Methotrexate ELISA kit

Sample

Serum, plasma and urine

Species Reactivity

Human; Mouse; Rat

Detection Method

cELISA

Intended Use

The Methotrexate ELISA kit is a complete kit for the quantitative determination of methotrexate in serum, plasma and urine samples. Please read the complete kit insert before performing this assay.

Contents of Kit

1. Methotrexate Microtiter Plate, One Plate of 96 Wells (Catalog No. 80-2678): A plate using break-apart strips coated with a methotrexate-BSA conjugate.
2. Methotrexate Antibody, Lyophilized (Catalog No. 80-2677): Lyophilized methotrexate rabbit polyclonal antibody.
3. Antibody Diluent, 6ml (Catalog No. 80-2680): Buffer for reconstitution of the methotrexate antibody.
4. Methotrexate Conjugate, 10ml (Catalog No. 80-2679): A blue solution of goat anti-rabbit IgG conjugated to HRP.
5. Methotrexate Standard, 2 vials, 1,000ng (Catalog No. 80-2676): Two vials containing 1,000ng each of lyophilized methotrexate.
6. Wash Buffer Concentrate, 100ml (Catalog No. 80-1287): Tris buffered saline containing detergents.
7. Assay Buffer 13, 1×, 50ml (Catalog No. 80-1500): Tris buffered saline containing proteins and detergents.
8. TMB Substrate, 10ml (Catalog No. 80-0350): A solution of 3,3',5,5' tetramethylbenzidine (TMB) and hydrogen peroxide. Protect from prolonged exposure to light.
9. Stop Solution 2, 10ml (Catalog No. 80-0377): A 1N solution of hydrochloric acid in water. Keep tightly capped. Caution: Caustic.
10. Methotrexate Assay Layout Sheet, 1 each (Catalog No. 30-0330)
11. Plate sealer, 3 each (Catalog No. 30-0012)

Storage

All components of this kit, are stable at 4°C until the kit's expiration date. Any unused reconstituted Antibody or Standard should be stored at -20°C as soon as possible after reconstitution. Repeated freeze thaws should be avoided.

Precision

Intra-assay precision was determined by assaying 20 replicates of methotrexate controls at two concentrations in a single assay.

Inter-assay precision was determined by measuring methotrexate controls at two concentrations in multiple assays (n=10) over several days.

Note: %CV values reflect the inherent variability imparted by the wide dynamic range of the assay. Increasing the number of replicates will reduce the variability of assigned values.

Sensitivity

This sensitivity was determined by interpolation from the average of 9 separate standard curves run with replicate data points at each concentration. The sensitivity was determined at 2 standard deviations below the average net OD of 18 zero standard replicates (2 per standard curve). The sensitivity or limit of detection of the assay is 0.087ng/ml.

General Description

Methotrexate is a drug used in the treatment of cancer and autoimmune disease. It is designed as an anti-folate 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 Methotrexate 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.

Standard Curve

The results shown below are for illustration only and should not be used to calculate results.

Typical standard curves fit to 4 parameter logistic equations are shown below. These curves must not be used to calculate methotrexate concentrations; each user must run a standard curve for each assay.

Units of Measure: Samples measured in the methotrexate ELISA can be expressed in terms of concentration by mass or molarity. Nanograms/ml units are converted to μM by dividing by the molecular weight (454.5 Da).