As shown in figure a, log values of concentrations of each standard sample were plotted against absorbance at 450 nM. Plot was generated via fitting the data into a non-linear regression curve in Graphic Prism 5. As shown in figure b., mouse growth hormone levels in wt and mutant littermates were measured at 450nm, followed by interpolation of the x value from 450 nm absorbance (Y value) using non-linear regression equation that was derived from generating the standard curve.
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To measure the concentration of growth hormone and reliably compare concentrations between wild type (wt) and mutant mice is not an easy task. Mouse growth hormone is secreted from the pituitary gland into circulation to affect the size of multiple organs during development. The release of murine growth hormone peaks at pubertal stage and gradually reduces to a constant level when mice reach adulthood. The level of growth hormone at adulthood is instrumental in maintaining homeostasis of various tissues; however, it is a challenge to choose the right assay to detect endogenous level of growth hormone, and especially hard to quantitatively compare serum levels of growth hormone from mice of different genetic backgrounds.
EMD Millipore’s Rat/Mouse Growth Hormone ELISA Kit directly targets these problems. It offers a fast, simple and very sensitive way to measure growth hormone levels in living mice. It only requires 10 ul of serum and takes only 3.5 hrs if you are following the steps exactly as listed in the instructions. It is sensitive enough to detect as little as 70pg/ml of growth hormone in 10ul of serum. If you are looking for a certain range of species tolerance, this ELISA kit works to detect both rat and mouse growth hormone. The kit includes sufficient reagents to run 96 assays simultaneously; on the other hand, it also offers flexible alternatives to run 12 strips of 8 wells, one at a time. The concept of this assay is based on the sandwich ELISA method which detects low levels of growth hormone with high specificity. The binding between growth hormone in serum and the anti-growth hormone antibody coated on the ELISA plate can be subsequently amplified and detected by an enzymatic (horseradish peroxidase [HRP]) colorimetric reaction that allows the user to quantitatively measure this binding by reading absorbance at a fixed wavelength in a plate reader.
ELISA
The purpose of my experiment is to compare mouse growth hormone levels at adulthood between wild type and mutant littermates in a FVB (an inbred mouse strain that carries homozygous Fv1b allele for sensitivity to the B strain of Friend leukaemia virus) background. Blood was drawn from wild type or mutant littermates and was allowed to coagulate at room temperature for 30 minutes, followed by a gentle and quick spin. Supernatant) was taken as serum input and diluted according to the manual.
Briefly – standards and diluted samples were applied to the wells on the ELISA plate, followed by adding detection antibody, enzymatic solution (HRP-based), HRP substrate and stop solution as described very clearly in the instructions. Read absorbance at suggested wavelength on the TECAN Infinite M200 Pro multimode microplate reader.
The protocol is very easy to follow. One thing that you need to take extra care with is that serum must be used within 30 minutes after blood withdraw and coagulation; otherwise results will fluctuate between experiments. In addition, assay standards in a powder format are limited; it is suggested to plan experiments well and freshly dissolve them in solution every time a new assay is performed. Since negative and positive controlsshould be used in each experiment, one 96-wellplate is ideal for runnning three independent experiments (32 wells/ each).
The manual is very easy to follow with no tricks; however, you do need to make serial dilutions to prepare the standards; wash buffer, etc. also requires dilution. As was stated in the instructions, every time a sample is run, you have to also run standards, positive and negative controls, which tremendously limits the wells available for your sample. I hope they can improve or optimize controls for this issue. In summary, this is a very robust growth hormone detection kit that allows you to measure a wide range of growth hormone variations between different genetic backgrounds and various treatment conditions.
Product Name: Rat/Mouse Growth Hormone ELISA KitSupplier Page