During breeding male stickleback use the spiggin protein as an underwater adhesive to build their nest. The physical properties of spiggin protein render it extremely difficult to purify as multimerization of the protein results in aggregate formation and precipitation. On the other hand, spiggin would be a valuable tool to be able to measure, since it is one of the few proteins that so far are known to be under androgenic control and therefore could be used as a measurable parameter for androgen exposure.
This paper presents the development of sensitive quantitative antibody based spiggin assays useful for the detection of androgenic and anti-androgenic substances in the environment using three-spined stickleback as a model. Furthermore, it describes the use of recombinant spiggin γ as a standard for spiggin immunoblotting. The isoform composition of expressed spiggin protein complexes may vary in kidney and in urinary bladder . Since the spiggin isoforms share the amino acids corresponding to the spiggin γ protein (Fig. 1), this isoform was chosen as a good candidate to use as a standard. The differences in the region for which the synthetic peptide KTK was raised is shown, with spiggin 2 and 3 lacking part of the sequence, whereas the sequence for the synthetic peptide HRD is present in all the sequences. The polyclonal native spiggin antibody used in the present study exhibits a high specificity as observed by ELISA and Western blot analysis. The ELISA showed that the spiggin antibody cross-reacts with the native adhesive protein in a linear range and in a concentration-dependent manner (Fig. 2). Spiggin polyclonal antibody against the native protein cross-reacted with native spiggin and produced several bands of different molecular weigh on the Western blot (Fig 4b). These bands correspond well with the expected sizes of the individual subunits and multimerized spiggin. Thus, the polyclonal antibody directed against native spiggin is able to cross-react with all spiggin subunits on Western blot analysis and also detects spiggin in ELISA analysis. Detection of native spiggin from the urinary bladder indicated a high level of multimerization with a strong band around 122 kDa and additional bands around 70 kDa and 52 kDa. In the kidney from female fish the protein is less abundant than in the males, including the multimerized isoforms. As in the males, bands corresponding to the sizes of all spiggin isoforms were detected in the kidney and the urinary bladder. (Fig. 4b). In contrast to males no multimerized spiggin could be identified in the urinary bladder of female stickleback. In male kidney there are bands corresponding to 230 kDa, 120 kDa, 100 kDa and 70 kDa. While the 230 kDa band corresponds to multimerized spiggin isoforms, the other bands correspond well with the calculated molecular weights of individual spiggin isoforms (Table 1).
The use of a synthetic peptide standard allows for quantification of spiggin and we have shown that the HRD peptide can be used in a competitive ELISA for detection of spiggin in methyltestosterone induced male and female three spined sticklebacks . In this study both peptide antibodies were shown to be able to identify and quantify spiggin in biological samples. By using KTK and HRD in an ELISA it was found that a three day 11-KT exposure gave rise to a 1.73 and 1.79 spiggin increase respectively, indicating that the two antibodies can be used individually to measure androgen induced spiggin production.
Due to the native spiggin proteins highly hydrophobic properties, which are instrumental for its use by stickleback males as underwater glue, it poses challenges in handling and detection. Therefore, the use of recombinant V5/His6-tagged spiggin γ as a standard for spiggin measurements offers an alternative in order to obtain quantitative measurements of spiggin in biological samples. Addition of V5/His6 epitopes to recombinant proteins has shown to increase their solubility  and recombinant spiggin can be produced in bacteria in necessary amounts and in a short period of time which makes it highly available.
On the western blot, the spiggin antibodies against the native protein and antibodies against the synthetic peptides cross-reacted with the recombinant protein in bacterial cell lysate enabling detection of a recombinant protein of approximately 50 kDa. The molecular weight of the recombinant protein on Western blot was less than the theoretical protein weight which is in agreement with previously reported results . Since E coli lack glycosylation properties, discrepancies in theoretical and experimental molecular weight for the recombinant spiggin could be explained by the physical properties of the spiggin. The high hydrophobicity of spiggin might also contribute to this phenomenon due to higher affinity to SDS .
Spiggin shows great potential as a screening tool for endocrine disruptors, and has been used for this purpose in kidney cell cultures , tissue slice cultures  and in whole animal exposures . However, there are many factors in the environment that might affect the production of the adhesive protein. For instance, parasite harbouring fish should be excluded to avoid errors, as infection of Schistocephalus solidus in male three-spined stickleback result in reduced levels of spiggin in the kidney . The correlation between total spiggin content in all three groups with KSI suggests that KSI measurements could be used as an initial rapid screening method to detect possible androgenic disturbances.
Due to the adhesive and multimerization properties of spiggin it is of interest to develop methods to prevent polymerisation of both native and recombinant protein upon purification. We have tested the effets of saline solution as solvent to simplify handling of samples. Adjustments of pH was not found to improve the solubility of spiggin whereas a saline solution kept spiggin soluble for several days in +4°C (data not shown). Even so, the positive effect of salinity on spiggin soulubility could not be used in the ELISA procedures since the small volumes of the samples added to the buffer used in ELISA results in low final concentrations of salt and a rapid mutlimerization of the protein in the wells.
The seasonal cycling in plasma levels of 11-ketotestosterone in the male three-spined stickleback show an marked increase in May with a peak from June to July, this peak is also mirrored in the kidney epithelial height, (KEH), which increases from approximately 15 μm to 40 μm in the spawning season [15, 16]. Seasonal variations in KSI showed an increase from March to April, peaking in May, followed by a decrease in June to July . Measurements of spiggin in conjunction with measurements of KSI would enable trend monitoring of local populations over time to obtain data on possible increases in accumulation of endocrine disrupting substances, and the appearance of previously undetected environmental androgens. The cloning and characterization of the stickleback nuclear androgen receptor  will be a useful tool for future work on identification of androgenic substances in the aquatic environment.
In the present study we have characterized three spiggin antibodies and demonstrated that they are highly specific and able to detect basal levels of spiggin in both male and female fish. All three antibodies were able to detect protein corresponding to all the known sizes of spiggin, suggesting that they will give a total spiggin value when used in ELISA assays. We have also developed two quantitative spiggin ELISA using peptide standards and have shown that posibility of producing recombinant spiggin protein for use as a standard with native spiggin antibodies.