Environmental Toxicology and Chemistry, Vol. 27, No. 7, pp. 1614-1620, 2008 O 2008 SETAC

Printed in the USA 0730.7268108 $12.00 + .O0

VALIDATION OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR MEASURING VITELLOGENIN IN CALIFORNIA HALIBUT (PARALICHTHYS CALIFORNICUS)

CELIA ~ÁZQUEZ-BOUCARD," MARIO BURGOS-ACEVES, FABIOLA ARCOS-ORTEGA, and GERARDO ANGUIANO-VEGA

Aquatic Organisms Reproduction and Toxicogenomic Laboratory, Department of Environmental Management and Conservation, Centro de Investigaciones Biológicas del Noroeste, La Paz, Baja California Sur, México

(Received 21 July 2006; Accepted 4 Fehruaty 2008)

Abstract-Vitellogenin (VTG) is the major protein present in the plasma of females undergoing oogenesis. In males, the VTG gene normally is suppressed; however, synthesis of VTG can be induced by exposure to xenoestrogenic compounds. In the present study, an enzyme-linked immunosorbent assay was developed and validated to evaluate VTG levels in the California halibut (Paralichthys cali+ornicu.s). Vitellogenin and lipovitellin (LV) were identified in the plasma of 17B-estradiol-induced females and in the ovaries of wild females, to our knowledge for the first time. Purified VTG from the plasma of induced females was obtained, and polyclonal antibodies against the LV of mature female ovaries was prepared and their specificity assessed by Western blot analysis. At Bahía Magdalena, Baja California Sur, México, quantitative measurements of VTG in the plasma of female specimens were made during one reproductive cycle.

Keywords-Paralichthys cn1ifi)rnicus Biomarker Vitellogenin Lipovitellin Enzyme-linked immunosorbent assay

INTRODUCTION The California halibut (Parulichthys californicus) is a com-

In recent years, the large volumes of pollutants spilled in coastal areas have led to rapid deterioration of some habitats, and these pollutants jeopardize the physiological performance of aquatic animals. The polluting agents that have received the most attention are the endocrine-disrupting compounds, which are characterized by their capacity to change the normal func- tioning of the endocrine system [l-31. Among the endocrine- disrupting compounds are xenoestrogens, which have the abil- ity to imitate the actions of natural hormones and cause an- tagonistic or competitive effects among natural horinones. Also, endocrine-disrupting compounds alter the synthesis, transport, metabolism, and secretion of natural hormones; the production and function of hormonal receptors severely affect the reproductive system of fish and other marine animals [4]. Examples of xenoestrogenic compounds are aromatic hydro- carbons, polychlorinated biphenyls, heavy metals, DDT and other pesticides, and plastic derivatives [5-71.

Detection of xenoestrogens in wild animals requires bio- markers that produce specific and predictable responses to es- trogenic stimulation. In teleost oviparous fish, vitellogenin (VTG) is a glycolipophosphoprotein synthesized in the liver in response to circulating estrogens, mainly 17P-estradiol (E,). It is released into the bloodstream and transported into the growing oocyte, where it is progressively sequestered by re- ceptors (endocytosis) [8,9]. The synthesis of VTG normally is restricted to adult females during follicular maturation be- fore ovulation; however, VTG production can be induced in males and immature females through exposure to xenoestro- gens. This reaction makes VTG a good biomarker to detect exposure of marine animals to xenoestrogenic compounds, as shown in invertebrates [ lo] , amphibians [ l 1 ,121, reptiles [13,14], and fish [15,16].

" To whom correspondence may be addressed (cboucard04@cibnor.mx).

Published on the Web 2/12/2008.

mercially important species along the west coast of North America froin the state of Washington, USA, to Bahía Mag- dalena on the Baja California Peninsula, México. It is found in areas highly impacted by agricultural and industrial activity. The benthic characteristics of this species place it in contact with contaminated sediments. Therefore, this species is an ap- propriate model for and indicator of environmental change. Other benthic species of the Soleidae family are already being used as indicator species for environmental monitoring pro- grams in Canada and the United States [17].

Paralichthys ca1ifornicu.v also is an important species for aquaculture, in which production success largely depends on control of the reproductive process of captive animals. Used as a biomarker for the presence of xenoestrogens in the en- vironment, the concentration of VTG in plasma is an indicator of female reproductive maturity and performance. This ex- plains why its measurement is vital for reproductive manage- ment in postlarvae production laboratories.

The aim of the present study was to develop an enzyme- linked immunosorbent assay (ELISA) to quantify VTG in the plasma of wild and captive P. ca1ifi)rrzicus to monitor con- tamination and support production in aquaculture facilities.

MATERIALS AND METHODS

Sampling

Mature and immature female and male California halibut (n = 82 animals) were collected once per month during the period from December 2001 to August 2002 in Bahía Mag- dalena. Classification of ovarian developmental stages was based on the size of oocytes (A. Burgos-Aceves. 2003. Master's thesis. Centro de Investigaciones Biológicas del No- roeste, La Paz, Baja California Sur, México). Three milliliters of blood were collected into Eppendorf tubes with 10 mM phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO, USA). Blood samples were left at 4OC, and the serum was separated by centrifugation at 5,000 g for 15 min

P. californicus vitellogenin E L I S A Environ. Toxicol. Chenz. 27, 2008 1615

at 4°C and stored at -80°C until analysis. Ovaries and livers were collected and stored on liquid nitrogen at -80°C until analysis. After two months, the ovaries were homogenized in equal volumes of extraction buffer (0.05 M Tris, 0.5 M NaCl, 5 mM ethylenediaminetetra-acetic acid; pH 7; 3: 1, vlv, buffer1 tissue) and protease inhibitor cocktail (0.003%; Sigma-Al- drich). The homogenate was centrifuged at 10,000 g for 15 min at 4'C. Approximately 0.5 g of liver was homogenized in three volumes of buffer: 100 mM NaH,PO,-H,O with 1 mM ethylenediaminetetra-acetic acid (pH 7) and 120 ~1 of 10 mM PMSE The homogenate was centrifuged at 7,000 g for 15 min at 4°C. Protease inhibitor was added to tissue samples im- mediately after the samples were removed from the freezer (i.e., before samples started to defrost).

Three i~nmature females were stimulated with E, (Sigma- Aldrich) by three intraniuscular injections (one per day) of E? dissolved in propylene glycol (1 mgl40 pl). The dose was 5 mglkg wet body weight. Blood samples were collected O and 8 d after induction in tubes with 10 mM PMSE Blood samples were left at 4"C, and the serum was separated by centrifugation at 5,000 g for 15 min at 4°C and stored at -80°C until analysis.

VTG and lipovitullin identijicution

Total protein concentrations were determined in plasma, ovary, and liver using the method described by Lowry et al. [18]. For identification of VTG in plasma and lipovitellin (LV) in ovary of wild and induced females, the plasma and ovarian tissues of mature and immature females were separated by native polyacrylamide gel electrophoresis (PACE; 6%) using Tris-glycine buffer (pH 8.8). Molecular weight of separated protein fractions was estimated using a high-molecular-weight control standard kit (Amersham Pharmacia Biotech, Piscata- way, NJ, USA) as follows: P-Macroglobulin, 820 kDa; apo- ferritin, 443 kDa; catalase, 240 kDa; ceruloplasmin, 132 kDa; and albumin, 67 kDa. Gels were stained with Coomassie blue R-250 (Bio-Rad, Hercules, CA, USA), Sudan Black B (Sigma- Aldrich), and Schiff's reagent (Sigma-Aldrich) to visualize proteins, lipids, and carbohydrates, respectively.

Production of anti-LV and anti-VTG irnrnune sera

Polyclonal antibodies were obtained from New Zealand rabbits. The ovaries of wild mature female P. californicus and the plasma of E,-induced female P. californicus were analyzed by preparative-PAGE (6%). A small gel portion was cut ver- tically in both extremes and stained with Coomassie blue R- 250. These portions were placed next to the rest of the gel that was not stained, and fractions corresponding to LV and VTG were excised from the gel. Then, these fractions were ho- mogenized with 0.85% (v/v) NaCl and injected into four rab- bits. Rabbits initially were immunized with approximately 50 p g of the protein emulsified in 1 m1 of Freund's complete adjuvant and 0.85% NaCI. Two booster injections of approx- imately 120 p g of protein in 1 m1 of Freund's incomplete adjuvant plus 0.85% NaCl were given 10 and 30 d after the first injection. The concentration of LV and VTG proteins that were injected into the rabbits was approximated (calculated from the concentration of total proteins analyzed by electro- phoresis), and the percentages of each fraction were calculated by measuring the bands size. Blood of rabbits were collected before the initial immunization (control serum) and 10 d after the last booster dose (50 ml). The antiserum was centrifuged at 2,500 g for 10 min and stored at -80°C.

VTG purijkution

17P-estradiol-treated P. californicus plasma was fraction- ated using a diethylaminoethyl chromatographic procedure in a diethylaminoethyl-Sepharose CL-6B column (10,000- 1,000,000 fractionation range; length, 100 cm; inner diameter, 1.8 cm; Fine Pharmacy Chemicals, Uppsala, Sweden). The equilibrium buffer was 100 mM Tris-HCI (pH 7.8) with 1 mM PMSF. Plasma from E,-treated females (150 ~ 1 ) was diluted in the same buffer. A linear gradient from 0 to 500 mM NaCl was run using a gradient volume of 177 m1 and a flow rate of 0.2 mllh. Effluent was collected in 2.5-m1 fractions, and the absorbance of each fraction was measured at 280 nm. The fractions were concentrated and analyzed by native and sodium dodecyl sulfate (SDS)-PACE.

Irnrnunologic procedures

The specificity of the antiserum against LV of P. calijor- nicus was tested by Western blot analysis. The plasma, ovary, and liver of females and the plasma and liver of males were examined by 12% SDS-PAGE. A solution of 0.5 M Tris-HC1 (pH 6.8), 1% SDS, I % 2-mercaptoethanol, 10% glycerol, and 0.05% bromophenol blue was used as dissociation buffer, and protein was transferred from the gel to polyvinylidene difluo- ride (Immobilon transfer membrane; Bio-Rad) with a mini- transblot electrophoresis transfer cell (Bio-Rad) using 25 mM Tris, 192 mM glycine, and 20% methanol buffer. Nitrocellulose paper was immersed in the following solutions: 5% Blotting- grade blocker (Bio-Rad) in Tris-borate-saline buffer (0.15 M NaC1, 10 mM Tris, and 10 mM borate), antiserum against LV of P. californicus (1:3,000), and goat anti-rabbit immuno- globulin (Ig) G alkaline phosphatase conjugate (1 :3,000). Col- or was developed using diaminobenzidine in Tris-borate-sa- line buffer.

Immunodiffusion precipitation was performed as described by Ouchterlony [19]. Agar gel (1%; thickness, 2 mm) was prepared on a glass slide. Lipovitellin antiserum and samples of plasma, liver, and ovary from mature females; ovary from immature females; plasma from the males; and purified VTG were put in separate wells (0.9 cm apart) and then placed in a humid chamber (4°C) for 48 h. After washing (three times for 6 h each) in 0.9% NaCl, the gel was stained with Coomassie blue R-250.

ELISA procedure

A noncompetitive ELISA, described previously by Váz- quez-Boucard et al. [20], was developed for the detection and quantification of P. californicus VTG. Samples of plasma, liver extracts, and the standard solution (purified P. califor- nicus VTG) were diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6). Five loadings per dilution were added in 96- well microplates at 150 p,l/well and incubated for 2 h at 37OC. Nonspecific binding of the antibody was determined by coating six wells with male P. calijornicus plasma. Plates were washed four times for 5 min each with 0.01 M phosphate-buffered saline containing 0.15 M NaCI, 0.027 M KC1, and 0.05% Tween (PBS-T) at pH 7.4 to remove excess unbound protein from the solid phase. To reduce nonspecific binding, al1 wells were blocked (2 h) with 0.5% bovine serum albumin in PBS- T, followed by four additional washes with PBS-T. The anti- serum against P. californicus LV was diluted at 1:3,000 (VI v) antibody1PBS-T buffer. A volume of 150 pl/well was added to the plates and incubated for 3 h at 37OC. After four washes

1616 Environ. Toxicol. Chem. 27, 2008 C. Vázquez-Boucard et al.

A B C D E F fixed concentration of antigen (purified VTG) and 13 concen-

Fig. 1. Native polyacrylamide gel electrophoresis: Identification of vitellogenin and lipovitellin (A) molecular mass markers, (B) proteins of plasma from immature females, (C) proteins of plasma from in- duced (17P-estradiol) females, (D) carbohydrates of plasma from in- duced females, (E) lipids of plasma from induced females, and (F) lipids of ovarian homogenate from mature female. Arrows indicate vitellogenin and lipovitellin.

for 5 min each with PBS-T, wells were incubated with goat anti-rabbit IgG peroxidase conjugate (Sigma-Aldrich) diluted 1:3,000 in blocking buffer for 2 h at 37OC, followed by four more washes for 5 min each with PBS-T. Immediately before use, 1 mglml of p-nitrophenyl phosphate substrate (Sigma- Aldrich) was prepared, and 150 ~1 were added per well. Plates were incubated for 30 min at room temperature. Absorbance was measured at 405 nm using a Multiskan MKFTM (Labsys- tems, Helsinki, Finland).

Validation of the ELISA method

Antigen optimal concentration. The working concentrations of the coating antigen were determined by testing six serial dilutions of plasma and liver tissue lysates (0.5-30 pg/ml of total proteins).

Optimal antibody concentratinn. The titer of the antiserum (anti-LV) was established using a noncompetitive ELISA of a

trations of P. californicus anti-LV antibody: 1:50, 1:100, 1:500, 1:750, 1:1,000, 1:5,000, 1:7,500, 1:10,000, 1:12,000, 1: 15,000, 1:20,000, 1 :25,000, and 1:30,000.

Standard curve. The standard curve was generated with serial dilutions of purified VTG (20, 40, 60, 80, 100, 200, 400, 600, and 800 nglml). The primary antibody anti-LV was di- luted 1:3,000 in PBS-T. The VTG concentration in P. cali- fornicus samples of liver and plasma were calculated based on linear regression from the standard curve of the primary antibody anti-LV.

Assay precision. The intra- and interassay coefficients of variation (CVs) were determined by running the standard curve five times on different days (interassay CV), and on each plate, every point of the standard curve was run eight times (intra- assay CV).

Parallelism. Serial dilutions (0.1-20 ~ g l m l of total proteins) of female plasma, ovary, and liver tissue homogenate as well as purified VTG and a control sample from the serial dilutions of male plasma were assayed by ELISA to assess parallelism with the standard curve. This indicated the specificity to the antibody (anti-LV) and, similarly, assured that the antibody recognized the antigen in the different tissues.

VTG concentration in E,-treated females

Vitellogenin plasma concentrations of females obtained by 8 d of stimulation with E, were evaluated by ELISA using the protocol described above. Native and SDS-PAGE of plasma was conducted to visualize the induction of VTG.

Statistical analvses

The relationship between ELISA optical density and con- centrations of purified VTG or diluted tissue samples was as- sessed by linear regression. Standard errors of the mean were calculated. The data were log,,, transformed for normalization [21]. A value of p < 0.05 was considered to be statistically significant. A one-way analysis of variance was used to eval- uate the effect of the variation of sampling date on the con- centrations of plasma and liver VTG. This was followed by a comparison of averages using the Tukey-Kramer test 1211. Parallelism between regression curves was assessed by a com- parison of slopes using analysis of covariance [22].

RESULTS

Identijication and purification of VTG and LV

Two fractions of VTG were identified by PAGE (6% gel) in the plasma of females induced with E, and of wild mature

FRACTlON A B

Fig. 2. Diethylaminoethyl-Sepharose CL-6B (Fine Pharmacy Chemicals, Uppsala, Sweden) elution profile of induced (17B-estradiol) samples: Purification of vitellogenin (A) polyacrylamide gel electrophoresis of peak 4 and (B) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of peak 4, showing the induced proteins.

P. cali@rnicus vitellogenin ELISA Environ. To,xicol. Chem. 27, 2008 1617

d e f

Fig. 3. Specificity of antibodies antilipovitellin and immuuologic identity between lipovitellin and vitellogenin of Puralichthys cu1ifornicu.s: (A ) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of (a) ovary from wild mature females, (b) liver from wild mature females, (c) liver from wild males, (d) plasma from wild males, (e) plasma from induced (17P-estradiol) females, and (f) molecular mass markers; (B) blot reaction with antibodies against lipovitellin (a) ovary from wild mature females, (b) liver from wild mature females, (c) liver from wild males, (d) plasma from wild males, and (e) plasma from induced (17P-estradiol) females; (C) double radial immunodiffusion regarding (a) plasma from males, (b) plasma from mature females, (c) ovary from mature females, (d) liver from mature females, (e) purified vitellogenin, and (f) ovary from immature females. Ab = antilipovitellin antibody.

females. In contrast, they were absent in noninduced and im- mature females. The gels dyed with Sudan black and periodic acid-Schiff showed the glycolipoprotein nature of the iden- tified fractions. Based on their relative mobility, the molecular weights of the induced proteins were approximately 380 and 550 kDa, respectively. A LV fraction (550 kDa) was identified in the ovary of mature females (Fig. 1). The LV protein and lipid fraction were present at al1 stages of gonad maturity but were more evident in hydrated oocytes (stage IV). Its glyco- protein nature was observed at al1 stages. Four protein peaks in the plasma of induced females were obtained by gel filtration chromatography. The electrophoresis of peak 4 showed two bands of 550 and 380 kDa, identified as VTG in the plasma

of induced females. These proteins were analyzed by SDS- PAGE, and seven subunits were detected (Fig. 2).

Anti-LV antibody production and charucterization

The ability of an anti-LV antibody to specifically interact with the substance to be assayed from plasma, liver, and ova- ries was determined by two immunochemical techniques. Im- munoblotting showed the specificity of antibodies to plasma, ovary, and liver of wild mature females and to plasma of induced females (Fig. 3A and B). The immune serum was characterized in a double radial immunodiffusion test. Purified VTG, plasma, ovaries, and liver of wild mature vitellogenic females was precipitated by anti-LV immune serum, as indi-

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16 18 Environ. Toxicol. Chem. 27, 2008 C. Vázquez-Boucard et al.

cated by the formation of a single precipitin line. The ends of the precipitin lines met to form a single continuous curve, showing total immunologic identity between LV and VTG, and they did not react with male and immature females (Fig. 3C).

Validution of the ELISA method

Ovarian and liver homogenate, plasma, and purified VTG from a vitellogenic female were diluted and titrated by ELISA to determine the concentrations that were suitable for quan- tifying LV and VTG in tissue samples. Optimal concentrations of total protein in the samples for antigen coating were as follows: Plasma (5-15 pglml of total proteins), and liver (15- 30 ~ g l m l of total proteins). An antibody dilution of 1:3,000 and an optimal antibody saturation time of 2 h were chosen after several preliminary tests. The purified VTGs were coated with increasing concentrations of 20, 60, 100, 200, 400, 600, and 800 nglml of total proteins. Serial dilutions of female plasma, purified VTG, and ovarian tissue at different concen- trations produced displacement curves with slopes that were not significantly different ( p < 0.05) from that of the standard curve, assuring that the samples could be run at different di- lutions. In contrast, the curve obtained from liver homogenates was significantly different (Fig. 4A). A negative reaction was observed in analyses of males, demonstrating nonspecific bind- ing.

The antiserum standardization curve was obtained with pu- rified VTGs from E,-induced females (Fig. 4B). Based on linear regression of the standard curve, the relationship be- tween VTG concentration and its optical density was described by the following equation: y = 0 . 0 0 1 4 ~ + 0.0549 (r2 = 0.997).

The assay was not sufficiently sensitive below 20 nglml, because not enough coated antigen was available for detection. In contrast, excess VTG and total protein were found above 800 nglml, and the assay was invalid because the plastic of well was saturated. The CV for assay reproducibility was less than 10%.

The use of anti-LV antibody for the evaluation of VTG in plasma and liver in P. ca1ifornicu.s was verified by comparing the results with anti-LV antibody and anti-VTG antibody ( r = 0.992) (Fig. 5).

VTG concerltrations

Table 1 shows VTG (plasma and liver) concentrations of wild P. californicus females during the sampled season. The highest VTG concentrations transported in the blood occurred between the months of January and July, with an average of 362.86 nglml.

Through ELISA analyses, the presence of VTG in the plas- ma of three induced females was evaluated quantitatively, showing an important positive response 8 d after stimulation (day 1, 0.008 2 0.003 ~ g l m l ; day 8, 0.418 2 0.020 pglml).

DISCUSSION

Identi$cation a n d characterization of VTG and LV in P. californicus

In the present atudy, VTG was identified, to our knowledge for the first time, in the plasma of females induced by application of E?, and LV was identified in the ovaries of wild mature P. culiji~rnicus females. The criteria used for their identification were the glycolipoprotein nature of the molecule as well as its absence in immature females and in nonhormonally induced females and males. Two fractions with molecular weights of

0.0 1 0 2 4 6 8 10 12 14 16

Concentration ( pglml)

VTG (nglml)

Fig. 4. (A) Parallelism of curves for (a) liver from wild mature females (V), (b) purified vitellogenin (VTG; m), (c) plasma from induced (17p-estradiol) females (A), and (d) ovary from wild mature females (e). Each point represents the mean optical density value from five assays. (B) Vitellogenin enzyme-linked immunosorbent assay stan- dard curve for Puralichthys californicus. Each point represents the optical density (mean t standard error, n = 5 assays).

380 and 550 kDa were identified in plasma. The presence of a VTG fraction in the plasma of teleost fish has been reported for common carp (Cyprinus carpio) [23], striped bass (Morone saxatilis) [24], Atlantic halibut (Hippoglossus hippoglossus) [9], and Arctic charr (Salvelinus alpinus) [25]. The molecular weights of the fraction identified in these studies were between 390 and 600 kDa. Other works indicate the presence of two types bf VTG in rainbow trout (Salmo gairdneri) [26], coho salmon (Oncorhynchus kisutch) [27], and barfin flounder (Ver- asper moseri) [8], with molecular weights of between 320 and 636 kDa for VTG, and between 35 and 530 kDa for VTG,. According to Ng and Idler [28], the size and number of VTG vary among species and genera without regard to phylogenetic relationship. Nevertheless, the results obtained in the present study are similar to those of Matsubara et al. [8], who observed two types of VTG in V. moseri, with molecular weights of 500 and 530 kDa. Those authors associated the presence of two different forms of VTG with distinct functions during oocyte maturation and embryonic development.

Antibody speci$city

To corroborate the specificity of the antibodies, Western blot analysis showed that the prepared antiovarian LV poly-

P. culifornicus vitellogenin ELlSA Environ. Toxicol. Chem. 27, 2008 1619

clonal antibodies recognized both VTG in liver and plasma and LV from the ovary of mature females. Conversely, the positive reaction observed with the radial immunodiffusion technique between the anti-LV antibody and each tissue in- volved in oogenesis (blood, ovary, and liver) showed the an- tibody specificity. In addition, ends of precipiting lines formed a single continuous curve, showing total immunologic identity between LV and VTG molecules [29] of the two tissues (blood and ovary). The immunologic identity between LV and VTG has been detected in the prawn Fenneropenaeus indicus [20] and in the isopod Orchestia gammarella [30].

y = -0 0216 + 1 3268 * x Correlation r = O 99204

Validation of the ELISA

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The ELISA developed in the present study showed ac- ceptable parameters of sensitivity, specificity, and precision [31], which are essential for measuring VTG in P. californicus. The immunologic identity of the LV and VTG were verified, demonstrating the possibility of using an antibody directly against the LV of ovary in the quantification of VTG in plasma. For validating the results obtained by ELISA using an anti- LV antibody and a standard curve anti-VTG, however, we prepared anti-VTG antibodies from E,-induced plasma, and

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.,

Table 1. Viteilogenin concentrations of blood of wild Paralichthys californicus females in Magdalena Bay, Baja California Sur, México"

O O O 2 O 4 O 6 0 8 1 0 1 2 1 4 1 6 1 8

Optical density (405 nrn) Ab,

Fig 5 Comparison by enzyme-linked immunoiorbent asay of purified vitellogenin concentration as evaluated u\ing two antibodies, antilipovitellin (Ab,) and antivitellogenin (Ab?) in Paral~chrh ,~ c a l ~ u r n ~ s u ~ The 95% confidente interval is ~hown

we compared their sensitivity with that of the anti-LV anti- bodies.

Parallelism of curves of purified VTG and crude extracts of ovary and plasma demonstrate that the response of the im- mune serum is proportional to the adsorbed antigen Concen- tration. The covariance test did not indicate a significant dif- ference in slopes (p = 0.001). The LV and VTG concentrations varied, but the behavior remained unchanged, with no apparent interference from other carried proteins. In the liver of mature females, the response of the anti-LV antibody was not pro- portional to the adsorbed antigen concentration. A great amount of proteins was synthesized and transformed in the liver, being the place of an important proteolytic activity, which is very difficult to totally inhibit during the extraction. The VTG molecules in liver could be partially degraded, and this could cause the absence of affinity between the liver and the others tissues analyzed. In males and induced females, the immune enzymatic reaction was negative, which indicates the absence of contaminating immunogenic proteins in the anti- LV serum.

Inconsistencies in the accuracy and reproducibility of this rnethod might be introduced by nonuniform absorption of an- tigen into the plastic surface during the coating phase. Al- though the absorption rate is difficult to control, plates with a high degree of homogeneity were used. The precision of the assay is sufficient to allow direct comparison of samples on

Month Samples Blood (nglml) Liver (nglg) the same plate and from different plates assayed at different - - times. This was evident from intra- and intertest variabilities

December 4 32.28 t 0.08 A January 8

8.7 A of less than 10%. 143.53 2 0.10 AB 75.85 f 0.09 B

~ e b r u a i ~ 7 274.25 f 0.14 B 28.84 t 0.12 B March 9 668.33 ? 0.06 B 56.23 0.09 B VTG concentrations April 7 512.1 1 t 0.27 B 38.01 t 0.23 B

2 A relationship between the increase in blood VTG concen-

M ~ Y 434.30 f 0.86 B 28.84 t 0.61 B June 12 353.35 i 0.26 A % A tration in females and the high reproductive activity was ob- July 10 154.21 t 0.19 A 13.48 i 0.17 A served from histological studies made in ovaries of fish sam- August 8 38.26 t 0.15 A 4.78 0.07 A pled between January and July (A. Burgos-Aceves. 2003. Mas- Analysis of ter's thesis. Centro de Investigaciones Biológicas del Noroeste,

variance 67 p < 0.05 p < 0.05 La Paz, Baja California Sur, México). Thus, the VTG con-

" Values are presented as the mean * standard error. Within each row, centration in fish blood can be used as an indicator of gonad uppercase letters indicate significant differences (p < 0.05). maturity to refine hormonal induction techniques in captive

1620 Environ. Toxicol. Chem. 27, 2008 C. Vázquez-Boucard et al.

females or as an indicator of disturbance in the reproductive process caused by xenoestrogens. The antibody produced dur- ing the present study can be used in the evaluation of changes in VTG concentration in wild P. californicus plasma.

In comparison to control plasma, E, treatment resulted in an increase in the VTG concentration in the blood of P. cal- ifornicus females. 17P-Estradiol has been used previously to promote VTG production in reptiles [32] and fish [33]. These results demonstrated that E, treatment induced the synthesis of two VTGs with biochemical characteristics similar to those of VTGs identified in the plasma of wild mature females (mo- lecular weights, 380 and 550 kDa).

In conclusion, an ELISA that can detect and quantify VTG in the plasma of wild organisms or those induced with E, was developed. The results presented here validate this ELISA for detection of VTG variation in male and female fish for en- vironmental biomonitoring and reproduction studies in aqua- culture centers.

Acknowledgement-The present study was funded by the Centro de Investigaciones Biologicas del Noroeste (La Paz, Baja California Sur, Mexico) and by grants to C. Vázquez-Boucard from Consejo Nacional de Ciencia y Tecnología, México (20007007). The authors are grateful to O. Saravia, C. Ceseña, J. Sandoval, E. Calvillo, J. Angulo, G. Colado, A. Cruz, M. Ramírez, and L. Ibarra. We are also grateful to the American Journal Experts Company, L. Mercier, and the reviewers for their valuable comments and suggestions. M. Burgos-Aceves was a student fellow of Centro de Investigaciones Biológicas del Noroeste, and the results presented here are part of his Bachelor and Master of Science theses under the direction of C. Vázquez-Boucard.

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