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ELISA titration of vitellogenin and vitellin in the freshwater prawn, Macrobrachium rosenbergii, with monoclonal antibody
Comp. Biochem. Physiol. Vol. 85B, No. 1, pp. 1-4, 1986 Printed in Great Britain
0305-0491/86 $3.00+ 0.00 Pergamon Journals Ltd
ELISA TITRATION OF VITELLOGENIN A N D VITELLIN IN THE FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII, WITH MONOCLONAL ANTIBODY E. DERELLE*, J. GROSCLAUDE~, J.-J. MEUSY*, H. JUNI~RA* and M. MARTIN* *Universit6 P. et M. Curie, Laboratoire de Sexualit6 et Reproduction des Invert6br6s, Biit. A, 4 place Jussieu, 75230 Paris Cedex 05, France. "H.N.R.A., Station de Virologie et d'Immunologie, route de Thiverval, 78850 Thiverval Grignon, France (Received 18 November 1985)
Abstract--1. A monoclonal antibody has been produced against viteUin of the freshwater prawn, Macrobrachium rosenbergii. 2. This antibody reacted against the light subunit of vitellin and vitellogenin (L-subunit: 84 kD; H-subunit: 92.2 kD). 3. An indirect ELISA test was developed for titrating vitellogenin in the haemolymph, thus allowing quantitative comparisons along the reproductive cycle.
INTRODUCTION
Viteilogenesis is a crucial period in the female reproductive cycle of Crustacea, characterized by a rapid yolk deposition. The major part of the yolk, vitellin, originates from the uptake of a seric precursor, vitellogenin, by the growing oocytes. Vitellogenin and vitellin in Crustacea are both lipo-glycocarotenoproteins of high molecular weight and cannot be distinguished one from another by conventional immunochemical techniques. Studies on viteliogenesis mechanisms and horm o n a l controls require a highly sensitive and specific quantitation of vitellogenin and vitellin. We have chosen the "enzyme linked i m m u n o s o r b e n t assay" or ELISA. In addition to its high sensitivity, this method avoids the use of radioisotopes and excessive consumption of reagents. ELISA and R I A assays for arthropod vitellogenin and vitellin have been developed among some insect species (Dumas et al., 1982; M a et al., 1984; Ferenz, 1978; Asher et al., 1983). We describe here a quantitative sandwich assay using rabbit polyclonal crude immunoglobulins against vitellogenic ovary extract of the freshwater prawn, Macrobrachium rosenbergii and a mouse antivitellin monoclonal antibody. We thus achieved a higher guarantee of specificity. Furthermore it will be possible to produce in the future unlimited quantity of this monoclonal antibody. MATERIALS AND METHODS Vitellin and vitellogenin preparation The animals were obtained from the Centre Oc~anologique du Pacifique, IFREMER (Tahiti, Polyn6sie Fran~aise). Vitellogenic ovaries were homogenized in 0.1 M sodium phosphate buffer, pH 7.2 (5 ml buffer for 1 g fresh tissue). After centrifugation (12,000 g, 20 min), the components of the supernatant were separated by polyacrylamide gel decC.B.P. 85/IB--A
trophoresis in tubes (acrylamide 6% in Tris-glycine buffer, pH 8.3). Vitellin appeared as an orange coloured band of low mobility. Vitellin was sliced out and eluted by electrodialysis, then dialysed against 0.01 M phosphate buffer. After lyophilization, the vitellin was solubilized and its protein content was estimated by the method of Lowry et al. (1951) with BSA as standard. Vitellogenin was enriched from crude haemolymph by the same electrophoretie procedure. Rabbit immunization and immunoglobulin preparation Two rabbits were injected intramuscularly with the antigenie mixture (supematant of viteUogenic ovary homogenate: 0.2 ml, PBS: 0.3 ml and incomplete Freund's adjuvant: 0.5 ml). Six weeks later, they were boosted intravenously with 0.05 ml of the vitellogenic ovary extract in PBS. After one week, blood was collected. During the same year, two more boosters were made, followed each time by blood sampling. Finally, all blood samples were pooled after being controlled by classical methods of double diffusion. The immunoglobulinswere precipitated by a satured ammonium sulphate solution (1:2) and centrifuged. The pellet was taken up by 0.01 M sodium phosphate buffer, pH 6.3, and dialysed against the same buffer. Haemolymph sampling The haemolymph (10 #1 per animal) was harvested every other day from the pericardial cavity with a microsyringe containing 10 #1 of 10% sodium citrate solution as anticoagulant. Monoclonal antibody production BALB/e mice were injected intraperitoneally with 7.5/~g of purified viteUin in complete Freund's adjuvant. Three weeks later, they were boostered intravenously with the same amount of antigen. Spleens were removed for cell fusion three days after booster. Cell fusion was carried out following the general procedure of K6hler and Milstein (1976) as modified by Nowinski et al. (1979) with SP20. Agl4 myeloma cells. The hybrid cells were selected in hypoxanthinazaserin medium (Zagury et al., 1979) with thymocytes from 3 weeks old mice
E. DERELLE et al.
2
(10 x 10 6 cells/ml). The supernatants of the wells containing developing cultures were tested 10 to 14 days after cell fusion for antibody production by direct ELISA. The microtitre plates (Linbro) were coated with purified vitellin (3 #g protein per ml 0.1 M sodium carbonate buffer, pH 9.6) for 1½hr at 37°C. The supernatants were diluted 1:1 with PBS, pH7.4, 0.1% Tween-20, 0.02% sodium azide and incubated in the wells for 1½hr at 37°C. Vitellin linked antibodies were revealed by an anti-mouse rabbit Ig alkaline phosphatase conjugate reacting with all murine IgG subclasses (Miles, 1:400). After pNPP addition (para-nitrophenylphosphate, 1 mg/ml in 0.05M carbonate buffer, pH 9.8, MgC12 10 -3 M), the yellow colour developed was measured by a Titertek ELISA reader at 405 nm. The positive cell lines were cloned by limiting dilution. The positive sub-clones were grown in aseites for the production of the final titration reagent. The cells were stored frozen in liquid nitrogen. Ig typing was performed by ELISA using rabbit immunoglobulins reacting with specific murine subclasses (Miles).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting Preparations of vitellin and vitellogenin, haemolymph samples from males and vitellogenic females, whole vitellogenic ovary extracts, and mol. wt standards (Bio-rad) were analysed in SDS--10% acrylamide slab gels according to Laemmli (1970). The transfer of proteins from gel onto nitrocellulose sheet (0.45/~m) was carried out following the procedure of Towbin et al. (1979). The sheet was then soaked for 1 hr at 37°C in blocking solution. After washing twice with PBS + TA (0.05% Tween-20 and 0.02% sodium azide), it was incu-
bated for 3 hr at 37°C with monoclonal antibody ascite diluted 1:100, The three time washed membrane was then incubated with the 1:250 solution of peroxidase-labelled sheep anti-mouse IgG (Institut Pasteur) for 1½hr at 37°C. Irnmunocomplexes were revealed according to Hawkes et al. (1982).
Double antibody sandwich ELISA After treatment by 1% glutaraldehyde, the microtitre plates were coated with rabbit Ig (5 #g/ml in 0.1 M sodium carbonate buffer, pH 9.6 during 2 hr at 37°C and one night at 4°C). After 3 washings with PBS, pH 7.4, + TA, different concentrations of purified vitellin used as standards were added on each microtitre plate and the incubation was performed during 1½hr at 37°C. These dilutions and those following were carried out in PBS + TA + 0.1% BSA. After 3 washings, the monoclonal antibody was diluted 1:1500 and the plate was incubated in the same conditions as before. Following 3 washings, alkaline phosphatase labelled rabbit antibodies developed against all IgG mouse subclasses (Sigma, 1:1000) were incubated during 1½hr at 37°C and one night at 4°C. After incubation and subsequent washings, the pNPP substrate was added. Results were read spectrophotometrically after 30 rain on Dynatech ELISA reader. The concentrations of vitellin or vitellogenin were calculated on Multics computer (INRA) using an iterative estimation program (Ross, 1982). We modelized the response by the logistic P1/I + exp - P2(logc - P3). The abcissa is the log of the concentration values (c), the ordinate is the optical density (P1). The abcissa of the inflexion point is P3, the slope for log c --- P3 is equal to PI.P2/4.
A MIW,
1
2
B 3
4
5
6
7
150
100
K
50
K
20
K
Fig. 1. A: SDS-PAGE electrophoresis (10% acrylamide) of crude vitellogenic ovary extract (I), enriched vitellogenin fraction (2), haemocyanin (3), and isolated vitellin (4). B: Immunoblotting pattern of enriched vitellogenin fraction (5), isolated vitellin (6), and crude vitellogenic ovary extract (7), obtained with clone # 19 McAb. H: heavy subunit; L: light subunit.
Vitellogenin and vitellin RESULTS
Hybrid cell lines One fusion gave five positive cultures against vitellin. Among them, clone 4~ 19 was selected for its high affinity in ELISA for the antigen, and subcloned. Ascitic reagent was prepared from several mice. The pool of the fluids constituted the ELISA reagent (clone ~ 19 McAb). It reacted also with the vitellogenin on plates coated with female haemolymph. Clone ~ 19 isotype was G1. Antigenic structure recognized by clone 4~19 McAb. On SDS-PAGE, vitellin and vitellogenin were separated into the same two polypeptide subunits. The light chain was little more intensely stained than the heavy chain (Fig. IA). Identical subunits were identified in vitellogenic female haemolymph and in vitellogenic ovary extract, but not in male haemolymph. By comparison with standard proteins, their mol. wts were estimated to be 92,200 Daltons (S.E. = 2950; n = 16) (H-subunit) and 84,000 Daltons (S.E. = 2600; n = 16) (L-subunit). After the transfer of proteins from the gel onto the nitrocellulose sheet and incubation with clone 19 McAb, the immunoenzymatic method revealed a positive reaction with the L-subunit exclusively (Fig. 1B). Vitellogenin titration As the monoclonal antibody prepared against purified vitellin recognized also vitellogenin, it has been used for viteliogenin titration by ELISA. A typical calibration curve with vitellin is shown in Fig. 2. Vitellogenin fluctuations in the haemolymph of a vitellogenic female are presented in Fig. 3. The concentration varied from a very low level--less than I mg/ml--at the beginning and at the end of the moulting/reproductive cycle to a high level--about 10-15 mg/ml--in D0-Dr stages. DISCUSSION AND CONCLUSIONS
It became necessary for developing studies on vitellogenesis in Crustacea to be able to quantify vitellogenin and vitellin with accuracy and reliability. Recently, Byard and Aiken (1984), searching for an index of ovarian growth in Homarus americanus,
1.5-
==
10-
"[ 64"
2-
x~viatton
o
~
---c
c
k Do
1~,
1~
t~
O0 Gr 61-~2 A~e 6
Molting Stages
Fig. 3. Typical haemolymphatic vitellogenin fluctuations during the moulting reproductive cycle in a vitellogenic female, have quantitated vitellogenin by electrophoregram scanning. Immunological techniques, such as ELISA, are valuable techniques in this regard. The McAb we have produced against purified vitellin of M. rosenbergii has given also a reaction with vitellogenin. Vitellin and vitellogenin of M. rosenbergii are both made up of two subunits of very close mol. wts (84,000 and 92,200 Daltons). The mol. wt of vitellin was estimated to about 330,000 Daltons by gel filtration chromatography on G200 Sephadex (P. Jugan, personal communication). The ratio of the subunits is not known. We have shown by using SDS-PAGE immunoblotting and ELISA that the clone # 19 McAb recognized the light subunit and the native protein as well. It can be used for titration of vitellogenin and vitellin but it must be reminded that the results are expressed in reference to a vitellin standard. Our titration procedure is highly reliable and sensitive. Small quantities of vitellogenin and vitellin, about 25 ng/ml, can be detected and a very satisfying accuracy is obtained up to 100-250 ng/ml. The examples of vitellogenin titration given here showed that the level of vitellogenin in haemolymph of a vitellogenic female fluctuated from about 0.1 mg/ml at the beginning of the reproductive/molting cycle to about 10-15mg/ml at D0-Dr, stages and fall down in D r - D 2 stages, just before exuviation and oviposition. Such a drastic decrease before oviposition has been related in H. americanus (Byard and Aiken, 1984). A following paper will give an extensive report of the variations of vitellogenin in the haemolymph and vitellin amount in the ovary during the reproductive cycle of M. rosenbergii. Acknowledgements--This work was supported by grants 822790 from PIROCEAN and 040678 UA from CNRS. We are grateful to Professor Y. Croisille for his helpful assistance during rabbit immunization against vitellogenic ovary extract and to S. Huet for Multics computer utilization.
00.5
REFERENCES o
o.bs
'o~t ' k~ Vitellin pg/ml
'
go
Fig. 2. A typical calibration curve obtained with viteUin standard.
Asher C., Ramachandran J. and Applebaum S. W. (1983) Determination of locust vitellogenin by radioimmunoassay with [3H]propionyl-vitellogenin. Gen. Comp. Endocrinol. 52, 207-213.
4
E. DERELLEet al.
Byard E. H. and Aiken D. E. (1984) The relationship between molting, reproduction, and a hemolymph female-specific protein in the lobster, Homarus americanus. Comp. Biochem. Physiol. 77A, 749-757. Dumas M., Buschor J. and Lanzrein B. (1982) Development of an enzyme linked immunosorbent assay (ELISA) for measurement of vitellogenin concentrations in cockroaches. Experientia 38, 874-875. Ferenz H.-J. (1978) Uptake of vitellogenin into developing oocytes of Locusta migratoria. J. Insect Physiol. 24, 273-278. Hawkes R., Niday E. and Gordon J. (1982) A dotimmunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119, 142-147. K6hler G. and Milstein C. (1976) Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Europ. J. lmmunol. 6, 511-519. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685. Lowry O. H., Rosebrough N.J., Farr A. L. and Randall R.
J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 19, 263-275. Ma M., Newton P. B., He G., Kelly T. J., Hsu H. T., Masler E. P. and Borkovec A. B. (1984) Development of monoclonal antibodies for monitoring Aedes atropalpus vitellogenesis. J. Insect Physiol. 30, 529-536. Nowinski R. C., Lostrom M. E., Tam M. R., Stone M. R. and Burnette W. N. (1979) The isolation of hybrid cell lines producing monoclonal antibodies against the p15 (E) protein of ecotropic routine leukemia viruses. Virology 93, 111-126. Ross G. J. S. (1982) Lawes Agricultural Trust, Rothamsted Experimental Station, UK (version 3.07). Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer from acrylamide gels to nitrocellulose sheets: procedure and some applications. Biochem. 76, 4350-4354. Zagury D., Phalente L., Bernard J., Hollande E. and Buttin G. (1979) Anti-peroxydase antibody-secreting hybrid lines. I. Identification, cloning and cell characterization. Eur. J. Immunol. 9, 1-16.
0305-0491/86 $3.00+ 0.00 Pergamon Journals Ltd
ELISA TITRATION OF VITELLOGENIN A N D VITELLIN IN THE FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII, WITH MONOCLONAL ANTIBODY E. DERELLE*, J. GROSCLAUDE~, J.-J. MEUSY*, H. JUNI~RA* and M. MARTIN* *Universit6 P. et M. Curie, Laboratoire de Sexualit6 et Reproduction des Invert6br6s, Biit. A, 4 place Jussieu, 75230 Paris Cedex 05, France. "H.N.R.A., Station de Virologie et d'Immunologie, route de Thiverval, 78850 Thiverval Grignon, France (Received 18 November 1985)
Abstract--1. A monoclonal antibody has been produced against viteUin of the freshwater prawn, Macrobrachium rosenbergii. 2. This antibody reacted against the light subunit of vitellin and vitellogenin (L-subunit: 84 kD; H-subunit: 92.2 kD). 3. An indirect ELISA test was developed for titrating vitellogenin in the haemolymph, thus allowing quantitative comparisons along the reproductive cycle.
INTRODUCTION
Viteilogenesis is a crucial period in the female reproductive cycle of Crustacea, characterized by a rapid yolk deposition. The major part of the yolk, vitellin, originates from the uptake of a seric precursor, vitellogenin, by the growing oocytes. Vitellogenin and vitellin in Crustacea are both lipo-glycocarotenoproteins of high molecular weight and cannot be distinguished one from another by conventional immunochemical techniques. Studies on viteliogenesis mechanisms and horm o n a l controls require a highly sensitive and specific quantitation of vitellogenin and vitellin. We have chosen the "enzyme linked i m m u n o s o r b e n t assay" or ELISA. In addition to its high sensitivity, this method avoids the use of radioisotopes and excessive consumption of reagents. ELISA and R I A assays for arthropod vitellogenin and vitellin have been developed among some insect species (Dumas et al., 1982; M a et al., 1984; Ferenz, 1978; Asher et al., 1983). We describe here a quantitative sandwich assay using rabbit polyclonal crude immunoglobulins against vitellogenic ovary extract of the freshwater prawn, Macrobrachium rosenbergii and a mouse antivitellin monoclonal antibody. We thus achieved a higher guarantee of specificity. Furthermore it will be possible to produce in the future unlimited quantity of this monoclonal antibody. MATERIALS AND METHODS Vitellin and vitellogenin preparation The animals were obtained from the Centre Oc~anologique du Pacifique, IFREMER (Tahiti, Polyn6sie Fran~aise). Vitellogenic ovaries were homogenized in 0.1 M sodium phosphate buffer, pH 7.2 (5 ml buffer for 1 g fresh tissue). After centrifugation (12,000 g, 20 min), the components of the supernatant were separated by polyacrylamide gel decC.B.P. 85/IB--A
trophoresis in tubes (acrylamide 6% in Tris-glycine buffer, pH 8.3). Vitellin appeared as an orange coloured band of low mobility. Vitellin was sliced out and eluted by electrodialysis, then dialysed against 0.01 M phosphate buffer. After lyophilization, the vitellin was solubilized and its protein content was estimated by the method of Lowry et al. (1951) with BSA as standard. Vitellogenin was enriched from crude haemolymph by the same electrophoretie procedure. Rabbit immunization and immunoglobulin preparation Two rabbits were injected intramuscularly with the antigenie mixture (supematant of viteUogenic ovary homogenate: 0.2 ml, PBS: 0.3 ml and incomplete Freund's adjuvant: 0.5 ml). Six weeks later, they were boosted intravenously with 0.05 ml of the vitellogenic ovary extract in PBS. After one week, blood was collected. During the same year, two more boosters were made, followed each time by blood sampling. Finally, all blood samples were pooled after being controlled by classical methods of double diffusion. The immunoglobulinswere precipitated by a satured ammonium sulphate solution (1:2) and centrifuged. The pellet was taken up by 0.01 M sodium phosphate buffer, pH 6.3, and dialysed against the same buffer. Haemolymph sampling The haemolymph (10 #1 per animal) was harvested every other day from the pericardial cavity with a microsyringe containing 10 #1 of 10% sodium citrate solution as anticoagulant. Monoclonal antibody production BALB/e mice were injected intraperitoneally with 7.5/~g of purified viteUin in complete Freund's adjuvant. Three weeks later, they were boostered intravenously with the same amount of antigen. Spleens were removed for cell fusion three days after booster. Cell fusion was carried out following the general procedure of K6hler and Milstein (1976) as modified by Nowinski et al. (1979) with SP20. Agl4 myeloma cells. The hybrid cells were selected in hypoxanthinazaserin medium (Zagury et al., 1979) with thymocytes from 3 weeks old mice
E. DERELLE et al.
2
(10 x 10 6 cells/ml). The supernatants of the wells containing developing cultures were tested 10 to 14 days after cell fusion for antibody production by direct ELISA. The microtitre plates (Linbro) were coated with purified vitellin (3 #g protein per ml 0.1 M sodium carbonate buffer, pH 9.6) for 1½hr at 37°C. The supernatants were diluted 1:1 with PBS, pH7.4, 0.1% Tween-20, 0.02% sodium azide and incubated in the wells for 1½hr at 37°C. Vitellin linked antibodies were revealed by an anti-mouse rabbit Ig alkaline phosphatase conjugate reacting with all murine IgG subclasses (Miles, 1:400). After pNPP addition (para-nitrophenylphosphate, 1 mg/ml in 0.05M carbonate buffer, pH 9.8, MgC12 10 -3 M), the yellow colour developed was measured by a Titertek ELISA reader at 405 nm. The positive cell lines were cloned by limiting dilution. The positive sub-clones were grown in aseites for the production of the final titration reagent. The cells were stored frozen in liquid nitrogen. Ig typing was performed by ELISA using rabbit immunoglobulins reacting with specific murine subclasses (Miles).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting Preparations of vitellin and vitellogenin, haemolymph samples from males and vitellogenic females, whole vitellogenic ovary extracts, and mol. wt standards (Bio-rad) were analysed in SDS--10% acrylamide slab gels according to Laemmli (1970). The transfer of proteins from gel onto nitrocellulose sheet (0.45/~m) was carried out following the procedure of Towbin et al. (1979). The sheet was then soaked for 1 hr at 37°C in blocking solution. After washing twice with PBS + TA (0.05% Tween-20 and 0.02% sodium azide), it was incu-
bated for 3 hr at 37°C with monoclonal antibody ascite diluted 1:100, The three time washed membrane was then incubated with the 1:250 solution of peroxidase-labelled sheep anti-mouse IgG (Institut Pasteur) for 1½hr at 37°C. Irnmunocomplexes were revealed according to Hawkes et al. (1982).
Double antibody sandwich ELISA After treatment by 1% glutaraldehyde, the microtitre plates were coated with rabbit Ig (5 #g/ml in 0.1 M sodium carbonate buffer, pH 9.6 during 2 hr at 37°C and one night at 4°C). After 3 washings with PBS, pH 7.4, + TA, different concentrations of purified vitellin used as standards were added on each microtitre plate and the incubation was performed during 1½hr at 37°C. These dilutions and those following were carried out in PBS + TA + 0.1% BSA. After 3 washings, the monoclonal antibody was diluted 1:1500 and the plate was incubated in the same conditions as before. Following 3 washings, alkaline phosphatase labelled rabbit antibodies developed against all IgG mouse subclasses (Sigma, 1:1000) were incubated during 1½hr at 37°C and one night at 4°C. After incubation and subsequent washings, the pNPP substrate was added. Results were read spectrophotometrically after 30 rain on Dynatech ELISA reader. The concentrations of vitellin or vitellogenin were calculated on Multics computer (INRA) using an iterative estimation program (Ross, 1982). We modelized the response by the logistic P1/I + exp - P2(logc - P3). The abcissa is the log of the concentration values (c), the ordinate is the optical density (P1). The abcissa of the inflexion point is P3, the slope for log c --- P3 is equal to PI.P2/4.
A MIW,
1
2
B 3
4
5
6
7
150
100
K
50
K
20
K
Fig. 1. A: SDS-PAGE electrophoresis (10% acrylamide) of crude vitellogenic ovary extract (I), enriched vitellogenin fraction (2), haemocyanin (3), and isolated vitellin (4). B: Immunoblotting pattern of enriched vitellogenin fraction (5), isolated vitellin (6), and crude vitellogenic ovary extract (7), obtained with clone # 19 McAb. H: heavy subunit; L: light subunit.
Vitellogenin and vitellin RESULTS
Hybrid cell lines One fusion gave five positive cultures against vitellin. Among them, clone 4~ 19 was selected for its high affinity in ELISA for the antigen, and subcloned. Ascitic reagent was prepared from several mice. The pool of the fluids constituted the ELISA reagent (clone ~ 19 McAb). It reacted also with the vitellogenin on plates coated with female haemolymph. Clone ~ 19 isotype was G1. Antigenic structure recognized by clone 4~19 McAb. On SDS-PAGE, vitellin and vitellogenin were separated into the same two polypeptide subunits. The light chain was little more intensely stained than the heavy chain (Fig. IA). Identical subunits were identified in vitellogenic female haemolymph and in vitellogenic ovary extract, but not in male haemolymph. By comparison with standard proteins, their mol. wts were estimated to be 92,200 Daltons (S.E. = 2950; n = 16) (H-subunit) and 84,000 Daltons (S.E. = 2600; n = 16) (L-subunit). After the transfer of proteins from the gel onto the nitrocellulose sheet and incubation with clone 19 McAb, the immunoenzymatic method revealed a positive reaction with the L-subunit exclusively (Fig. 1B). Vitellogenin titration As the monoclonal antibody prepared against purified vitellin recognized also vitellogenin, it has been used for viteliogenin titration by ELISA. A typical calibration curve with vitellin is shown in Fig. 2. Vitellogenin fluctuations in the haemolymph of a vitellogenic female are presented in Fig. 3. The concentration varied from a very low level--less than I mg/ml--at the beginning and at the end of the moulting/reproductive cycle to a high level--about 10-15 mg/ml--in D0-Dr stages. DISCUSSION AND CONCLUSIONS
It became necessary for developing studies on vitellogenesis in Crustacea to be able to quantify vitellogenin and vitellin with accuracy and reliability. Recently, Byard and Aiken (1984), searching for an index of ovarian growth in Homarus americanus,
1.5-
==
10-
"[ 64"
2-
x~viatton
o
~
---c
c
k Do
1~,
1~
t~
O0 Gr 61-~2 A~e 6
Molting Stages
Fig. 3. Typical haemolymphatic vitellogenin fluctuations during the moulting reproductive cycle in a vitellogenic female, have quantitated vitellogenin by electrophoregram scanning. Immunological techniques, such as ELISA, are valuable techniques in this regard. The McAb we have produced against purified vitellin of M. rosenbergii has given also a reaction with vitellogenin. Vitellin and vitellogenin of M. rosenbergii are both made up of two subunits of very close mol. wts (84,000 and 92,200 Daltons). The mol. wt of vitellin was estimated to about 330,000 Daltons by gel filtration chromatography on G200 Sephadex (P. Jugan, personal communication). The ratio of the subunits is not known. We have shown by using SDS-PAGE immunoblotting and ELISA that the clone # 19 McAb recognized the light subunit and the native protein as well. It can be used for titration of vitellogenin and vitellin but it must be reminded that the results are expressed in reference to a vitellin standard. Our titration procedure is highly reliable and sensitive. Small quantities of vitellogenin and vitellin, about 25 ng/ml, can be detected and a very satisfying accuracy is obtained up to 100-250 ng/ml. The examples of vitellogenin titration given here showed that the level of vitellogenin in haemolymph of a vitellogenic female fluctuated from about 0.1 mg/ml at the beginning of the reproductive/molting cycle to about 10-15mg/ml at D0-Dr, stages and fall down in D r - D 2 stages, just before exuviation and oviposition. Such a drastic decrease before oviposition has been related in H. americanus (Byard and Aiken, 1984). A following paper will give an extensive report of the variations of vitellogenin in the haemolymph and vitellin amount in the ovary during the reproductive cycle of M. rosenbergii. Acknowledgements--This work was supported by grants 822790 from PIROCEAN and 040678 UA from CNRS. We are grateful to Professor Y. Croisille for his helpful assistance during rabbit immunization against vitellogenic ovary extract and to S. Huet for Multics computer utilization.
00.5
REFERENCES o
o.bs
'o~t ' k~ Vitellin pg/ml
'
go
Fig. 2. A typical calibration curve obtained with viteUin standard.
Asher C., Ramachandran J. and Applebaum S. W. (1983) Determination of locust vitellogenin by radioimmunoassay with [3H]propionyl-vitellogenin. Gen. Comp. Endocrinol. 52, 207-213.
4
E. DERELLEet al.
Byard E. H. and Aiken D. E. (1984) The relationship between molting, reproduction, and a hemolymph female-specific protein in the lobster, Homarus americanus. Comp. Biochem. Physiol. 77A, 749-757. Dumas M., Buschor J. and Lanzrein B. (1982) Development of an enzyme linked immunosorbent assay (ELISA) for measurement of vitellogenin concentrations in cockroaches. Experientia 38, 874-875. Ferenz H.-J. (1978) Uptake of vitellogenin into developing oocytes of Locusta migratoria. J. Insect Physiol. 24, 273-278. Hawkes R., Niday E. and Gordon J. (1982) A dotimmunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119, 142-147. K6hler G. and Milstein C. (1976) Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Europ. J. lmmunol. 6, 511-519. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685. Lowry O. H., Rosebrough N.J., Farr A. L. and Randall R.
J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 19, 263-275. Ma M., Newton P. B., He G., Kelly T. J., Hsu H. T., Masler E. P. and Borkovec A. B. (1984) Development of monoclonal antibodies for monitoring Aedes atropalpus vitellogenesis. J. Insect Physiol. 30, 529-536. Nowinski R. C., Lostrom M. E., Tam M. R., Stone M. R. and Burnette W. N. (1979) The isolation of hybrid cell lines producing monoclonal antibodies against the p15 (E) protein of ecotropic routine leukemia viruses. Virology 93, 111-126. Ross G. J. S. (1982) Lawes Agricultural Trust, Rothamsted Experimental Station, UK (version 3.07). Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer from acrylamide gels to nitrocellulose sheets: procedure and some applications. Biochem. 76, 4350-4354. Zagury D., Phalente L., Bernard J., Hollande E. and Buttin G. (1979) Anti-peroxydase antibody-secreting hybrid lines. I. Identification, cloning and cell characterization. Eur. J. Immunol. 9, 1-16.