Vitellogenin (VTG) ELISA Kit

Antigen: Vitellogenin (VTG)

Synonyms: vg4, wu:fb59b02, wu:fb59d07, wu:fb59e10, si:rp71-23d18.3, vg5, MGC114012, wu:fi49a01, zgc:114012, si:rp71-23d18.5, vtg b1, Vg, GB13999, vg6, wu:fb62e09, si:rp71-23d18.1, vg7, MGC158513, wu:fb62f05, si:dkey-4c23.1, vtga2, VTG, vtg1

Reactivity: Medaka

Application: ELISA

Catalog no.: ABIN368677

Additional Information

Alternative name: Vitellogenin

Description: In recent years, influence of Endocrine Disrupting Chemicals (EDC) upon living organisms has been one of the hot topics in the environmental science. However, evaluation methods of chemical compound species that may have EDC effects has not yet been established, and it is absolutely necessary to take action to complete such a task. Vitellogenin (Vg), a precursor of yolk protein, is a female-specific protein that is present only in the blood of higher oviparous vertebrate organisms. Vitellogenin is normally expressed in the liver under regulation of estrogen (female sex hormone) during an egg formation process, and when it reaches a growing oarian follicle via blood stream it contributes to the egg yolk formation. Since estrogen is known to have an effect upon male species to induce unusual expression of Vg, an ELISA screening method detecting Vg in EDC exposed male animals is the most reliable bio-screening assay system.

Synonyms: vg4, wu:fb59b02, wu:fb59d07, wu:fb59e10, si:rp71-23d18.3, vg5, MGC114012, wu:fi49a01, zgc:114012, si:rp71-23d18.5, vtg b1, Vg, GB13999, vg6, wu:fb62e09, si:rp71-23d18.1, vg7, MGC158513, wu:fb62f05, si:dkey-4c23.1, vtga2, VTG, vtg1

Application Details

Principle: This kit is based on the ELISA method which is used with specific anti- medaka polyclonal antibody. Anti- medaka polyclonal antibody is coated on the microplate, so that Vg in the sample and standard is captured. After reacting biotin-conjugated anti-medaka polyclonal antibody and HRP-conjugated streptavidin, the complex of coated-antibody-antigen-antibody is formed. Finally, you could measure the Vg concentration depend on the coloring strength which has developed with enzymatic reaction.

Protocol: (1) Preparation of reagents to be used: (a) Wash solution: Make sure that the solution does not contain any crystallized material,upon reaching room temperature. Working solution is prepared by dilution of 30 mL of the stock wash solution with distilled deionized water to 570 mL. This solution is stable for 14days when stored in a refrigerator. (b) Antibody-coated microtiter plate Apply 300uL in each well and wait another 10 to 60 minutes. (c) Standard dilution: Dilute the concentrated standard solution (64ng/mL) to make 32 16 8 4 2 1 ng/mL with Dilution solution. (d) Biotin conjugated anti-medaka Vg polyclonal antibody (x100). The stock Biotin conjugated anti-medaka Vg polyclonal antibody can be prepared for 96 well reaction upon dilution of 60uL stock solution with 6mL Dilution solution just before use. (e) HRP-conjugated Streptavidin (x500). The stock HRP-conjugated Streptavidin can be prepared for 96 well reaction upon dilution of 12uL stock solution with 6mL Dilution solution just before use. (f) Coloring solution: Dissolve two OPD tablets with 13 mL of Substrate buffer. (2) Preparation of samples to be used: The homogenaize of Medaka liver should be diluted more than 10 times with Dilution solution. (3) Assay procedure: (a) Discard the wash solution from the wells completely, but do not keep wells in dry condition for reliable data. (b) Apply 50uL of each concentration of standard solution and samples into the wells and incubate for 2 hour. (c) After incubation, discard the reaction solution and wash with 300uL wash solution. Repeat this washing procedure another 2 times. (d) Apply 50uL Biotin conjugated anti-medaka Vg polyclonal antibody and incubate for 1 hour. (e) After the incubation, wash the well 3 times with wash solution (See (c)). (f) Apply 50uL HRP-conjugated streptavidin and incubate for 1 hour. Substrate buffer should be restored to room temperature from refrigerator at this time. (g) After the incubation, wash the well 3 times with wash solution (See (c)). (h) Apply 100uL Coloring solution into each well for 5 minutes in room temperature. (i) Apply 50uL of Stop solution for stopping the enzymatic reaction. (j) Read absorbance with a microplate reader at 490nm or 492nm. (k) Measure the medaka Vg concentration using standard curve.

Application Notes: (a) The Reagents should be stored at recommended temperature. (b) Do not use the reagents which is expired the date of usage. (c) Do not leave the standard, medaka Vg, for long time under room temperature. (d) Biotin conjugated anti-medaka Vg polyclonal antibody and HRP-conjugated streptavidin is not stable under sun light, so that do not leave it for long time under sun light. (e) The apparatus for making coloring solution should be clean one. (f) OPD (o-phenylendiamin) is harmful, so that take good care to it. (g) Stop solution, 1N H2SO4, is strong acid, so that take good care to it. (h) The kit is constructed with well-adjusted combination for each lot. Replace combination among different lots may cause unexpected results. (i) This kit is only for research use. Do not use for medicine or any other things. (j) When using the reagents, take care to avoid them from touching to skin,mucous membrane, clothes, and getting into eye. (k) If the reagents happen to get into eye or mouth, wash out them and go to see a doctor if you need. (l) After using the kit, wash your hand very carefully. (m) If you find that the packages of the reagents are broken or something trouble, do not use them. (n) When you store the reagents, make sure to avoid them from vaporizing, falling down. (o) After using the reagents, the packages should be discarded under established rule. (p) We can not guarantee for any other usage of the products.

Components: (1) Antibody-coated microtiter plate (8 wellsx12) 1 plate. (2) The standard, Medaka Vg 64ng/mL 300uL x 2. (3) Dilution solution 30mL x 2. (4) Biotin conjugated anti-medaka Vg antibody (x100) 80uL x 1. (5) HRP-conjugated streptavidin (x500) 20uL x 1. (6) OPD (o-phenylendiamin) tablets 2 tab. (7) Substrate buffer 15 mL x 1. (8) Stop solution 6 mL x 1. (9) Wash solution (x20) 30 mL x 1

Material not included: (1) A microplate reader. (2) A micropipet. (3) A microplate washer

Storage: Store at -80 degrees Celsius

Research Area: Hormones

Restrictions: For Research Use only

Images

Standard Curve