CA1178886A - Process for recognising females in the course of vitellogenesis in oviparous animals, by an immunoagglutination test - Google Patents
Process for recognising females in the course of vitellogenesis in oviparous animals, by an immunoagglutination testInfo
- Publication number
- CA1178886A CA1178886A CA000393566A CA393566A CA1178886A CA 1178886 A CA1178886 A CA 1178886A CA 000393566 A CA000393566 A CA 000393566A CA 393566 A CA393566 A CA 393566A CA 1178886 A CA1178886 A CA 1178886A
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- CA
- Canada
- Prior art keywords
- vitellogenin
- test
- females
- blood
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
Abstract
ABSTRACT OF THE DISCLOSURE
The invention relates to a process for recognising females in the course of vitellogensis in oviparous animals , consisting in detecting th vittelogenin specifically present in the blood of females, by an immunoagglutination test made on a sample of blood serum of the animals. This process allows a simple, rapid determination of the sex of oviparous animals, particularly fish, well before the appearance of the morpho-logical features usually used for recognising the sexes.
The invention relates to a process for recognising females in the course of vitellogensis in oviparous animals , consisting in detecting th vittelogenin specifically present in the blood of females, by an immunoagglutination test made on a sample of blood serum of the animals. This process allows a simple, rapid determination of the sex of oviparous animals, particularly fish, well before the appearance of the morpho-logical features usually used for recognising the sexes.
Description
-il'7~8t~6 The present invention relates to a simple, rapld and speciflc test for recognising the sex of oviparous animals, without injuring them.
Sex recognition, particularly in fish, is useful to 5 ecologists for determlning the breeding potential of the popu-lations. Management of the breeding stocks intended for fish-farming is also optimised thereby.
Outside the period of reproduction, the search for the sex-determining criterla from morphological features is a 10 long and haræardous operation. On the other hand, it is possible to envisage a sex-determlning technique based on the bio-chemical recognition of a protein specific to one of the sexes.
In 1932, Sasaki proposed sex recognltion of chlcken by using, as sex-determining criterion, the presence of a plas-15 matic protein precursor of the vitellin reserves of the egg. Morerecently, an equivalent proteln (vltellogenin) has been demon-strated in salmon and trout by techniques of electrophoresis and immunodiffusion.
Although these techniques are sufficiently sensitive to 20 enable the vitellogenin to be detected in the course of the annual cycle, the difficulty of carrying them out in the field precludes their being considered as being capable of furnishing the potential users with immediate responses.
The sex-determining process according to the invention 25 consists in detecting the vitellogenin specifically present In the blood of females at a certain moment of the cycle of reproductior~
i. e. in the course of vitellogenesis, by an lmmunoagglutination test made on a sample of serum or blood of the animal; thls sample ls brought into presence with a support sensitized by 30 antlvitellogenin antibodies, and the formation of a precipitate (positive test) indicates the presence of vitellogenin.
The immunoagglutination test is based on the reaction between the antigen (vitellogenin in the medium to be tested) and its specific antibody fixed on a support having a relatively large 35 specific surface such as tanned sheep erythrocytes or particles of latex.
8~6 The purpose of the support is to render the antigen-antlbody reactlon vlsible. Although this always takes place whatever the ratio oI the antigen-antibody concentrations, it is vlsible only in a reduced range of the concentration ratios, 5 called "zone of equivalence". In fact, the visualisation of the reaction corresponds to a precipitation of the antigen-antibody complex due to the formation of a sufficient density of bridges between the antlbodies by the antigens. The accompanying Figure illustrates this phenomenon.
This Figure shows:
- at a), the different products present: 1) the support (latex balls), 2) the antibody, 3) the antigen;
- at b), the sensitised support, i. e. coated with the antibody;
- at c), if the antigen is in excess: there is saturatlon by the antigen of the sites of connection (antibody) on the support;
- at d), if the antibody is in excess: there are only very few sites of antibody-antigen connection on the support.
In the two cases c) and d) where one of the reagents 20 ls in excess, the density of the bridges formed is too low to form a precipitant network, and the reaction appears negative to the observer.
- On the other hand, at e), which shows what occurs in the "zone of equivalence", the number of bridges formed between the anti-25 bodles by the antigens is sufficient to provoke a visible preci-pitation (agglutination): the reaction appears positive.
Consequently, for a given concentration of antibodies on the support, an apparently negative reaction Inay result from an absence of antigen, but also from an excess concentratlon of 30 antlgen In the tested medium, In this case, to decide between these two possibilities (absence or excess of antigen), there is adcled to the reagents already present,known serum giving a posi-tive reaction (i. e. containlng antigen): if a precipitation is pro-duced, there was absence of antigen in the medium tested. In the 35 contrary case, the antigen~vasin excess with respect to the con-centration of antibody chosen.
8~16 To carry out the agglutination test, the antiboay specific of the vitellogenin, with which an adequate support will be sensi-tised, must be available. Antisera containing antivitellogenin ~'-globulins (anti-VgP) were prepared by injection into 5 rabblts of pure vitellogenin (VgP) isolated from the blood of oestrogenlsed fish ( particularly of the Salmonidae family) Obtainln~ of pure vitello~zenin:
In the Salmonidae family, the oestrogenisatlon of the male and of the emale, whatever the time of the reproductive 10 cycle, induces a hepatic synthesis of vitellogenin and an increase in the plasmatlc concentration of this protein (Bailey, 1957, Takashima et al., 1972). This method was applied to female rainbow trouts which were treated with oestradiol-17/3 by lntraperitoneal injection for four weeks, at a rate of one injection 15 of 0. 5 mg/kg every other day. At the end of the treatment, the animals were sacrificed and the blood collected at the caudal artery. Purification of the vitellogenln is inspired from the tech-nique of Hara and Hirai (1978): after coagulation, the serum is separated by centrifugation and dialysed three days against one 20 hundred volumes of distilled water renewed each day. The pre-cipltate formed is separated by centrifugation (300 g, 4C, one hour). The sediment is redissolved in a tris 0. 05 M NaCl M
buffer, pH 9. 2. The insoluble matter is eliminated by centrifu-gation. The supernatant matter is deposited on a column of 25 sepharose 6B (lm x 1. 6 cm) The vitellogenin in the fractions issuing from chromato-graphy was recognised by electrophoresls on 5'10 polyacrylamide gel (14 cm long) according to the technique of Ornstein ancl Davis (1964) modified by readjusting the pH of the migration bufer to 30 9. 2 by N NaOH. Sera of females in the course of vitellogenesis and of males are used as reference. The richest fractions are collected together and dlalysed against distilled water. The pre-cipltate is taken up in the tris 0 05M NaCl M buffer, at pH 9. 2, then rechromatographed over sepharose 6B and dialysed under the 35 same conditions as hereinabove.
The concentration of the final solution of vitellogenin (VgP) was determined by the method of Lowry et al (1951) , 8~6 Preparation of the antivitello~enin ~-~lobulins 0. 5 mg of VgP, in the presence of complete Freund adjuvant, is injected into rabbits by the subcutaneous route, and this injection is repeated every fifteen days for three months.
5 ~fter this period, the animals are bled, and the anti-VgP
~ -globulins are isolated from the serum by precipitation with amnlonium sulfate at 33% saturation; the precipitate is centri-fuged, dialysed against distilled water and lyophilised Preparation of the sensitised support:
Latex balls (Difco 0. 81) were used, but any other con-ventional adsorption support may be used. The lyophilised anti-VgP ^~-globulins are redissolved in a veronal buffer ~0. 05M) CaC12 (5mM). The antibody is fixed on the latex balls according to the technique of McCarthy (1975) in which the glycine-NaCl 15 buffer has been replaced by a veronal buffer at pH 8. 2. The tests were made by sensitising the balls by solutions containing the anti-VgP ~r -globulin s at concentrations 4 and 9 times higher than the concentration of the ~r-globulins in the serum.
A~zlutination test:
The agglutlnation test is carried out by bringing into presence, on a trim~ed glass slide, a drop of the medium to be tested, a drop of veronal buffer at pH 8~ 2 and a drop of the sensitised latex ball suspension. The mixture is homogenised and stirred slowly. The positive reaction, characterised by the 25 appearance of aggregates of balls, is developed in 2 to 3 minutes.
When the reaction ls negative, control positive serum is added and 1 to Z minutes are allowed to lapse to determine whether there ls an excess (negative response) or an absence of vitellogenln (positive response), No difference was observed between the reactions of the serum and of the plasma of the same animal The whole blood may also be used to carry out the test. However, the blood provokes coagulations which may be confused with the specific agglutination and this difficulty is eliminated by adding heparin 35 (300 UI/ml) to the reaction buffer. For the same résponse, a quantity of blood one and a half to two times greater than that of 11788~6 the corresponding sera must be used.
Validation of the test:
-The validation of the responses to the agglutinatlontest was sought on the one hand by immunodiffusion on 0. 8%
5 agarose gel in veronal buffer 0. 05M, pH 8. 3, and on the other hand by carrying out the test on the plasma of animals whose se~ was previously determined by observation of the gonads.
The tests were made on two species:
- fario trouts (Salmo trutta): males and females were killed 10 monthly in the course of their first reproductive cycle. The females were characterised either from the gonadosomatic ratios (RCS) or from the precise stage and the diameter of the ovocytes in the course of the final phases of the cycle, according to the criteria defined by Jalabert (1978): end of vitellogenesis 15 (FV), maturing, ovulated. Plasma of each animal was obtained after blood sampling before slaughter. This plasma is kept frozen until the test.
- Altantic salmo_: come from catches by sport fishermen during the whole legal fishing season. The whole blood was fro-20 zen in situ, the serum being separated by centrifugation only afterit has arrived in the laboratory. The animals were characterised by by thelr gonado-somatic ratio and by the mean diameter of the ovocytes calculated from 48 ovocytes taken on six different ovi-gerous slides.
25 Sensitivitv of the test:
Table I hereinafter shows, for concentrations of vitello-genin varying from 120 to 0. 05 mg/ml, the responses obtained on the one hand wlth the lmmunoagglutination test according to the lnvention using two concentrations of ~-globulins for sensitising 30 the latex balls, and on the other hand wlth the immunodiffusion te st.
The table shows that two different ranges of concentra-tion of vitellogenin leading to positive responses correspond to the two concentrations of sensitisation anti-VgP (9 times and 4 times 35 greater than that of the whole antiserum). The sensitivity of res-ponse for media with low concentration of vitellogenin is increased 8~6 -b -by reducing the quantity of sensitlsing antibodies.
On the two sides of the positive response range, a negative reaction may be due either to an excess or to a lack of vitellogenin; hence the necessity,in the case of a negative reaction, 5 of the addition to the reactlon medium of a known positive serum, to decide between the two hypotheses.
The agglutination test according to the invention was applied to the detection of the vitellogenin in the course of the reproductive cycle of the two species of fish indicated hereinabove.
10 Fario trout: The results obtained are shown in Table II herein-after, Any any time of the cycle, no positive reaction was demonstrated Irom the male sera.
On the other hand, in the female, the appearance of 15 vitellogenln may be detected in a few animals from April 12 and becomes general on May 26, whilst the phase of rapid growth aof the gonad and the ovocytes has not begun. During a period of one or two post-ov~ation months, no positive response was obtained.
On a larger sample, the immunodiffusion test confirmed that Z0 all the females possess circulating vitellogenin around mid-May.
Atlantic salmon Catches were spread out from the end of March to the end of June, at which period no secondary sexual character enabled the sexes to be differentiated The vitellogenin is detected In all females by immuno-25 agglutination, whilst the RGS's are low and the diameters of theovocytes do not vary substantially during the fishing season. None of the males responded positively.
- Thus, the study carried out on a complete cycle in the farlo trout shows that the sensitivity of the lmmunoagglutinatlon 30 test allows a qualltatlve detection of the vltellogenin from the month of April and constitutes a sufficiently reliable process for sex determination as frorn the month of May. The use of the process according to the invention therefore enables stocks of breeding females to be constituted in Iish Iarms several months before the 35 app~ ance of the morphologlcal features generally used for recog-nising the sexes, 8~36 ~ or the Atlantic salmon, earlier studies indicate the presence of vitellogenin in the females at dlfferent stages at ascent and on spa~hning grounds. The study carried out with the agglutination test gives the same results: all the females fished 5 between the beginning of March and mid-June respond positively;
vitellogenesis is apparently therefore started at the moment of entry into fresh water. The carrying out of the process according to the invention at the traps is therefore suitable for determining the sex ratio and reproductive potential of a population of salmons.
The concentrations of sensitising antibodies have enabled detection limits to be obtained, compatible with the rates of circulating vitellogenin in the majority of the animals starting vitellogenesis. A greater sensitivity of the test may be obtained by reducing the quantil;y of antibodies fixed on the balls. These lS condltions should ~make lt possible to extend the ranges o positlve responses towards the low concentrations of vitellogenin, and to increase the percentages of sex determination at the be-ginning of cycle. However, lt should be specified that, the more the test ls sensitive, tne more the response times increase and 20 the less clear is read-out. Thus, the definition of the optimal sensitising antib~dy/support ratios must be determined in each pa rti cula r ca s e .
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Sex recognition, particularly in fish, is useful to 5 ecologists for determlning the breeding potential of the popu-lations. Management of the breeding stocks intended for fish-farming is also optimised thereby.
Outside the period of reproduction, the search for the sex-determining criterla from morphological features is a 10 long and haræardous operation. On the other hand, it is possible to envisage a sex-determlning technique based on the bio-chemical recognition of a protein specific to one of the sexes.
In 1932, Sasaki proposed sex recognltion of chlcken by using, as sex-determining criterion, the presence of a plas-15 matic protein precursor of the vitellin reserves of the egg. Morerecently, an equivalent proteln (vltellogenin) has been demon-strated in salmon and trout by techniques of electrophoresis and immunodiffusion.
Although these techniques are sufficiently sensitive to 20 enable the vitellogenin to be detected in the course of the annual cycle, the difficulty of carrying them out in the field precludes their being considered as being capable of furnishing the potential users with immediate responses.
The sex-determining process according to the invention 25 consists in detecting the vitellogenin specifically present In the blood of females at a certain moment of the cycle of reproductior~
i. e. in the course of vitellogenesis, by an lmmunoagglutination test made on a sample of serum or blood of the animal; thls sample ls brought into presence with a support sensitized by 30 antlvitellogenin antibodies, and the formation of a precipitate (positive test) indicates the presence of vitellogenin.
The immunoagglutination test is based on the reaction between the antigen (vitellogenin in the medium to be tested) and its specific antibody fixed on a support having a relatively large 35 specific surface such as tanned sheep erythrocytes or particles of latex.
8~6 The purpose of the support is to render the antigen-antlbody reactlon vlsible. Although this always takes place whatever the ratio oI the antigen-antibody concentrations, it is vlsible only in a reduced range of the concentration ratios, 5 called "zone of equivalence". In fact, the visualisation of the reaction corresponds to a precipitation of the antigen-antibody complex due to the formation of a sufficient density of bridges between the antlbodies by the antigens. The accompanying Figure illustrates this phenomenon.
This Figure shows:
- at a), the different products present: 1) the support (latex balls), 2) the antibody, 3) the antigen;
- at b), the sensitised support, i. e. coated with the antibody;
- at c), if the antigen is in excess: there is saturatlon by the antigen of the sites of connection (antibody) on the support;
- at d), if the antibody is in excess: there are only very few sites of antibody-antigen connection on the support.
In the two cases c) and d) where one of the reagents 20 ls in excess, the density of the bridges formed is too low to form a precipitant network, and the reaction appears negative to the observer.
- On the other hand, at e), which shows what occurs in the "zone of equivalence", the number of bridges formed between the anti-25 bodles by the antigens is sufficient to provoke a visible preci-pitation (agglutination): the reaction appears positive.
Consequently, for a given concentration of antibodies on the support, an apparently negative reaction Inay result from an absence of antigen, but also from an excess concentratlon of 30 antlgen In the tested medium, In this case, to decide between these two possibilities (absence or excess of antigen), there is adcled to the reagents already present,known serum giving a posi-tive reaction (i. e. containlng antigen): if a precipitation is pro-duced, there was absence of antigen in the medium tested. In the 35 contrary case, the antigen~vasin excess with respect to the con-centration of antibody chosen.
8~16 To carry out the agglutination test, the antiboay specific of the vitellogenin, with which an adequate support will be sensi-tised, must be available. Antisera containing antivitellogenin ~'-globulins (anti-VgP) were prepared by injection into 5 rabblts of pure vitellogenin (VgP) isolated from the blood of oestrogenlsed fish ( particularly of the Salmonidae family) Obtainln~ of pure vitello~zenin:
In the Salmonidae family, the oestrogenisatlon of the male and of the emale, whatever the time of the reproductive 10 cycle, induces a hepatic synthesis of vitellogenin and an increase in the plasmatlc concentration of this protein (Bailey, 1957, Takashima et al., 1972). This method was applied to female rainbow trouts which were treated with oestradiol-17/3 by lntraperitoneal injection for four weeks, at a rate of one injection 15 of 0. 5 mg/kg every other day. At the end of the treatment, the animals were sacrificed and the blood collected at the caudal artery. Purification of the vitellogenln is inspired from the tech-nique of Hara and Hirai (1978): after coagulation, the serum is separated by centrifugation and dialysed three days against one 20 hundred volumes of distilled water renewed each day. The pre-cipltate formed is separated by centrifugation (300 g, 4C, one hour). The sediment is redissolved in a tris 0. 05 M NaCl M
buffer, pH 9. 2. The insoluble matter is eliminated by centrifu-gation. The supernatant matter is deposited on a column of 25 sepharose 6B (lm x 1. 6 cm) The vitellogenin in the fractions issuing from chromato-graphy was recognised by electrophoresls on 5'10 polyacrylamide gel (14 cm long) according to the technique of Ornstein ancl Davis (1964) modified by readjusting the pH of the migration bufer to 30 9. 2 by N NaOH. Sera of females in the course of vitellogenesis and of males are used as reference. The richest fractions are collected together and dlalysed against distilled water. The pre-cipltate is taken up in the tris 0 05M NaCl M buffer, at pH 9. 2, then rechromatographed over sepharose 6B and dialysed under the 35 same conditions as hereinabove.
The concentration of the final solution of vitellogenin (VgP) was determined by the method of Lowry et al (1951) , 8~6 Preparation of the antivitello~enin ~-~lobulins 0. 5 mg of VgP, in the presence of complete Freund adjuvant, is injected into rabbits by the subcutaneous route, and this injection is repeated every fifteen days for three months.
5 ~fter this period, the animals are bled, and the anti-VgP
~ -globulins are isolated from the serum by precipitation with amnlonium sulfate at 33% saturation; the precipitate is centri-fuged, dialysed against distilled water and lyophilised Preparation of the sensitised support:
Latex balls (Difco 0. 81) were used, but any other con-ventional adsorption support may be used. The lyophilised anti-VgP ^~-globulins are redissolved in a veronal buffer ~0. 05M) CaC12 (5mM). The antibody is fixed on the latex balls according to the technique of McCarthy (1975) in which the glycine-NaCl 15 buffer has been replaced by a veronal buffer at pH 8. 2. The tests were made by sensitising the balls by solutions containing the anti-VgP ~r -globulin s at concentrations 4 and 9 times higher than the concentration of the ~r-globulins in the serum.
A~zlutination test:
The agglutlnation test is carried out by bringing into presence, on a trim~ed glass slide, a drop of the medium to be tested, a drop of veronal buffer at pH 8~ 2 and a drop of the sensitised latex ball suspension. The mixture is homogenised and stirred slowly. The positive reaction, characterised by the 25 appearance of aggregates of balls, is developed in 2 to 3 minutes.
When the reaction ls negative, control positive serum is added and 1 to Z minutes are allowed to lapse to determine whether there ls an excess (negative response) or an absence of vitellogenln (positive response), No difference was observed between the reactions of the serum and of the plasma of the same animal The whole blood may also be used to carry out the test. However, the blood provokes coagulations which may be confused with the specific agglutination and this difficulty is eliminated by adding heparin 35 (300 UI/ml) to the reaction buffer. For the same résponse, a quantity of blood one and a half to two times greater than that of 11788~6 the corresponding sera must be used.
Validation of the test:
-The validation of the responses to the agglutinatlontest was sought on the one hand by immunodiffusion on 0. 8%
5 agarose gel in veronal buffer 0. 05M, pH 8. 3, and on the other hand by carrying out the test on the plasma of animals whose se~ was previously determined by observation of the gonads.
The tests were made on two species:
- fario trouts (Salmo trutta): males and females were killed 10 monthly in the course of their first reproductive cycle. The females were characterised either from the gonadosomatic ratios (RCS) or from the precise stage and the diameter of the ovocytes in the course of the final phases of the cycle, according to the criteria defined by Jalabert (1978): end of vitellogenesis 15 (FV), maturing, ovulated. Plasma of each animal was obtained after blood sampling before slaughter. This plasma is kept frozen until the test.
- Altantic salmo_: come from catches by sport fishermen during the whole legal fishing season. The whole blood was fro-20 zen in situ, the serum being separated by centrifugation only afterit has arrived in the laboratory. The animals were characterised by by thelr gonado-somatic ratio and by the mean diameter of the ovocytes calculated from 48 ovocytes taken on six different ovi-gerous slides.
25 Sensitivitv of the test:
Table I hereinafter shows, for concentrations of vitello-genin varying from 120 to 0. 05 mg/ml, the responses obtained on the one hand wlth the lmmunoagglutination test according to the lnvention using two concentrations of ~-globulins for sensitising 30 the latex balls, and on the other hand wlth the immunodiffusion te st.
The table shows that two different ranges of concentra-tion of vitellogenin leading to positive responses correspond to the two concentrations of sensitisation anti-VgP (9 times and 4 times 35 greater than that of the whole antiserum). The sensitivity of res-ponse for media with low concentration of vitellogenin is increased 8~6 -b -by reducing the quantity of sensitlsing antibodies.
On the two sides of the positive response range, a negative reaction may be due either to an excess or to a lack of vitellogenin; hence the necessity,in the case of a negative reaction, 5 of the addition to the reactlon medium of a known positive serum, to decide between the two hypotheses.
The agglutination test according to the invention was applied to the detection of the vitellogenin in the course of the reproductive cycle of the two species of fish indicated hereinabove.
10 Fario trout: The results obtained are shown in Table II herein-after, Any any time of the cycle, no positive reaction was demonstrated Irom the male sera.
On the other hand, in the female, the appearance of 15 vitellogenln may be detected in a few animals from April 12 and becomes general on May 26, whilst the phase of rapid growth aof the gonad and the ovocytes has not begun. During a period of one or two post-ov~ation months, no positive response was obtained.
On a larger sample, the immunodiffusion test confirmed that Z0 all the females possess circulating vitellogenin around mid-May.
Atlantic salmon Catches were spread out from the end of March to the end of June, at which period no secondary sexual character enabled the sexes to be differentiated The vitellogenin is detected In all females by immuno-25 agglutination, whilst the RGS's are low and the diameters of theovocytes do not vary substantially during the fishing season. None of the males responded positively.
- Thus, the study carried out on a complete cycle in the farlo trout shows that the sensitivity of the lmmunoagglutinatlon 30 test allows a qualltatlve detection of the vltellogenin from the month of April and constitutes a sufficiently reliable process for sex determination as frorn the month of May. The use of the process according to the invention therefore enables stocks of breeding females to be constituted in Iish Iarms several months before the 35 app~ ance of the morphologlcal features generally used for recog-nising the sexes, 8~36 ~ or the Atlantic salmon, earlier studies indicate the presence of vitellogenin in the females at dlfferent stages at ascent and on spa~hning grounds. The study carried out with the agglutination test gives the same results: all the females fished 5 between the beginning of March and mid-June respond positively;
vitellogenesis is apparently therefore started at the moment of entry into fresh water. The carrying out of the process according to the invention at the traps is therefore suitable for determining the sex ratio and reproductive potential of a population of salmons.
The concentrations of sensitising antibodies have enabled detection limits to be obtained, compatible with the rates of circulating vitellogenin in the majority of the animals starting vitellogenesis. A greater sensitivity of the test may be obtained by reducing the quantil;y of antibodies fixed on the balls. These lS condltions should ~make lt possible to extend the ranges o positlve responses towards the low concentrations of vitellogenin, and to increase the percentages of sex determination at the be-ginning of cycle. However, lt should be specified that, the more the test ls sensitive, tne more the response times increase and 20 the less clear is read-out. Thus, the definition of the optimal sensitising antib~dy/support ratios must be determined in each pa rti cula r ca s e .
11 J~86 E
Q, ,~, o ~
~ ~ ~ X ~ o~ ~ ~ E ,~
``-" 11'-ib~886 _ ~_ ~
CL, h ~ ~ ~ ~ ~ ~
O
V ¢ O + + + + + +
h 5 ~ _ ~ ~o ~
_ ~ X O h I ~ ~ ~ _ +
~ ~0 ~ ~ ~
~ O ,L h ~ U~
O '~- ~n + + + + + + + + + + + + + _ ~
~ . cX Iq~,~ ~ ^J ~.
. ~ O ~ -- + ' + + + + h ~1~ I ~ ? D ,C o ~ D ¦
~ , _ ~ ~0 l ~ 0~ C~^ O^ O- 0 0~ 0- O^ 0~ O^ C
b + ~ + ~ + ~ + ~ + ~ + ~ + ~ + ~ + ~ + ~ o . l ~ 1 ~ ~_ O ~;~. V V ~/ V v V c~ o ~ ~ ~ ~ h . l `$ i~ ~ ~ ~ C`l ~`1 ~) ~ `;t O
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Claims (8)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Process for recognising females in the course of vitellogenesis in oviparous animals, consisting in detecting the vitellogenin specifically present in the blood of females, comprising the step of carrying out an immunoagglutination test on a sample of blood or of serum of the animal, which is brought into presence with a support sensitised by anti-vitellogenin gamma-globulins, the formation of a precipitate in the sample tested indicating the presence of vitellogenin.
2. The process of claim 1, wherein, when the test is negative, known serum containing vitellogenin is subsequently added to the tested sample to determine between an absence of vitellogenin (formation of a precipitate) and an excess of vitellogenin (absence of precipitate) in the initial sample.
3. The process of claim 1 or claim 2, wherein, when the test is carried out on a blood sample, heparin is added to the reaction buffer.
4. The process of claim 1, wherein the support is sensitised, by a process known per se, with the aid of solutions of anti-vitellogenin gamma-globulins of variable concentrations, the sensitivity of the test being greater for the media poor in vitellogenin as this concentration is weaker.
5. The process of claim 4, wherein the concentration of the sensitising antibodies is 4 to 9 times greater than that of the antibodies in the whole antiserum.
6. The process of claim 2, wherein the females in the course of vitellogenesis in fish and in particular in the Salmonidae family are detected.
7. The process of claim 6, wherein the gamma-globulins used for sensitisation of the support are obtained in conventional manner from an antiserum from rabbits having received injections of vitellogenin.
8. The process of claim 7, wherein the vitellogenin injected in rabbits is extracted from the blood of oestrogenised Salmonidae, and purified by chromatography.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8100103 | 1981-01-06 | ||
| FR8100103A FR2497576B1 (en) | 1981-01-06 | 1981-01-06 | METHOD OF RECOGNIZING FEMALE VITELLOGENESIS IN OVIPARATED ANIMALS BY AN IMMUNOAGGLUTINATION TEST |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1178886A true CA1178886A (en) | 1984-12-04 |
Family
ID=9253895
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000393566A Expired CA1178886A (en) | 1981-01-06 | 1982-01-05 | Process for recognising females in the course of vitellogenesis in oviparous animals, by an immunoagglutination test |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0056018B1 (en) |
| JP (1) | JPS57136164A (en) |
| AT (1) | ATE16049T1 (en) |
| CA (1) | CA1178886A (en) |
| DE (1) | DE3266717D1 (en) |
| FR (1) | FR2497576B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10107801B2 (en) | 2013-03-14 | 2018-10-23 | Horiba Abx Sas | Flow assay method for an object of interest |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2196426A (en) * | 1986-09-02 | 1988-04-27 | Aquabio Ltd | Fish disease diagnosis |
| ATE216081T1 (en) | 1996-10-04 | 2002-04-15 | Embrex Inc | METHOD FOR SEX DETERMINATION OF BIRDS IN OVO |
-
1981
- 1981-01-06 FR FR8100103A patent/FR2497576B1/en not_active Expired
-
1982
- 1982-01-04 AT AT82400001T patent/ATE16049T1/en not_active IP Right Cessation
- 1982-01-04 DE DE8282400001T patent/DE3266717D1/en not_active Expired
- 1982-01-04 EP EP82400001A patent/EP0056018B1/en not_active Expired
- 1982-01-05 CA CA000393566A patent/CA1178886A/en not_active Expired
- 1982-01-05 JP JP57000841A patent/JPS57136164A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10107801B2 (en) | 2013-03-14 | 2018-10-23 | Horiba Abx Sas | Flow assay method for an object of interest |
Also Published As
| Publication number | Publication date |
|---|---|
| DE3266717D1 (en) | 1985-11-14 |
| FR2497576B1 (en) | 1985-06-28 |
| FR2497576A1 (en) | 1982-07-09 |
| EP0056018A3 (en) | 1982-08-04 |
| ATE16049T1 (en) | 1985-10-15 |
| EP0056018B1 (en) | 1985-10-09 |
| EP0056018A2 (en) | 1982-07-14 |
| JPS57136164A (en) | 1982-08-23 |
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