Vitellin is the most important nutrient and energy source for the development of oocytes as well as developing embryos.The quality and quantity of vitellin in the mature ovary play a vital role in the crustacean reproduction and offspring quality.The swimming crab,Portunus trituberculatus,is a commercially important fisheries resource and mariculture species in East Asian countries.Because of overfishing,destruction of coastal spawning and nursery grounds and marine pollution,the landing of this crab has shown a declined trend in East China Sea since 1990s.The decline in natural stock and increase in market demands have driven substantial aquaculture interests for the crab species.However,the seed production of the crab is dependent on wild-caught broodstock,which could become a constraint to the sustainable development of this crab aquaculture.Our previous studies have shown pond-reared female broodstock have poor ovarian development and worse reproductive performance compared to wild females.Therefore,it is very urgent to understand the mechanism of vitellin synthesis and accumulation for female P.trituberculatus. The present study was conducted to purify vitellin from mature ovary of female P.trituberculatus by gel filtration chromatography,and the elution process was monitored at wavelength of 280 nm and 470 nm,respectively.The number and molecular weight of subunits were identified by sodium dodecyl sulphatePAGE(SDS-PAGE)and marker protein,respectively.Then,the rabbit polyclonal vitellin anti-serum was prepared and purified.Based on the vitellin anti-body,we optimized the reaction parameters of Enzyme-Linked Immunoabsorbent Assay(ELISA),and then the stable and standard ELISA was developed for female P.trituberculatus.The results showed that vitellin was divided into three major polypeptides with molecular weight of 102,75,and 66 ku,respectively by the denatured SDS-PAGE.The Western-blotting confirmed all three major polypeptides had specific reactivity with vitellin anti-body.The higher linear correlationship could be found on the sample direct coating ELISA than the antibody sandwich coating ELISA.Then,the optimal dilution rate of purified vitellin antibody and coating period were shown to be close to 1∶90 000 and 8 hours at 4 ℃,respectively.At 37 ℃,the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour,respectively,while the best color development solution was around 20 minutes.Furthermore,based on these parameters,the standard linear equation,y=0.000 9x+0.399 1(R²=0.986 1,x and y represent Vn concentration and OD₄₅₀value,respectively),was established for the determination of female P.trituberculatusvitellin concentration with the valid range of 200-900 ng/mL.The susceptibility of this ELISA was around 14.9 ng/mL for Vn contents.Finally,the ELISA was used to determine ovarian Vn contents for the identification of validity of this assay.The results demonstrated the mean coefficients of variation of intraassay and interassay were 3.59% and 3.10%,respectively.In conclusion,the developed ELISA of this study is precise,stable and repeatable.