Human TGF-β1 ELISA Kit,Absin_specification/price/image

Product Detail
Company Profile

Product Specification

Usage

Need to bring your own test equipment
1. Microplate reader (can measure the absorption value of 450nm detection wavelength and 540nm or 570nm correction wavelength)
2. High precision liquid dispenser and disposable suction head
3. Distilled water or deionized water
4. Washing bottle (spray bottle), multi-channel plate washer or automatic plate washer
5. 500mL cylinder

1, preparation before the experiment
1. Sample collection and storage
① Cell culture supernatant: particles should be removed by centrifugation; Test the samples immediately. If the sample is not tested in time after collection, it is recommended that the sample be divided according to the dosage and stored in the refrigerator at -20 ° C to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.
② Serum: Samples were collected using a serum separation tube (SST) and samples were left at room temperature for 30 minutes. The samples were centrifuged at 1000g for 15 min. Serum was immediately removed and tested immediately. If the sample is not tested in time after collection, it is recommended to repack according to a single dosage and freeze in ≤ -20℃ refrigerator to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.
③ Plasma: Plasma was collected using EDTA, heparin, or citric acid as an anticoagulant, centrifuged at 1000g for 15 min within 30 min of collection, and tested immediately. If the samples are not detected in time after collection, it is recommended to separate the samples according to the single dosage and freeze them in &le. -20℃ refrigerator to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.

2. Reagent preparation (Place all reagents and samples at room temperature for 15 minutes before use. It is recommended that all experimental samples and standards do double hole detection )
1× Washing liquid preparation: kit of concentrated washing liquid is 25 & times; Mother liquor, diluted to 1× with distilled water before use; Working liquid. : take 10 ml concentrated washing liquid + 240 ml distilled water constant volume to 250 ml, actual operation can be calculated first use, and then to preparation.
②1× Dilute with buffer preparation: kit for the enrichment of dilute with buffer, should take 49.3 mL of 1 & times; Add 0.7mL of concentrate dilution buffer to the washing solution, and mix it up to 1× Working solution.
③ Detection of antibodies: The dry powder was centrifuged to the bottom of the tube and 110uL of dilution buffer was used (1×) Dissolve and let stand at room temperature for 5 minutes to obtain 100× Mother liquor; Dilute to 1× before use; Working solution. Calculate the desired volume by using 100uL per well. example: 10 Wells were used, then take 10uL of 100 times the working concentration of the test antibody, using dilution buffer (1×) Constant volume to 1mL, get 1mL of 1× The working concentration of the detected antibody.
④SA-HRP: SA-HRP is 40× Mother liquor, use dilution buffer before use (1×) Dilute to make 1× Working solution, 100uL required per well. example: used 10 holes, then take 25uL of 40× Mother liquor +975uL dilution buffer (1×) Constant volume to 1mL to obtain 1× of 1mL; The working concentration of the detected antibody.
⑤ Chromogenic agent: according to 100uL per well, calculate the amount needed for this test, take out the corresponding volume of chromogenic agent, avoid light; The removed chromogenic agent is only used on the same day.
⑥ Standards: lyophilized standards with dilution buffer (1×) Redissolve, redissolve volume 1000uL, to obtain a concentration of 2000pg/mL standard mother liquor. Gently shake for at least 5 minutes, it is fully dissolved. Add 300uL dilution buffer (1×) to each dilution tube. . The standard mother liquor is diluted according to the picture below, and each tube must be fully mixed before pipetting to the next tube. No dilution of standard mother liquor can be used as a standard curve peak (2000 pg/mL), dilute with buffer (1 & times) Can be used as a standard curve zero (0 pg/mL).

Before the sample on the activation:

cellular supernatant	Serum/plasma
100 ul cells that add 20 ul supernatant fluid sample activation fluid or（1 N HCl）	40uL serum/plasma added to 20uL sample activation solution or（1 N HCl）
Blending and incubation for 10 min at room temperature	Blending and incubation for 10 min at room temperature
Add 20 ul activated terminated liquid or（1.2 N NaOH/0.5 M HEPES）	Add 20uL activation termination solution or (1.2 N NaOH/0.5 M HEPES)
mixing	mixing
Can be directly add sample detection	The activated sample was diluted 20-fold in the sample diluent before the experiment began
Is required for a return to the standard curve to calculate calculate dilution ratio (x 1.4)	Return to the standard curve to calculate to calculate when diluted multiples (x 40)

Note: Fetal calf and calf serum contain high levels of TGF-β 1, it has been reported in the literature to be as high as about 16ng/ml. The highest level of TGF-&beta was 1600pg/ml in the culture supernatant containing 10% fetal bovine serum. Blank medium control group can be added as background deduction, or serum-free medium can be used to culture cells directly.

2. Operation steps
1. Ready for all the needed reagent and standard;
2 Remove the microplate from the sealed bag that has been balanced to room temperature, and put the unused slat back into the aluminum foil bag and re-seal it;
3. Add 300uL washing solution to the microplate, let it soak for 30 seconds, discard the washing solution and pat the microplate dry on absorbent paper, please use immediately do not let the microplate dry;
4. Add different concentrations of standard, experimental samples or quality control into the corresponding Wells, 100uL for each well. The Wells were sealed with plate adhesive and incubated at room temperature for 2 hours.
5 Suck the liquid out of the plate and wash the plate using a bottle washer, a multi-channel plate washer, or an automatic plate washer. Add 300uL of washing liquid to each well, and then suck the washing liquid out of the plate. Repeat 3 times. Every time you wash the plate, try to absorb the residual liquid to help you get a good test result. At the end of the last plate wash, please blot all the liquid in the plate or invert the plate and pat all the residual liquid in the absorbent paper;
6. Add 100uL detection antibody to each microwell. Seal the reaction Wells with sealer tape and incubate for 2 hours at room temperature;
7. Repeat the plate washing operation of step 5;
8. Add 100 ULSA-HRP to each microwell and incubate for 20 minutes at room temperature. Be careful to avoid light;
9. Repeat step 5 to wash the plate;
10. Add 100uL of color development solution to each microwell, incubate at room temperature for 5-30 minutes, pay attention to avoid light;
11. Add 50uL of termination solution to each microwell, and the color of the solution in the well will change from blue to yellow. If the color of the solution turns green or the color change is inconsistent, tap the microplate to mix the solution evenly;
12. Within 30 minutes after the termination solution is added, the absorbance value at 450nm is measured using a microplate reader and 540nm or 570nm is set as the correction wavelength. If the dual wavelength correction is not used, the accuracy of the results may be affected;
13 Calculation results: The corrected absorbance values (OD450-OD540/OD570), the compound reading were averaged for each standard and sample, and then the average zero standard OD value was subtracted. Standard curves were created by 4-parameter logic (4-PL) curve fitting using computer software. Alternatively, a curve can be generated by plotting the logarithm of the concentration of the standard against the logarithm of the corresponding OD value, and the best fit line can be determined by regression analysis. This process produces an adequate but less accurate fit to the data. If the sample is diluted, the concentration should be multiplied by the dilution.

Note: The standard curve data provided in are for reference only, and the sample content should be calculated according to the standard curve drawn in the same test.

2. Kit parameters
1. Sensitivity: human TGF-β The minimum detectable dose of 1 (MDD) general is less than 9.3 pg/mL. The lowest detectable value is the corresponding concentration calculated from the mean of the zero-point absorbance values of 20 standard curves plus two standard deviations.
2. Correction: the ELISA kit by high purity recombinant CHO cells expressed TGF - & beta; 1 protein correction. Linear:
3. 6 different samples with a high concentration of TGF - & beta; 1, and then use thinner (1 & times) Measure linearity by dilating the sample to within the detection range.

4, common problem resolution
1. The white board (no color), after the completion of color

NO.	Cause	Solution
1	Kit stored improperly; A mixture of different kit reagent	Buy a new kit, pay attention to the storage conditions; Do not mix
2	Endow low temperature, short time	If the temperature is too low, the incubation time is prolonged and the color development time is prolonged
3	Wrong addition or omission of reagents	In strict accordance with the manual steps to add the correct reagents
4	Used to configure the solution container not clean, or there is something wrong with the water	The use of clean containers and qualified distilled water
5	In the process of washing the plate, the soaking time is long, the number of washing the plate is too much, and the impact of washing the plate is large	In strict accordance with the manual operation
6	The temperature of the reagent was not uniform	All reagents were equilibrated at room temperature for 30 minutes
7	Detection of antibody and/or HRP concentrations was too low	Do not dilute at will according to the instructions

2. Flower plate (blank, negative and positive controls were normal, but the OD value of sample Wells was significantly higher)

NO.	Cause	Solution
1	Fewer washing, inadequate	Wash according to instructions
2	The substrate 3,3',5,5' -tetramethylbenzidine (TMB) is contaminated or exposed to metal ions or oxidants	The use of clean containers and when making up qualified distilled water; Avoid light preservation
3	High incubation temperature and/or excessive incubation time	Control incubation and the enzymatic reaction temperature and time
4	Sample did not change when the spear head, cause cross contamination	Change the tip of each sample
5	Near the hole cross contamination	Vertical clappers, using the appropriate legal pad, avoiding the hole into confetti
6	Samples are endogenous interfering substance	Possible infectious agents were speculated and treated accordingly
7	Sample hemolysis, storage for too long, incomplete agglutination, contaminated by bacteria, blood vessels to add impact	Avoid hemolysis, contamination, too long storage and other phenomena

Theory

Double antibody sandwich enzyme-linked immunosorbent assay was used in this kit. Specific anti-human TGF-beta1 antibody was precoated on a high-affinity microplate. After incubation, the TGF-beta1 present in the sample was combined with the solid-phase antibody and the detection antibody to form immune complexes. After washing to remove the unbound material, the horseradish peroxidase-labeled Streptavidin-HRP was added. After washing, the chromogenic substrate was added and the color was developed in the dark. The reaction was terminated by adding the termination solution, and the absorbance value was measured at 450nm wavelength (reference correction wavelength 540nm or 570nm).

Composition

Component	Size	Once opened, diluted or heavy soluble reagent expiration date
Human TGF-β1 Microplate	1	Unused slats can be stored at 2-8 ° C for up to 30 days after being sealed back in aluminum foil bags with desiccants
Human TGF-β1 standard substance	2	Dissolves, calculate the dosage repackaging, can be in - 20 ° C storage for 14 days
Human TGF-β1 detect antibody	1	Enrichment dissolved after the volume, can be in 2-8 ° C storage for 14 days
40×SA-HRP	1	40× concentration can be stored at 2-8°C; 1 x working concentration is not recommended to save
10 x condense dilute with buffer	1	After opening, can be in 2-8 ° C stored for 30 days
Color-substrate solution	1
Stop buffer	1
20 x inspissation washing buffer	1
Sealing film	3	Room temperature storage, to avoid contamination, do not repeat use

Background

The transforming growth factors beta1, 2, and 3 (TGF-beta1, TGF-beta2, and TGF-beta3) are highly pleiotropic cytokines that are secreted by virtually all cell types. TGF-β molecules have been proposed to act as cellular switches to regulate processes such as immune function, proliferation and epithelial-mesenchymal transition. Targeted deletion of these genes in mice suggests that each TGF-β isoform has some nonredundant function: Tgf-β1 is involved in hematopoiesis and endothelial cell differentiation. TGF-beta2 affects the development of the heart, lung, craniofacial, limb, eye, ear and genitourinary system. TGF-β3 may affect the occurrence of pa and lung development. The full in vitro biological activity of TGF-beta5 has not been explored. In inhibitory bioassay, however, have found TGF - walk, TGF - beta 2, TGF - beta3 and TGF - beta5 interchangeable, to a great extent and TGF - beta5 is expected to show a series of other members of the family of TGF - beta similar activity. To date, TGF-beta5 production has been demonstrated only in Xenopus laevis
. TGF-β ligands are initially synthesized as precursor proteins that undergo proteolytic cleavage. The mature segment forms active ligand dimers through a disulfide-rich core composed of a characteristic "cysteine knot". TGF - beta signal transduction begins with combined with auxiliary receptor beta chitosan (also called TGF - beta RIII) and type II serine/threonine kinase receptor (called TGF - beta RII) compounds. This receptor then phosphorylates and activates the type I serine/threonine kinase receptor ALK-1 or TGF-βRI (also known as ALK-5). Activated type I receptors phosphorylate and activate Smad proteins that regulate transcription. Different responses to TGF-β can be observed under different circumstances using other signaling pathways that are not related to Smad.

General Notes

1. Please use the kit within the validity period.
2. Components of different kits and different batch kits should not be mixed.
3. If the sample value is greater than the highest value of the standard curve, the sample should be diluted with (1×) The samples were diluted and retested. "If the cell culture supernatant sample needs to be diluted in a distributed manner, cell culture medium may be used for intermediate dilutions, except for the last step when diluent is used."
4. The difference of the test results can be caused by a variety of factors, including the operation of the experimenter, the use of the pipettor, the washing technique, the reaction time or temperature, the storage of the kit, etc.
5. The termination solution in the kit is acidic. Please protect your glasses, hands, face and clothes when using it.
6. For scientific research only, not for in vitro diagnosis.

Storage Temp.

Unopened kit, 2-8° C Storage

Test Range

31.3pg/mL-2000pg/mL

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.