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Detection of snail hemocyanin in the whole hemolymph by serologically specific electron microscopy
hnmunologv Letters. 6 (1983) 323 325 Elsevier
DETECTION
OF SNAIL HEMOCYANIN
SEROLOGICALLY
SPECIFIC
IN THE WHOLE ELECTRON
HEMOLYMPH
BY
MICROSCOPY
J o s 6 0 . GASPAR, L. O. S. BER1AM*, Lficia P. S. AIROLDI* and A. R. OL1VEIRA* A~¢~o de Virologia, Instituto AgronC)mico, Caixa Postal 28, 13100 Campinas SP," and *Deparlamento de Bioqu[mica, UNICA MP, 13100 CamphTas, SP, Brasil (Received 18 May 1983) (Accepted 20 May 1983)
1. Summary
3. Materials and methods
This study demonstrates the applicability of the serologically specific electron microscopy (SSEM) technique in the detection of hemocyanin molecules in the whole hemolymph of the snail, Megalobulio mulus ovatus. The results are positive and easily reproducible. The SSEM might be useful as a technique for taxonomic studies of snails as well as to study structural aspects of their hemocyanin molecules.
3. I. Hemocyanin purification Hemolymph was obtained from 3-day-fasting snails. It was centrifuged at 5000 rpm for 20 min and the supernatant was treated with ammonium sulfate [9]. The hemocyanin (Hcy) precipitate was centrifuged and redissolved in an appropriate volume of 0.05 M Tris-HC1 buffer, pH 7.4, containing 20 mM CaCI2. It was then submitted to an ultracentrifugation at 78,000 × g for 270 rain and the pellet was resuspended in the same buffer and recentrifuged under the same conditions.
2. Introduction
The serologically specific electron microscopy (SSEM) [1], based on the specific attachment of virus particles to antibody-coated grids of electron microscopy, has been utilized to detect viruses of plants in tissue extracts [2-6]. This technique associated with the "decoration" method, has also been used to determine the serological relationship between different viruses [7,8]. This paper presents the detection and identification by the SSEM of hemocyanin molecules from the whole hemolymph and purified preparations of the Brazilian terrestrial snail, Megalobulimulus ovatus.
Key words: Megalobulimulus ovatus hemocyanin snail serologically specific electron microscopy hemolymph antigen antibody reaction 0165 2478/83/$3.00 © 1983 Elsevier Science Publishers B.V.
3.2. Antiserum preparation For antisera preparation the Hcy pellet was resuspended to a final concentration of 10 mg/ml in phosphate buffer (0.01 M, pH 7.0) plus 0.85% (w/v) NaCI. Three rabbits weighing about 3 kg received two intralymphnode injections [10] of the emulsified antigen (lmcomplete Difco Adjuvant) 15 days apart. 31ood samples taken daily and were tested by the ~gar double-diffusion technique. Antisera from the fifth day after the first antigen injection and from several other bleedings were tested by the SSEM. 3.3. S S E M preparation Copper electron microscopy grids were coated with a film of Parlodion 0.4% in amyl acetate and then carbon-coated. The grids were (a) floated on drops of whole-hemocyanin antiserum (Hcy-As) diluted 1:500 in Sorensen's phosphate buffer (0.06 M, 323
+~i~~i~i+~+
Figs. 1 4. Serologically specific electron microscopy preparation of hemocyanin molecules. Each one 120,000 ;'~. Eig. 1: purified hemocyanin on a normal serum-coated grid. Fig. 2: purified hemocyanin on a Hcy-As-coated grid. Fig. 3: whole hemolymph tm a Hcy-As coated grid. Fig. 4: antibody-coated hemocyanin molecules (arrows).
324
pH 6.5) for 30 min at room temperature and then (b) washed with 5 drops of the same buffer. Later, they were (c) drained with filter paper and (d) transferred to drops of antigen (whole hemolymph or purified hemocyanin diluted with 0.05 M Tris-HCl buffer, pH 7.4, containing 20 mM CaCI2, to approximately 60 g g / m l ) for 30 rain at room temperature. Afterwards they were (e) washed consecutively with 5 drops of Sorensen's phosphate and 5 drops of distilled water and (f) negatively stained with uranyl acetate 2%, and examined in a Siemens Elmiskop I electron microscope. To "decorate" the hemocyanin molecules, grids that passed through the a d stages were floated on drops of the same antiserum for 2 h at 4 ° C and then passed through the e f stages. Other variations with dilutions and time of reaction were not tried. To obtain the relative molecule values mentioned later, molecules were counted on a 5.5 × 7.0 cm area of negatives taken at 5300 ×. Five negatives from different parts of each grid were utilized.
4. Results and discussion The objective of this study was to verify the applicability of the S S E M technique in the detection of hemocyanin molecules in purified preparations and whole hemolymph. The results (Figs. 1 3) were positive and easily reproducible. The number of hemocyanin molecules trapped on grids coated with specific antiserum were at least 40 times greater for purified preparations and 20 times greater for whole hemolymph when compared with grids coated with normal serum. Nevertheless, in preparations with whole hemolymph, many other molecules were trapped, producing a background on the grids not removed with successive buffer water washings. This
drawback can be avoided by utilizing a purified antiserum from which the non-specific antibodies were removed. Fig. 4 shows hemocyanin molecules "decorated" (coated) with antibodies, demonstrating the specific nature of ihe antigen antibody reaction. The results indicate the possibility of using the S S E M , combined with decoration, as a technique for taxonomic studies of snails based on the serological reaction of their hemocyanin molecules. The S S E M can also be utilized in the observation of sites of specific antibody attachment to the hemocyanin molecules utilizing antiserum preparated against sub-fractions of the molecule. So, hemocyanin structure, localization of antigenic determinants, and assembly pathways of its molecules, can be similarly studied as with viruses [ l 1,12].
References [1] Derrick, K. S. (1973) Virology 56, 652 653. [2] Derrick, K. S. and Brlansky, R. H. (1976) Phytopathology 66, 815 820. [3] Brlansky, R. H. and Derrick, K. S. (1979) Phytopathology 69, 96 100. [4] Milne, R. G. and l_uisoni. E. (1977) in: Rapid Immune Electron Microscopy ot Virus Preparations (Maramorosch, K. and Koprovski, H. Eds.) Methods en Virology, Vol. Vl, pp. 265 281, Academic Press, New York. [5] Roberts, 1. M. and Harrison, B. D. (1979) Ann. Appl. Biol. 93, 289 297. [6] Paliwal, Y. C. (1977) Phytopath. Z. 94, 8 15. [7] Delecolle, B. and Lot, H. (1981) Agronomie I, 763 770. [8] Roberts, 1. M., Tamada, T. and Harrison, B. D. (1980) J. Gen. Virol. 47, 209 213. [9] Heirwegh, K., Borginon, H. and Lontie, R. (1961) Biochim. Biophys. Acta 48, 517 526. [10] Oliveira, A. R. (1975) Summa Phytopath. 1, 61 64. [1 I] Luisoni, E., Milne, R. G. and Boccardo, G. (1975) Virology 68, 86 96. [12] Yanagida, M. and Ahmad-Zadeh, C. (1970)J. Mol. Biol. 51,411 421.
325
DETECTION
OF SNAIL HEMOCYANIN
SEROLOGICALLY
SPECIFIC
IN THE WHOLE ELECTRON
HEMOLYMPH
BY
MICROSCOPY
J o s 6 0 . GASPAR, L. O. S. BER1AM*, Lficia P. S. AIROLDI* and A. R. OL1VEIRA* A~¢~o de Virologia, Instituto AgronC)mico, Caixa Postal 28, 13100 Campinas SP," and *Deparlamento de Bioqu[mica, UNICA MP, 13100 CamphTas, SP, Brasil (Received 18 May 1983) (Accepted 20 May 1983)
1. Summary
3. Materials and methods
This study demonstrates the applicability of the serologically specific electron microscopy (SSEM) technique in the detection of hemocyanin molecules in the whole hemolymph of the snail, Megalobulio mulus ovatus. The results are positive and easily reproducible. The SSEM might be useful as a technique for taxonomic studies of snails as well as to study structural aspects of their hemocyanin molecules.
3. I. Hemocyanin purification Hemolymph was obtained from 3-day-fasting snails. It was centrifuged at 5000 rpm for 20 min and the supernatant was treated with ammonium sulfate [9]. The hemocyanin (Hcy) precipitate was centrifuged and redissolved in an appropriate volume of 0.05 M Tris-HC1 buffer, pH 7.4, containing 20 mM CaCI2. It was then submitted to an ultracentrifugation at 78,000 × g for 270 rain and the pellet was resuspended in the same buffer and recentrifuged under the same conditions.
2. Introduction
The serologically specific electron microscopy (SSEM) [1], based on the specific attachment of virus particles to antibody-coated grids of electron microscopy, has been utilized to detect viruses of plants in tissue extracts [2-6]. This technique associated with the "decoration" method, has also been used to determine the serological relationship between different viruses [7,8]. This paper presents the detection and identification by the SSEM of hemocyanin molecules from the whole hemolymph and purified preparations of the Brazilian terrestrial snail, Megalobulimulus ovatus.
Key words: Megalobulimulus ovatus hemocyanin snail serologically specific electron microscopy hemolymph antigen antibody reaction 0165 2478/83/$3.00 © 1983 Elsevier Science Publishers B.V.
3.2. Antiserum preparation For antisera preparation the Hcy pellet was resuspended to a final concentration of 10 mg/ml in phosphate buffer (0.01 M, pH 7.0) plus 0.85% (w/v) NaCI. Three rabbits weighing about 3 kg received two intralymphnode injections [10] of the emulsified antigen (lmcomplete Difco Adjuvant) 15 days apart. 31ood samples taken daily and were tested by the ~gar double-diffusion technique. Antisera from the fifth day after the first antigen injection and from several other bleedings were tested by the SSEM. 3.3. S S E M preparation Copper electron microscopy grids were coated with a film of Parlodion 0.4% in amyl acetate and then carbon-coated. The grids were (a) floated on drops of whole-hemocyanin antiserum (Hcy-As) diluted 1:500 in Sorensen's phosphate buffer (0.06 M, 323
+~i~~i~i+~+
Figs. 1 4. Serologically specific electron microscopy preparation of hemocyanin molecules. Each one 120,000 ;'~. Eig. 1: purified hemocyanin on a normal serum-coated grid. Fig. 2: purified hemocyanin on a Hcy-As-coated grid. Fig. 3: whole hemolymph tm a Hcy-As coated grid. Fig. 4: antibody-coated hemocyanin molecules (arrows).
324
pH 6.5) for 30 min at room temperature and then (b) washed with 5 drops of the same buffer. Later, they were (c) drained with filter paper and (d) transferred to drops of antigen (whole hemolymph or purified hemocyanin diluted with 0.05 M Tris-HCl buffer, pH 7.4, containing 20 mM CaCI2, to approximately 60 g g / m l ) for 30 rain at room temperature. Afterwards they were (e) washed consecutively with 5 drops of Sorensen's phosphate and 5 drops of distilled water and (f) negatively stained with uranyl acetate 2%, and examined in a Siemens Elmiskop I electron microscope. To "decorate" the hemocyanin molecules, grids that passed through the a d stages were floated on drops of the same antiserum for 2 h at 4 ° C and then passed through the e f stages. Other variations with dilutions and time of reaction were not tried. To obtain the relative molecule values mentioned later, molecules were counted on a 5.5 × 7.0 cm area of negatives taken at 5300 ×. Five negatives from different parts of each grid were utilized.
4. Results and discussion The objective of this study was to verify the applicability of the S S E M technique in the detection of hemocyanin molecules in purified preparations and whole hemolymph. The results (Figs. 1 3) were positive and easily reproducible. The number of hemocyanin molecules trapped on grids coated with specific antiserum were at least 40 times greater for purified preparations and 20 times greater for whole hemolymph when compared with grids coated with normal serum. Nevertheless, in preparations with whole hemolymph, many other molecules were trapped, producing a background on the grids not removed with successive buffer water washings. This
drawback can be avoided by utilizing a purified antiserum from which the non-specific antibodies were removed. Fig. 4 shows hemocyanin molecules "decorated" (coated) with antibodies, demonstrating the specific nature of ihe antigen antibody reaction. The results indicate the possibility of using the S S E M , combined with decoration, as a technique for taxonomic studies of snails based on the serological reaction of their hemocyanin molecules. The S S E M can also be utilized in the observation of sites of specific antibody attachment to the hemocyanin molecules utilizing antiserum preparated against sub-fractions of the molecule. So, hemocyanin structure, localization of antigenic determinants, and assembly pathways of its molecules, can be similarly studied as with viruses [ l 1,12].
References [1] Derrick, K. S. (1973) Virology 56, 652 653. [2] Derrick, K. S. and Brlansky, R. H. (1976) Phytopathology 66, 815 820. [3] Brlansky, R. H. and Derrick, K. S. (1979) Phytopathology 69, 96 100. [4] Milne, R. G. and l_uisoni. E. (1977) in: Rapid Immune Electron Microscopy ot Virus Preparations (Maramorosch, K. and Koprovski, H. Eds.) Methods en Virology, Vol. Vl, pp. 265 281, Academic Press, New York. [5] Roberts, 1. M. and Harrison, B. D. (1979) Ann. Appl. Biol. 93, 289 297. [6] Paliwal, Y. C. (1977) Phytopath. Z. 94, 8 15. [7] Delecolle, B. and Lot, H. (1981) Agronomie I, 763 770. [8] Roberts, 1. M., Tamada, T. and Harrison, B. D. (1980) J. Gen. Virol. 47, 209 213. [9] Heirwegh, K., Borginon, H. and Lontie, R. (1961) Biochim. Biophys. Acta 48, 517 526. [10] Oliveira, A. R. (1975) Summa Phytopath. 1, 61 64. [1 I] Luisoni, E., Milne, R. G. and Boccardo, G. (1975) Virology 68, 86 96. [12] Yanagida, M. and Ahmad-Zadeh, C. (1970)J. Mol. Biol. 51,411 421.
325