Rat 5-Hydroxytryptamine （5HT） ELISA Kit

（96T）

1           This immunoassay kit allows for the in vitro quantitative determination of rat 5HT concentrations in serum, plasma.

2           Expiration date six months from the date of manufacture

 

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5-Hydroxytryptamine, also known as serotonin is a monoamine neurotransmitter. It is found extensively in the gastrointestinal tract of animals, and about 80 to 90 percent of the human body's total serotonin is located in the enterochromaffin cells in the gut, where it is used to regulate intestinal movements. The remainder is synthesized in serotonergic neurons in the central nervous system （CNS） where it has various functions, including the regulation of mood, appetite, sleep, muscle contraction, and some cognitive functions including memory and learning.

The microtiter plate provided in this kit has been pre-coated with a goat-anti-rabbit antibody. Standards or samples are then added to the appropriate microtiter plate wells with a HRP-conjugated 5HT and antibody preparation specific for 5HT and incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of 5HT in the samples is then determined by comparing the O.D. of the samples to the standard curve.

0.64 ng/ml-160 ng/ml. The standard curve concentrations used for the ELISA’s were 160 ng/ml, 40 ng/ml, 10 ng/ml, 2.48 ng/ml,

0.64 ng/ml.

This assay recognizes rat 5HT. No significant cross-reactivity or interference was observed.

The minimum detectable dose of rat 5HT is typically less than

0.4 ng/ml. The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined as the lowest protein concentration that could be differentiated from zero.

1.    Unopened test kits should be stored at 2-8?C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit, provided it is stored as prescribed above. Refer to the package label for the expiration date.

2.    Opened test plate should be stored at 2-8?C in the aluminum foil bag with desiccants to minimize exposure to damp air. The kits will remain stable until the expiring date shown, provided it is stored as prescribed above.

3.    A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

 

1         Bring all reagents and plate to room temperature for at least 30 minutes before use. Unused wells need store at 2-8℃and avoid sunlight.

2         Wash Buffer If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have compley dissolved. Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer.

 

1          Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.

2          Pipettes and pipette tips.

3          Deionized or distilled water.

4          Squirt bottle, manifold dispenser, or automated microplate washer.

5          An incubator which can provide stable incubation conditions up to 37°C±0.5°C.

 

1          Serum Use a serum separator tube （SST） and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediay or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

2          Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediay or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

 

1. Set a Blank well without any solution. Add 50μl of Standard or Sample per well. Standard need test in duplicate.

2. Add 50μl of HRP-conjugate to each well （not to Blank well）, then 50μl Antibody to each well. Mix well and then incubate for 1 hour at 37°C.

3. Fill each well with Wash Buffer （about 200μl）, stay for 10 seconds and Spinning. Repeat the process for a total of three washes. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

4. Add 50μl of Substrate A and 50μl Substrate B to each well, mix well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark.

5. Add 50μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.

6. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.

 

Average the duplicate readings for each standard, Blank, and sample and subtract the optical density of Blank. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic （4-PL） curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the 5HT concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

1          The kit should not be used beyond the expiration date on the kit label.

2          Do not mix or substitute reagents with those from other lots or sources.

3          If samples generate values higher than the highest standard, dilute the samples with the appropriate Diluent and repeat the assay.

4          Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.

5          This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

 

1          Centrifuge vials before opening to collect contents.

2          To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.

3          When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision.

4          To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Sealers can not be reused.

5          Substrate Solution should remain colorless or light blue until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless or light blue to gradations of blue.

6          Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.