Lates calcarifer, commonly known as Asian sea bass or locally called
siakap, is a catadramous fish (grows to maturity in fresh or brackish waters
and spawns in the sea) in family Latidae of order Perciformes. It is one of
the nine Lates species from family Latidae which is widely distributed
in coastal Australia, Southeast and Eastern Asia and India (Luna,
2008). This species are farmed in cages, salt water ponds as well as fresh
water (Webster and Lim, 2002). In recent years, sea bass
has gained growing importance in aquaculture both as recreational and commercial
fish, with a high and fairly stable price (Luna, 2008).
Sea bass production and consumption in Malaysia has increased dramatically over
the years with the estimated aquaculture production of 194, 623.11 tonnes.High
demand of L. calcarifer in aquaculture industry has triggered research
scientists studying the reproductive physiology of this species. Susca
et al. (2001) concluded that the reproductive physiology of many
fish species has been studied by analyzing the vitellogenin (vtg) levels in
the blood plasma which is important for broodstock management for spawning purpose
in captive condition.
The vtg, classified as phosphoglycolipoprotein is a precursor of egg yolk,
synthesized in female oviparous vertebrate in preparation for spawning under
the control of natural occurring steroid hormone 17-β estradiol (E2)
(An et al., 2007; Palumbo
et al., 2009; Meucci and Arukwe, 2005; Ngamniyom
and Panyarachun, 2011). The vtg is transported to the follicle in the ovary
via bloodstream, where it is progressively sequestered into developing oocyte
and offspring in the reproductive stage (Kordes et al.,
2002; Li et al., 2005; Prakash
et al., 2007). Its synthesis (vitellogenesis) is controlled by hormone
and estradiol is reported as a common hormonal stimulus to trigger the vtg synthesis
in many fish species (Nishi et al., 2002). Lomax
et al. (1998) noted that plasmatic vtg concentration in English sole
(Pleuronectes vetulus) was dramatically increased in female fish during
reproductive period prior to spawning, when estrogen levels were elevated. However,
vtg levels were decreased upon spawned out spawners (Methven
et al., 1992). Therefore, the measurement of vtg levels in Asian
sea bass (Lates calcarifer) would provide a useful tool in determining
the maturity status of this economically important species in captive and wild
condition (Kishida and Specker, 2000). Vtg is normally
absent in female blood during non-reproductive period and in male as well as
juvenile fish. However, Prakash et al. (2007)
and Selcer et al. (2006) proved that these organisms
produced vtg via inducing with synthetic estrogens, 17-β estradiol (E2)
in murrel and Morelets crocodile (Crocodylus moreletii).
Several direct and indirect immunoassays were developed for measuring vtg levels,
namely Radioimmunoassay RIA (Tao et al., 1993),
immunodiffusion (Mananos et al., 1994), alkali-liable
phosphate (Periera et al., 1992) and Enzyme Linked
Immunosorbent Assay (ELISA). Among these methods, ELISA was highlighted as the
most favourable tool used for plasmatic vtg measurement considering its high
sensitivity, simple and rapid (Mananos et al., 1994;
Lomax et al., 1998; Ebrahimi,
2007; An et al., 2007). Since, then, ELISA
was developed and established among several teleost vtg of freshwater and marine
fish such as English sole (Lomax et al., 1998),
sea bream (Mosconi et al., 1998), Japanese medaka
(Nishi et al., 2002), carp (Matsumoto
et al., 2002), Feathed minnow (Eidem et al.,
2006) and Mossambicus tilapia (Swart and Pool, 2009).
Recently, several ELISA for measuring vtg have been standardized and developed in a commercial available kit. However, the antibody does not display a good cross-reactivity among different species and a new assay is generally needed for specific species. Since, there was no apparent antibody has a binding properties to L. calcarifer vtg, polyclonal antibody against purified vtg was developed to study the cross-reactivity in ELISA.
The present study aimed to develop and validate a sensitive and rapid biochemical method capable for quantifying vtg levels in the maturing females plasma of L. calcarifer. The sensitive and accurate measurement of ELISA has a broad application in fisheries and aquaculture industry. The potential ELISA kit developed can help farmers to measure vtg levels of L. calcarifer in determining sex and sexual maturity for spawning purpose.
MATERIALS AND METHODS
Fish: Two-year-old juvenile Asian sea bass (Lates calcarifer) (initial mean weight 1.5±0.5 kg, length 168±15 cm) were obtained from commercial supplier and transported to the hatchery (Marine Science Station, Port Dickson, Universiti Putra Malaysia, UPM). They were held in 10 tonne tank with aeration and flow through sea water and fed daily with chopped fresh fish.
Preparation of Lates calcarifer vtg: Each fish was given a series intraperotoneal (i.p.) injections (2 mg kg-1 body weight) of 17-β E2 (Nacalai tesque, Japan), dissolved in a solution of ethanol and 0.9% NaCl in a ratio of 1:9, v/v (ethanol:0.9% NaCl solution, 1 mL). Three times of injections were given at two days intervals. Three days following the last injection, blood was obtained from caudal vessel with a 5 mL heparinized syringes and directly centrifuged at 12000 rpm for 30 min (4°C). Prior to purification, plasma was stored in -80°C in small aliquots.
Vtg purification: The procedure used for purification of plasmatic vtg
was modified from with modifications. One mL of aliquots plasma was purified
through gel filtration chromatography using Sepachryl HR-300 column (GE HEealthcare
BioScience, Upsalla, Sweeden), eluted with 360 mL 0.05 M Tris-HCl pH 8.0 (Nacalai
tesque, Japan). Elution profile was monitored at 280 nm and the fractions around
peak were collected as purified L. calcarifer vtg. The liquid was further
concentrated using Vivaspin centrifuge tube (30 kDa molecular weight cut-off,
GE Healthcare BioScience, Upsalla, Sweeden) according to the methods proposed
by manufacturer. Finally, the purified vtg concentration was determined by Bradford
assay (1976) using Bovine Serum Albumin (BSA) (Sigma Diagnostics, USA) as
standard. Freshly purified vtg was used to generate polyclonal antibody and
as a standard to develop ELISA. Working solutions of vtg for standard curve
were prepared at various concentration of purified vtg (Meucci
and Arukwe, 2005).
Antibody production: Polyclonal antibody against purified vtg (antigen) were generated in New Zealand white rabbits (body weight 3.5±0.5 kg, n = 4). For the priming, rabbits were immunized intradermal and intramuscularly with purified vtg, 0.05 mg mL-1 protein emulsified with Freunds Complete Adjuvant (FCA, Calbiochem, Darmstadt, Germany) in a ratio 1:1 v/v (1 mL). Rabbits were boosted up by three additional injections (0.02 mg mL-1 protein) emulsified with Freunds Incomplete Adjuvant (FIA). Blood was collected at weeks 4, 6 and 10 and the serum (anti-vtg) was assayed for reactivity towards vtg by screening ELISA. When antibody titer was sufficient, blood (~10 mL) was withdrawn from ear artery and allowed to clot overnight. Serum was separated by centrifuging the blood at 12000 g, 4°C for 30 min and stored in aliquots. Serum was then used in the development and validation of ELISA as primary antibody.
Development of an Enzyme Linked Immunosorbent Assay (ELISA): A checkerboard titration assay was used to select optimal working concentration of coating vtg and primary antibody for ELISA procedure. Purified vtg (1000 to 31.2 ng mL-1) were prepared by diluting in 0.05 M carbonate buffer, pH 9.6.
Ninety six microtiter plates (Nunc-Immuno Plate MaxiSorpTM) were coated with serial dilutions of purified vtg (200 μL per well) and incubated overnight at 4°C. For Non-Specific Binding (NSB), three wells were treated with coating buffer only. The plates were then blocked with blocking solution (1% skim milk in PBST, 300 μL per well) for two hours at 37°C. Following washes, plates were incubated with serial dilutions of primary antibody (1:500 to 1:8000 in blocking buffer, 200 μL per well) and incubated at 37°C for two hours. After four times washing with PBST, each well received 200 μL of secondary antibody, goat anti-rabbit IgG conjugated to horseradish peroxidase (prepared in dilution of 1:2000 in blocking buffer) and incubated two hours at 37°C. For substrate development, each well received 100 μL of Tetramethylbenzidine (TMB, Calbiochem, US) and incubated for 30 min. The reaction was stopped by adding 50 μL of 1 M H2SO4 to each well. The absorbance of 405 nm was read using microtiter plate reader (Thermoscientific, Finland).
Parameters such as the dilution of polyclonal antibody (anti-vtg), HRP conjugated secondary antibody, incubation time were optimized to develop a sensitive ELISA.
Antigen coating: Ninety six-well microtiter plates (Nunc-Immuno Plate
MaxiSorpTM) with 100 μL of purified vtg (250 ng mL-1,
diluted in coating buffer), sealed and incubated overnight at 4°C. For non-specific
binding, eight wells were coated with plasma from male samples at the same concentration.
The plates were then inverted to empty the contents and washed four times (1
min soak) with 100 μL of PBS-T (Phosphate buffered saline, 0.05% Tween
20, Calbiochem, Darmstadth, Germany) to remove unbound antigen. To reduce non-specific
binding, the plates were blocked (200 μL well-1) with blocking
buffer (1% skim milk in PBST), followed by four washing as described above.
Pre-incubation of samples and standards: Samples and standards were diluted in assay buffer (1% skim milk in PBST). Standards were prepared at initial concentration of 1000 ng mL-1 and serially diluted at two fold. The concentrations ranged from 1000 to 31.25 ng mL-1. In parallel, samples of E2-induced, vitellogenic female and male plasma were diluted in assay buffer in a ratio 1:100 to 1:1000000. Standards and samples were then mixed (1:1, v/v) with primary antibody diluted to 1:1000 in assay buffer. They were pre-incubated for 2 h at 37°C or overnight at 4°C.
Primary antibody incubation: Following pre incubation, standards and samples were dispensed in triplicate (150 μL well-1) into the coated plate and incubated for 2 h at 37°C. The plates were then washed as described above.
Secondary antibody incubation: Each well received 100 μL of Horse-Reddish Peroxidase conjugated goat anti-rabbit IgG (Nacalai tesque, Japan) diluted in 1:2000 in assay buffer. Plates were incubated for 45 min at 37°C followed by four washing with PBST.
Substrate incubation: For colourization, each well received 100 μL of Tetramethylbenzidine (TMB) substrate solution (Calbiochem, Damstadt, Germany). After 30 min incubation, colour development was stopped by addition of 100 μL of 0.05 M sulphuric acid, H2SO4 into each well. The absorbance of 405 nm was read using ELISA microplate reader (Thermo Fisher Scientific, Finland).
Expression of results: The proportion of antibody bound was calculated
using the formula Lomax et al. (1998):
where, OD is the absorbance of a sample or standard, OD0 is the
absorbance of zero standards and NSB is non-specific binding value. Binding
percentage values were logit transformed according to the formula below:
Parallelisms between the samples were assessed by using analysis of covariance
(Lomax et al., 1998). The amount of vtg in plasma
samples were calculated by interpolating from linear portion of the plasma dilution
curve to the calibration curve.
Standard curve: Standard curve were generated with serial dilutions of purified vtg (31.2, 62.5, 125, 250, 500 and 1000 ng mL-1 protein). The primary antibody was diluted 1:1000 in PBST. The vtg concentration in plasma of L. calcarifer was calculated based on linear regression from the standard curve.
Assay precision: The precision of the ELISA was assessed through measurement of intra and inter-assay coefficient of variations (CVs) by running the standard curves with different concentration on different days (inter-assay, N = 5). On each plate, every point of standard curve was run in nine replicates (intra-assay). To quantify assay variations, the CV was calculated using the following formula:
Antibody specificity: Serial dilutions of vitellogenic females
plasma and males plasma (10-2 to 10-7) as well as
vtg standard were assayed by ELISA to assess parallelism with standard curve.
This indicated the specificity of plasma samples to the antibody (anti-vtg).
Validation of ELISA method
Vtg measurement in L. calcarifer plasma of adult vitellogenic females
and males: Plasmatic vtg concentration for matured females (n = 5, body
weight of 6.3±0.5 kg) and male (n = 5, body weight of 4.0±0.5
kg) obtained during the breeding season in captive condition were determined
using developed ELISA methods.
Antigen: Asian sea bass plasmatic vtg of E2-treated was
purified by gel filtration chromatography using Sepachryl HR-300 column for
use as antigen in polyclonal antibody production. The protein concentration
of purified vtg was 0.059 mg mL-1 as determined by Bradford assay.
Optimal condition of competitive ELISA: A competitive, antibody-capture
ELISA was developed for Asian sea bass, L. calcarifer vitellogenin utilizing
the polyclonal antibody and purified L. calcarifer vtg as standard. ELISA
checkerboard titration assay of coating vtg and antiserum dilutions were shown
in Fig. 1. Six different combinations of vtg concentration
(31.2, 62.5, 125, 250, 500 and 1000 ng mL-1) and 5 serial dilutions
of antibody (1:500, 1:1000, 1:2000, 1:4000 and 1:8000) were evaluated. The results
were summarised in Fig. 2. The combination which yielded an
OD near 1 was selected as a workable ELISA (Swart and Pool,
2009). For routine assay conditions, an antiserum dilution of 1:1000 and
250 ng mL-1 coating vtg was selected for the use in ELISA.
The standard curve of purified vtg revealed a typical sigmoid pattern with
measurable ranges of 31.2 to 1000 ng mL-1 (Fig. 3a).
For assay detection limit, it was estimated to be 31.2 ng mL-1 which
corresponds to the vtg concentration of 93.6% antibody binding.
||Schematic for checkerboard titration. Shaded area resulted
in an OD of 0.917 after 30 min color development
||Optimal assay concentrations for vtg and antiserum. Vtg coating
of 250 ng mL-1 and antibody dilution of 1:000 was selected for
routine condition of ELISA
The upper and lower limits of ELISA detection were 38 and 93.6%, respectively.
The assay characteristics could be modified by changing the pre-incubation and
Parallelism of standard curves: In order to test the specificity of
the ELISA assay, parallelism of multiple binding curves (vitellogenic female,
E2-treated and control male plasma) was first verified using an analysis
of covarians F-test on mean squares to assess similarity of slopes. Standard
preparations of various antigens were compared to assess the antiserum (polyclonal
antibody) ability to recognize vtg in native form. Figure 4a
showed different displacement curves of E2-treated, vitellogenic
females and male plasma dilutions. No significant displacement was found with
male plasma. Figure 4b showed the linearization (logit transformation).
ANCOVA of E2-treated and vitellogenic female revealed a good parallelism
with the vtg standard curves (Fig. 3b), (Fobs =
0.562<Fcrit = 3.178 for 2,51 df, p>0.05) within the working
ranges of the assay, while unexposed male showed little cross reactivity with
vtg antibody, showing the specificity of L. calcarifer vtg (Shao
et al., 2005; Prakash et al., 2007).
Antibody specificity: Several dilutions of plasma from control male and natural vitellogenic females assayed to confirm the specificity of ELISA. Results showed that, there was no cross-reactivity observed in the male plasma as shown in Fig. 4.
Assay precision: Reproducibility of the assay was confirmed by evaluating
inter (N = 5) and intra assay (N = 9). Intra-assay variation was less than 9.4,
whereas inter-assay variation was less than 7.5 for 90% binding and less than
12.1 for 80 and 50% binding (Table 1).
||Binding displacement curve and linearization of vtg standard
used in competitive ELISA. (a) Representative binding (B%) of vtg standards
ranging from 31.2 to 1000 ng mL-1 (b) Logit transformation of
the binding curve (s = -0.8616, R2 = 0.9736). Each point represents
the mean of triplicate determinations
|| Characteristics of Lates calcarifer vtg competitive
|For routine measurement 250 ng mL-1 of antigen
concentration and 1:1000 of anti-serum dilution was selected. CV = Coefficient
These ELISA performance characteristics were generally similar to those reported
from other fish species (Korsgaard and Pedersen, 1998;
Fenske et al., 2001; Kordes
et al., 2002; Shao et al., 2005).
||Bindig displacement curves from serial dilutions of plasma
vitellogenic females and E2-treated juvenile L. calcarifer. (a) Binding
curves (B%). (b) Linearization of the plasma binding curves by logit transformation
The CVs for assay reproducibility is <15%. ELISA sensitivity, defined as
imprecision of measuring of zero dose was 6.9 ng mL-1. The value
was derived by plotting the graph of relationship between vtg dose (ng mL-1)
versus SD of versus vtg concentration (Sherry et al.,
Vtg measurement in plasma of matured females and males in captive condition:
ELISA was validated by measuring vtg in plasma of natural vitellogenic females
and matured males L. calcarifer (Fig. 6) using vtg
standard curve (Fig. 5) which ranged from 31.2 to 1000 ng
mL-1 (R2 = 0.979, y = 0.0008x+0.3614). Vtg concentrations
of matured female samples ranged from 0.9 to 1.54 mg mL-1. There
was no vtg detected in any of the male plasma (Fig. 6). Results
showed that vtg levels in natural vitellogenic females were higher, indicating
that the fish were ready to undergo spawning.
|| Vtg standard curves of the ELISA
||Vtg concentration for the plasma of vitellofenic females and
matured males in captive condition. (Bars represent the Mean±0.4
standard error). The vtg was not detected in plasma samples of males (n
The present study described development and validation of competitive ELISA
utilizing polyclonal antibody for quantifying vtg concentration in L. calcarifer,
as a measure of their maturational status. ELISA was reported as the most optimum
immunological methods proposed for detecting plasma vtg as it combined reasonable
sensitivity and speed (Heppel et al., 1999).
Purified vtg was used as a standard to generate the assay. The assay was used
to determine plasmatic vtg levels in females and males of L. calcarifer
taken during the breeding season in captive condition. Jackson
and Sullivan (1995) observed that ELISA assay enabled fishery biologist
to identify gender and maturational status of individual fish broodstock in
striped bass, white perch and white bass.
Purified L. calcarifer vtg was used to generate standard curve in this
assay. Constructed ELISA produced a linear standard curve of vtg concentration.
The result revealed that the vtg ELISA was dose dependent and sensitive enough
to be used for detecting plasmatic vtg levels. Similar finding was also noted
by Selcer et al. (2006) for vtg measurement in
Morelets crocodile. The developed assay was sensitive enough (6.9 ng mL-1)
to study the vtg patterns in female L. calcarifer during the reproductive
season. The lowest sensitivity of ELISA assay was 2.0 to 3.0 ng mL-1
in Danio rerio Hamilton-Buchanan. Sherry et al.
(1999) suggested that the sensitivity of the assay can be improved by optimizing
the incubation periods of primary and secondary antibody as well as adjusting
the anti-vtg or coated vtg-ratios.
For assay validation, vtg levels were evaluated in matured females and males
in captivity. Results demonstrated that there was no vtg detected in males compared
to matured female. This is in agreement with finding of Chang et al.
(1995) which also did not detected any vtg levels in males protandrous black
porgy, Acanthopagrus schlegeli. The vtg concentration found in females
L. calcarifer are comparable to those observed in other fish species
as noted by Selcer et al. (2006). High vtg levels
in pre spawning and spawning seasons might be correlated with sex reversal in
certain fish species (Chang et al. (1995).
Accuracy, precision, sensitivity and specificity determine reliability of assay
which ensures concentration is valid and provide identical result with the true
(Prakash et al., 2007). The repeated measurements
of vtg standard demonstrated precision with inter and intra-assay coefficient
of variations (Cvs) falls within the acceptable ranges as reported in several
other findings (Mananos et al., 1994; Mosconi
et al., 1998; Bulukin et al., 2007;
Swart and Pool, 2009). High degree of homogeneity plates
were used to avoid inconsistencies in the reproducibility and accuracy of the
developed assay. The sensitivity of the assay (<10 ng mL-1) was
almost equal to that ELISA reported form other studies (Mosconi
et al., 1998; Korsgaard and Pedersen, 1998;
Fenske et al., 2001; Shao
et al., 2005). In this study, the result demonstrated that polyclonal
antibody did not cross-react with any plasma protein in non-estrogenised L.
calcarifer, thus, confirmed that the protein, vtg are female specific. Parallelism
curves of vtg standard, natural vitellogenic females and E2-treated
demonstrated that the response of plasma dilutions were proportional to the
adsorbed antigen concentration. The covariance (ANCOVA) did not analyze the
significant difference in slopes (p>0.05), demonstrating the specificity
Meucci and Arukwe (2005) concluded that vtg acts as
sex determinant in dimorphism fish species such as groupers. Hence, it can be
applied in Asian sea bass species, since, this species also considered as dimorphism
fish. Information about vtg was useful for screening fish maturity status (Kishida
and Specker, 2000).
The ELISA developed in the present study resulted in acceptable parameters of specificity, precision and sensitivity which are needed for measuring L. calcarifer vtg. The availability of the assay capable of measuring vtg in plasma of naturally vitellogenic and estradiol treated juvenile in captive condition. It also facilitates further research on regulatory mechanisms of vitellogenesis and reproductive physiology of L. calcarifer.
We thank Mr. Mohd Kufli from Chenur Supplier Sdn. Bhd. for his guidance and providing accommodation during polyclonal antibody production. Special thank you is extended to Dr. Yasser Abdelhadi from Department of Aquaculture, Universiti Putra Malaysia (UPM) for his critical review and comments of the manuscript. This research was supported by Ministry of Science, Technology and Innovation (MOSTI), project no: UKM-MGI-NBD0007-2007, Vot no: 5487718.