ELISA/assay

Methotrexate ELISA kit

DEIA-XYZ209

96T

Methotrexate ELISA kit

MTX

cELISA

MTX

Human

Quantitative

Serum, Plasma

96T

1. The optimal storage temperature of the kit is 2-8 °C, do not freeze. 2. Unused ELISA strips must be sealed and stored at 2-8 °C.

MTX

Human

The Methotrexate ELISA kit is a colorimetric competitive immunoassay kit with results in 1.5 hours. Note: For research use only. Unless otherwise specified expressly on the packaging, all products sold here under are intended for and may be used for research purposes only and may not be used for food, drug, cosmetic or household use or for the diagnosis or treatment of human beings.

Methotrexate is a drug used in the treatment of cancer and autoimmune disease. It is designed as an antifolate to inhibit the metabolism of folic acid. Two distinct mechanisms of action have been described for methotrexate. In cancer treatments, methotrexate competitively inhibits the dihydrofolate reductase (DHFR) by blocking folate binding. DHFR converts dihydrofolate to active tetrahydrofolate. Inhibition of DHFR results in inhibition of the synthesis of purine and pyrimidine bases effectively limiting DNA and RNA synthesis and cancer cell growth. In autoimmune disease and specifically in the treatment of rheumatoid arthritis, methotrexate appears to impact several pathways resulting in inhibition of T cell activation. The effects include suppression of T cell expression of intercellular adhesion molecules, inhibition of methyl transferase activity and increased CD95 sensitivity leading to apoptosis in active T cells. Monitoring methotrexate levels is important to assure appropriate levels are maintained during therapy or treatment. High levels of methotrexate can lead to toxicity and potential renal failure as well as immunosuppression. Additionally, methotrexate is known to interact with a wide variety of drugs leading to additional complications. Determining the presence of methotrexate in samples from subjects in blinded research studies can assist in the interpretation of study results. Methotrexate is established as one of the most effective and safe therapeutics for rheumatoid arthritis. The safety profile assures that methotrexate will continue to be used in new studies in combination with other new or established drugs. The same is true in its use as a cancer therapeutic. The ELISA enables monitoring levels of methotrexate in both preclinical and clinical research. The methotrexate assay is also appropriate for the detection of methotrexate contamination after its use as a selective agent for recombinant protein production in mammalian cell lines.

The Methotrexate ELISA kit is a complete kit for the quantitative determination of methotrexate in serum and plasma samples. Please read the complete kit insert before performing this assay. The methotrexate ELISA uses a methotrexate monoclonal antibody to bind methotrexate in the sample or standard competitively to that pre-bound to the wells as a bovine serum albumin (BSA) conjugate. Anti-methotrexate antibody bound to methotrexate in the sample or standard are washed away while those captured by the immobilized methotrexate are detected with a secondary antibody horseradish peroxidase (HRP) conjugate. The assay is developed with tetramethylbenzidine (TMB) substrate and the resulting absorbance is measured with a microplate reader at 450nm. The intensity of the yellow color is inversely proportional to the concentration of methotrexate.

1. Methotrexate Microtiter Plate, One Plate of 96 Wells A plate using break-apart strips coated with a methotrexate BSA conjugate 2. Methotrexate Antibody Solution, 7mL Methotrexate monoclonal antibody 3. Enzyme Conjugated Secondary Antibody Solution, 12 ml Rabbit anti-mouse IgG conjugated to HRP. 4. Methotrexate Standard Solution, 1ppm, 1mL 5. Methotrexate Standard Solution, 100ppb, 1mL 6. Methotrexate Standard Solution, 20ppb, 1mL 7. Methotrexate Standard Solution, 4ppb, 1mL 8. Methotrexate Standard Solution, 0.8ppb, 1mL 9. Methotrexate Standard Solution, 0.16ppb, 1mL 10.Methotrexate Standard Solution, 0 ppb, 1mL 11. 20× concentrated wash solution, 50 ml Phosphate buffered saline containing detergents. 12. 2× Sample Diluent, 50mL Phosphate buffered saline. 13. TMB Substrate, 6ml × 2 A solution of 3,3',5,5' tetramethylbenzidine (TMB) and hydrogen peroxide. Protect from prolonged exposure to light. 14. Stop Solution, 7ml A 2M solution of sulphuric acid in water. Keep tightly capped. Caution: Caustic.

1. Deionized or distilled water. 2. Precision pipets for volumes between 50 μl and 1,000 μl. 3. Repeater pipet for dispensing volumes between 50 and 100 μl. 4. Disposable beakers for diluting buffer concentrates. 5. Graduated cylinders. 6. Adsorbent paper for blotting. 7. Microplate reader capable of reading at 450nm, preferably with correction between 620nm and 630nm.

The Methotrexate ELISA is compatible with serum and plasma samples from human. Samples diluted sufficiently into Sample Diluent (see Reagent Preparation) can be read directly from a standard curve. the minimum recommended dilutions for validated matrices of sample is 1:20 (e.g. 50uL serum/plasma sample+950uL Sample Diluent). Samples must be stored frozen at or below -20°C. Excessive freeze/thaw cycles should be avoided. Prior to assay, frozen samples should be brought to 4°C slowly and gently mixed. Samples may be clarified by centrifugation to reduce risk of matrix interference. Other Sample Types The methotrexate ELISA kit may be appropriate for testing biological matrices from other species that have not been validated and may be compatible with other buffer matrix formulations. It is recommended that any matrix of interest undergo testing to determine the minimum dilution in Sample Diluent to eliminate matrix interference.

Solution 1: 1× Sample Diluent Dilute the 2× Sample Diluent with deionized water diluent in the volume ratio of 1:1, called Sample Diluent (10ml 2× Sample Diluent + 10ml deionized water). Solution 2: 1× Wash solution Dilute the 20× concentrated wash solution with deionized water in the volume ratio of 1:19, which will be used for washing the plates.

Procedural Notes 1. Do not mix components from different kit lots or use reagents beyond the kit expiration date. 2. Allow all reagents to warm to room temperature for at least 30 minutes before opening. 3. Pre-rinse the pipet tip with reagent, use fresh pipet tips for each sample, standard and reagent. 4. Pipet standards and samples to the bottom of the wells. 5. Add the reagents to the side of the well to avoid contamination. 6. This kit uses break-apart microtiter strips, which allow the user to measure as many samples as desired. Unused wells must be kept desiccated at 4°C in the sealed bag provided. The wells should be used in the frame provided. 7. Prior to addition of conjugate and substrate, ensure that there is no residual wash buffer in the wells. Any remaining wash buffer may cause variation in assay results. Procedure 1. Take all reagents out at room temperature (20-25°C) for more than 30min, homogenize before use. 2. Get the microwells needed out and return the rest into the zip-lock bag at 2-8°C immediately. 3. The wash solution should be brought to room temperature (20-25°C) before use. 4. Number: Number every microwell position and all standards and samples should be run in duplicate. Record the standards and samples positions. 5. Add standard solution/sample and antibody solution: Add 50μl of standard solution or prepared sample to corresponding wells. Add 50μl of antibody solution to each well, mix gently by shaking the plate manually and incubate for 30min at room temperature (20-25°C) with cover. 6. Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 300μl of diluted wash solution atinterval of 10s for 5 times. Absorb the residual water with absorbent paper (the rest air bubble can be eliminated with unused tip). 7. Add enzyme conjugate: Add 100μl of enzyme conjugate to each well, mix gently by shaking the plate manually and incubate for 30min at room temperature (20-25°C) with cover. 8. Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 300μl of diluted wash solution at interval of 10s for 5 times. Absorb the residual water with absorbent paper. 9. Coloration: Add 100μl of substrate solution to each well. Mix gently by shaking the plate manually and incubate for 15 min at room temperature (20-25°C) with cover . 10. Measure: Add 50μl of the stop solution to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm, preferably with correction between 620 and 630 nm. (Read the result within 5min after addition of stop solution. )

Percentage absorbance The mean values of the absorbance values obtained from the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied by 100%. Absorbance (%)=(B/B 0 )*100% B ——absorbance of standards or samples B 0 ——absorbance of zero standard (0ng/ml)

Intra-plate coefficient of variation: Inter-plate variation coefficient: <10%

0.16 ppb

1. The mean values of the absorbance values obtained for the standards and the samples will be reduced if the reagents and samples have not been regulated to room temperature (20-25°C). 2. Do not allow microwells to be dry between steps to avoid unsuccessful repetitiveness and operate the next step immediately after tap the microwells holder. 3. Mix the homogenate and elute the plate adequately. 4. Avoid the stop solution touching skin for the 2M H 2 SO 4 . 5. Don't use the kits out of date. Don't exchange the reagents of different batches, or else it will drop the sensitivity. 6. Storage constitution: Keep the ELISA kits at 2-8°C without frozen. Avoid direct sunlight during all incubations. Covering the microtiter plates is recommended. 7. The reagents go bad: Substrate solution should be abandoned if its color has changed. The reagents may be turn bad if the absorbance value of the zero standard is less than 1 (A450nm 8. The coloration reaction needs 15min after the addition of substrate solution; But you can prolong the incubation time ranges from 15min to 12min if the color is too light to be determined. On the contrary, shorten the incubation time properly. 9. The best reaction temperature is room temperature (20-25°C), temperature too high or too low both will lead to the changes of sensitivity and absorbance values.

The Methotrexate ELISA kit is a colorimetric competitive immunoassay kit with results in 1.5 hours. Note: For research use only. Unless otherwise specified expressly on the packaging, all products sold here under are intended for and may be used for research purposes only and may not be used for food, drug, cosmetic or household use or for the diagnosis or treatment of human beings.

1. Methotrexate Microtiter Plate, One Plate of 96 Wells 2. Methotrexate Antibody Solution, 7mL 3. Enzyme Conjugated Secondary Antibody Solution, 12 ml 4. Methotrexate Standard Solution, 1ppm, 1mL 5. Methotrexate Standard Solution, 100ppb, 1mL 6. Methotrexate Standard Solution, 20ppb, 1mL 7. Methotrexate Standard Solution, 4ppb, 1mL 8. Methotrexate Standard Solution, 0.8ppb, 1mL 9. Methotrexate Standard Solution, 0.16ppb, 1mL 10.Methotrexate Standard Solution, 0 ppb, 1mL 11. 20× concentrated wash solution, 50 ml 12. 2× Sample Diluent, 50mL 13. TMB Substrate, 6ml × 2 14. Stop Solution, 7ml

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet