminating the lengthy step of signal recovery (Brigati et al., 1983). In addition, the use of biotinylated DNA probes has eliminated inconveniences such as the potential hazards of cumulative exposure to radioactivity and the problems of radioactive waste disposal. Still, the average amount of time spent on the entire in situ HIV technique requires two working days (Singer et al., 1990). The major part of this time is usually attributed to the several hours, or more commonly, the overnight incubation period necessary for satisfactory covalent binding of the labeled probe with the target viral sequences. Such a time-lag is a major drawback for wider applications of the in situ techniques, especially for diagnostic applications. Two recently published protocols (Coates et al., 1987; Boon and Kok, 1989) describe the use of microwaves for rapid detection of hybridized biotin-labeled DNA probes. However, to our knowledge, there are no reported attempts to reduce the duration of the hybridization process itself. Two standard, isotopic and nonisotopic, in situ hybridization techniques for the detection of HIV transcripts were compared to a newly developed hybridization technique based on the use of a microwave oven. Although all three techniques were equally sensitive there is a dramatic difference when one compares the gain in time. Instead of overnight incubation the microwave-based procedure requires only 30 seconds. In addition to the use of microwave irradiation there were several steps in the protocol where modifications were made. During preliminary trials it became clear that successful hybridization required an optimal fixation procedure which would not only preserve the cellular morphology but would also allow easy access of the bulky 9-kb probe to the viral sequences located within the host cell. The most commonly used fixative - formaldehyde - does not provide adequate permeabilization of the cell membrane and also creates a problem of nonspecific binding due to the cross-linking nature of the alkylation. A number of cumbersome postfixation steps is usually required to eliminate these shortcomings. However, membrane detergents, proteases, and acetic anhydride used for these aims have an adverse effect on cell morphology. These inconveniences prompted us to search for a simpler fixation procedure. After several trials a procedure similar to the one used for chromosome hybridization (Pinkel, personal communication; Pinkel et al., 1986) has been tested and
56
adopted as very simple and adequate for routine use. It is also relevant that suspending cells in a mild hypotonic solution before fixation resulted in an expanded or ‘swollen’ cytoplasm and contributed to better resolution of the intracellular signal. It also became apparent that even in the absence of stringency conditions there was no significant background in microwaved slides. It is possible that 3 billion/s oscillations generated by an average 600-W magnetron effectively eliminates non-covalent binding associated with background staining (Boon and Kok, 1989). Thus, the hybridization reaction can be followed immediately by biotin detection steps. All these technical tips would have little relevance if the application perspectives of the technique are limited. It was originally believed that HIVinfected cells are found at very low frequency (1 in 10 OO&lOO 000 cells) in peripheral blood (Harper et al., 1986). Later it was demonstrated (Harnish et al., 1987) that the occurrence of virus-infected cells in seropositive infants detected by flow cytometry ranges from 4 to 13%. These numbers were about the same, independent of the stage of AIDS. In other studies (Ho et al., 1989; Spear et al., 1990) it was estimated that in symptomatic and asymptomatic patients at least 1 in 400-1000 circulating mononuclear cells harbored HIV-l. These and a recent study using confocal microscopy (Lewis et al., 1990) suggest that the frequency of HIV-carrying cells in the blood of infected individuals is much higher than originally estimated. Therefore, it is possible that this in situ HIV detection in addition to research applications can also be used routinely for viral diagnosis. In conclusion, using microwave irradiation we were able to develop a simple and rapid in situ hybridization method. In addition to being rapid and sensitive this technique provides an extremely good localization of the signal and low background. The total amount of time required for detection of viral sequences in infected cells is less than one hour, as opposed to two days or more by standard protocols. It is hoped that the advantages of this approach will be appreciated and that they will stimulate widespread use of the microwave oven for hybridization purposes.
Acknowledgements The fun of experimenting with microwaves was inspired by Dr. Ariyasu who brought a microwave oven into the lab to melt agarose solutions. It was due to this interest that we subsequently discovered the unique ‘Surrealistic Microwave Cookbook’ by Boon and Kok and really appreciated their goodhumored way of exposing elaborate technical details. Initial steps in the development of this technique were supported by Hayashibara International Cancer Research Program. Further financing was provided primarily by grants from CONRAD No. CSA- 88-037, The Rockefeller Foundation No. GAHS 8938, No. GAPS 8921 and National Institutes of Health No. HD 27433.
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