The Methotrexate ELISA kit is a colorimetric competitive immunoassay kit with results in 1.5 hours. 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.
Contents of Kit
No.
Components
Size
Storage Conditions
1
Methotrexate Microtiter Plate
One Plate of 96 Wells
2-8°C
2
Methotrexate Antibody Solution
7mL
2-8°C
3
Enzyme Conjugated Secondary Antibody Solution
12 ml
2-8°C
4
Methotrexate Standard Solution 1ppm
1mL
2-8°C
5
Methotrexate Standard Solution 100ppb
1mL
2-8°C
6
Methotrexate Standard Solution 20ppb
1mL
2-8°C
7
Methotrexate Standard Solution 4ppb
1mL
2-8°C
8
Methotrexate Standard Solution 0.8ppb
1mL
2-8°C
9
Methotrexate Standard Solution 0.16ppb
1mL
2-8°C
10
Methotrexate Standard Solution 0 ppb
1mL
2-8°C
11
20× concentrated wash solution
50 ml
2-8°C
12
2× Sample Diluent
50mL
2-8°C
13
TMB Substrate
6ml × 2
2-8°C
14
Stop Solution
7ml
2-8°C
Storage
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.
Precision
Intra-plate coefficient of variation: <10% Inter-plate variation coefficient: <10%
Sensitivity
0.16 ppb
General Description
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.
Citations
Publication ()
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Background
Methotrexate is widely used in the treatment of various medical conditions, including autoimmune diseases and certain types of cancer. As an immunosuppressant and antimetabolite, methotrexate works by inhibiting the activity of an enzyme called dihydrofolate reductase. This enzyme plays a crucial role in the synthesis of DNA, RNA, and proteins. By interfering with these processes, methotrexate disrupts the rapid growth and division of cells, including immune cells and cancer cells. In the treatment of autoimmune diseases, such as rheumatoid arthritis, psoriasis, and systemic lupus erythematosus, methotrexate helps to reduce inflammation and suppress the overactive immune response. It can alleviate symptoms like joint pain, swelling, and skin lesions associated with these conditions. Methotrexate is also used as a chemotherapy agent in the treatment of certain types of cancer, such as leukemia, lymphoma, and solid tumors. In cancer treatment, methotrexate acts by inhibiting the growth and division of rapidly proliferating cancer cells, ultimately leading to their destruction.
Figure 1. Methotrexate mechanism of action. (Source: Arbel, Y. et al., 2018)
High therapeutic concentrations of methotrexate may cause hepatic and pulmonary abnormalities and myelosuppresion. In addition, nephrotoxicity may occur due to urinary deposition of MTX and its insoluble metabolites 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N10-methylpteroic acid (DAMPA). Therefore, therapeutic drug monitoring of methotrexate is needed to monitor its pharmacokinetics and reduce the risk of toxicity. Methotrexate ELISA Kit (DEIA-XYZ209) is specifically designed for the quantitative detection of methotrexate in biological samples, enabling efficient and convenient testing that allows laboratories to process multiple samples simultaneously and obtain reliable results.
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References
Risk factors associated with delayed methotrexate clearance and increased toxicity in pediatric patients with osteosarcoma
PEDIATRIC BLOOD & CANCER
Authors: Young, Elizabeth P.; Cheng, W. Susan; Bernhardt, Melanie B.; Wang, Lisa L.; Rainusso, Nino; Foster, Jennifer H.
High-dose methotrexate (HD-MTX; 12 g/m(2)) is part of standard therapy for pediatric osteosarcoma (OS). Risk factors associated with MTX toxicity in children with OS are not well defined. We investigated the association between peak MTX levels (four-hour) and delayed MTX clearance or treatment toxicity. Information was retrieved from electronic medical records of 33 OS patients treated with HD-MTX at Texas Children's Hospital from 2008 to 2015. We found that the four-hour MTX level did not contribute to toxicity or delayed MTX clearance. We demonstrated that certain demographic characteristics are associated with delayed clearance and increased toxicity.
Achieving sustained minimal disease activity with methotrexate in early interleukin 23-driven early psoriatic arthritis
RMD OPEN
Authors: den Braanker, Hannah; Wervers, Kim; Mus, Adriana M. C.; Bangoer, Priyanka S.; Davelaar, Nadine; Luime, Jolanda; Tchetverikov, Ilja; Hazes, J. M. W.; Vis, Marijn; Lubberts, Erik; Kok, Marc R.
Objectives Methotrexate (MTX) is currently the recommended first-line therapy for treating psoriatic arthritis (PsA), despite lacking clear evidence. No estimates of efficacy of MTX in usual care and no clear MTX responsive clinical or laboratory variables are currently available. This study describes the response to MTX monotherapy in newly diagnosed patients with PsA in usual care. Second, we compared clinical variables and cytokine profiles in patients responding and not responding to MTX monotherapy. Methods We used data collected in the Dutch southwest Early Psoriatic Arthritis cohoRt study to select patients with PsA with oligoarthritis or polyarthritis, and at least 1 year follow-up. We analysed disease activity at 6 months of patients who started MTX monotherapy and still used MTX monotherapy 1 year after diagnosis. Cytokine profiles were determined at baseline and after 3 and 6 months with a bead-based multi-immunoassay. Results We identified 219 patients of which 183 (84%) patients started MTX monotherapy within 6 months after diagnosis. 90 patients used MTX monotherapy throughout the first year of which 44 patients (24%) reached minimal disease activity(MDA) at 6 months, decreasing to 33 patients (18%) after 1 year. Non-responders had significantly higher concentrations of interleukin (IL) 23 and IL-10 before and during MTX therapy. Conclusions Our results showed that only 18% of patients with PsA are in sustained MDA after 1 year of MTX monotherapy and non-responders more often had IL-23driven disease. Our results indicate the need for more treatto-target and personalised therapy strategies in PsA.
Analytical methodologies for determination of methotrexate and its metabolites in pharmaceutical, biological and environmental samples
Methotrexate (MTX) is a folate antagonist drug used for several diseases, such as cancers, various malignancies, rheumatoid arthritis (RA) and inflammatory bowel disease. Due to its structural features, including the presence of two carboxylic acid groups and its low native fluorescence, there are some challenges to develop analytical methods for its determination. MTX is metabolized to 7-hydroxymethotrexate (7-OH-MTX), 2,4-diamino-N10-methylpteroic acid (DAMPA), and the active MTX polyglutamates (MTXPGs) in the liver, intestine, and red blood cells (RBCs), respectively. Additionally, the drug has a narrow therapeutic range; hence, its therapeutic drug monitoring (TDM) is necessary to regulate the pharmacokinetics of the drug and to decrease the risk of toxicity. Due to environmental toxicity of MTX; its sensitive, fast and low cost determination in workplace environments is of great interest. A large number of methodologies including high performance liquid chromatography equipped with UV–visible, fluorescence, or electrochemical detection, liquid chromatography-mass spectroscopy, capillary electrophoresis, UV–visible spectrophotometry, and electrochemical methods have been developed for the quantitation of MTX and its metabolites in pharmaceutical, biological, and environmental samples. This paper will attempt to review several published methodologies and the instrumental conditions, which have been applied to measure MTX and its metabolites within the last decade.
Application and pharmacological mechanism of methotrexate in rheumatoid arthritis
Methotrexate (MTX) has been used for the treatment of rheumatoid arthritis (RA) for about forty years and to date MTX remains the part of global standard of treatment for RA. The efficacy of MTX in RA is the result of multiple mechanisms of action. In order to summarize the possible pharmacological mechanisms of MTX in the treatment of RA, this review will elaborate on folate antagonism, promotion of adenosine accumulation, regulation of inflammatory signaling pathways, bone protection and maintenance of immune system function.
COA
Publication 
Figure 1. Methotrexate mechanism of action.
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