A kit for detection of myostatin or any other protein with a paper dip assay
Growth is regulated by many genes such as growth hormone, growth hormone receptor, insulin like growth factors and their binding proteins, bone morphogenetic proteins, myostatin and its receptors etc. Of all the factors, growth hormone, growth hormone receptor, insulin like growth factors and their binding proteins, bone morphogenetic proteins have positive effects on growth while myostatin and its receptors have negative effect on growth in all animals including poultry. Myostatin also called growth differentiating factor 8 (GDF8) is a member of transforming growth factor-β family expressed primarily in muscle tissues and involved in regulating development at embryonic stage and tissue homeostasis in adult stage. Myostatin regulates growth negatively by limiting muscular growth during pre-hatch and post hatch period. It is known that higher myostatin expression inhibits growth in animals and low level of myostatin in serum directly associated with higher body weights in animals. Myostatin is detected normally by ELISA/Western blotting. Detection of myostatin by ELISA, Western blotting etc. is laborious, time consuming, requirement of sophisticated instrument and do not involve any simple technique. Thus, it was utmost important to have a kit, which should be simple, quick, being applicable in the field. With our kit, level of myostatin is detected and the growth pattern associated with the level of myostatin, such as higher or lower body weight at juvenile as well as adult stage in birds may be assessed. In addition, with this kit, we can also detect any other proteins using the protein specific primary antibodies where other components are the same. We can nullify the limitations of ELISA/Western blotting by using this paper dip assay to quantify the level of protein. Technology Details:
Paper dip assay kit is very simple, quick to detect, economically viable, applicable without using any equipment, detectable with naked eye, not required any expertise, applicable at any age group animals etc. The methodology of the kit has been delineated below. • Blood or tissue samples are to be collected from animal. Serum are to be isolated from blood by keeping blood sample for 1to 2 hours. • Serum samples are to be diluted in different dilutions such as 1:25, 1:50, 1:100, and 1:200, 1:400 with PBS. • A volume of 50 µl diluted serum samples are to be taken in a 1.5ml tube. A volume of 100µl IgG for myostatin or any other protein are to be added in each tube; a volume of 100 µl conjugate is added in each tube; a volume of 200µl DAB is added in each tube and incubated at room temperature for 5 minutes. • Along with test serum, two controls; one containing 50 µl PBS in a tube as a control and other control does not have either serum or PBS, wherein one control has 50µl PBS, 100µl primary antibody, 100µl conjugate and 200µl DAB and the other control has100µl primary antibody, 100 µl conjugate and 200µl DAB and both controls are also incubated for 5 minutes at room temperature. • A strip of a particular type of paper with a length of 5 cm and width of 0.5cm is prepared. • The paper strip is dipped in each tube (Diluted serum sample mix tubes and two controls) for 5 to 10 sec; taken out and dried in air. • Observe appearance of band, if any, on the strips will indicate the level of protein and appearance of band beyond dilution of 1:100 will indicate higher level of myostatin or other protein. In case of myostatin, appearance of band beyond dilution of 1:100 will indicate slower growth rate in animals. Advantages:
• Paper-dip method used is very quick to detect. The whole process of detection needs 10 minutes provided enough serum samples are available. • The present method is cheap and economically viable. • This method is applicable without using any equipment. • In this method, the result can be detectable with naked eye. • The present kit is simple and one does not require any expertise to perform this. • Another advantage of this kit is that one can detect level of myostatin and future growth such as high or low body weight at juvenile as well as adult stage in birds. • The kit is cost effective and easy to use as compared to other techniques such as ELISA and Western blotting. • With this kit, one person can screen a large number of samples per day.