Sample Type 50 ul HT0570 Human Anti-tissue transglutaminase IgG CROSS REACTS WITH Human PRODUCT OVERVIEW The ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of samples in serum, plasma, cell culture supernatants and urine. INTENDED USE This Human TTG-IgG ELISA kit is intended for laboratory research use only and not for use in diagnostic or therapeutic procedures. The stop solution changes color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of Human TTG-IgG in the sample, this Human TTG-IgG ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of optical density versus Human TTG-IgG concentration. The concentration of the samples is then determined by comparing the O.D. of the samples to the standard curve. STORAGE INSTRUCTIONS Store at 4°C COMPONENTS Microtiter Plate Enzyme Conjugate Standards Substrate A Substrate B Stop Solution Wash Solution Balance Solution SAFETY NOTES 1. This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin or mucous membranes. In the case of contact, rinse the affected area with plenty of water. Observe all federal, state and local regulations for disposal. 2. All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of infectious agents.
As an analytic biochemistry assay, ELISA involves detection of an 'analyte' (i.e. the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the 'analysis' (i.e. controlled sequence of biochemical reactions that will generate a signal which can be easily quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction chamber or well needed to keep the reactants contained.
As a heterogenous assay, ELISA separates some component of the analytical reaction mixture by adsorbing certain components onto a solid phase which is physically immobilized. In ELISA, a liquid sample is added onto a stationary solid phase with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated and washed followed by some optical change (e.g. color development by the product of an enzymatic reaction) in the final liquid in the well from which the quantity of the analyte is measured. The qualitative 'reading' usually based on detection of intensity of transmitted light by spectrophotometry, which involves quantitation of transmission of some specific wavelength of light through the liquid (as well as the transparent bottom of the well in the multiple-well plate format). The sensitivity of detection depends on amplification of the signal during the analytic reactions. Since enzyme reactions are very well known amplification processes, the signal is generated by enzymes which are linked to the detection reagents in fixed proportions to allow accurate quantification - thus the name 'enzyme linked'.
The analyte is also called the ligand because it will specifically bind or ligate to a detection reagent, thus ELISA falls under the bigger category of ligand binding assays. The ligand-specific binding reagent is 'immobilized', i.e., usually coated and dried onto the transparent bottom and sometimes also side wall of a well (the stationary 'solid phase 'solid substrate' here as opposed to solid microparticle/beads that can be washed away), which is usually constructed as a multiple-well plate known as the 'ELISA plate'. Conventionally, like other forms of immunoassays, the specificity of antigen-antibody type reaction is used because it is easy to raise an antibody specifically against an antigen in bulk as a reagent. Alternatively, if the analyte itself is an antibody, its target antigen can be used as the binding reagent.
1. Bring all kit components and samples to room temperature before use.
2. Bring microtiter plate to room temperature before opening. Remove the desired number of well strips and immediately reseal and store at 2-8°C.
3. Dispense5 µL of BALANCE SOLUTION into 50 µL experimental samples.
NOTE: This step is required only when the sample is cell culture supernatant, body fluid or tissue homogenate.
4. Dilute 10 mL of WASH SOLUTION concentrate (100×) with 990 mL of deionized or distilled water. If crystals have formed in the concentrate warm to room temperature and mix to dissolve.
1. Add 50 µL of SAMPLE or STANDARD to the appropriate wells in the supplied microtiter plate. Note that wells have been pre-blocked and no additional blocking steps are required.
2. Incubate 1 hour at room temperature.
3. Empty wells and wash 3-5 times with 300-400 µL 1X WASH SOLUTION per well.
4. Empty final wash and add 100 µL of CONJUGATE per well and mix well. Cover and incubate 1 hour at 37°C in a humid chamber.
5. Wash each well 5 times with 1X WASH SOLUTION. After the last wash invert the plate and blot dry by tapping on absorbent paper. Note: Hold the sides of the plate frame firmly when washing to assure that all strips remain securely in the frame. Complete removal of the liquid at each step is essential for good performance.
6. Add 50 µL SUBSTRATE A to each well followed by addition of 50 µL SUBSTRATE B. Cover and incubate 10-15 minutes at room temperature. SUBSTRATE is light sensitive. Keep out of direct sunlight or cover with foil.
7. Add 50 µL of STOP SOLUTION to each well. Mix well.
8. Immediately read the optical density (O.D.) at 450 nm.
9. Subtract the mean blank value from each SAMPLE or STANDARD value and calculate the mean for duplicate (or greater) wells.
10. Construct the standard curve using graph paper or statistical software.
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