Golimumab (Simponi®) (mAb-based)

The drug Golimumab (trade name Simponi®) is a human monoclonal antibody that binds to both the soluble and transmembrane bioactive forms of human TNF-α. This interaction prevents the binding of TNF-α to its receptors, thereby inhibiting the biological activity of TNF. Golimumab has been proven effective in the treatment of Rheumatoid Arthritis (RA), Ankylosing Spondylitis (AS), Psoriatic Arthritis (PsA) or Ulcerative Colitis (UC). Antibodies to Golimumab were detected in 57 (4%) of Golimumab-treated patients across the Phase 3 RA, PsA, and AS trials through Week 24. Similar rates were observed in each of the three indications. Patients who received Golimumab with concomitant Methotrexate (MTX) had a lower proportion of antibodies to Golimumab than patients who received Golimumab without MTX (approximately 2% versus 7%, respectively). Of the patients with a positive antibody response to Golimumab in the Phase 2 and 3 trials, most were determined to have neutralizing antibodies to Golimumab as measured by a cell-based functional assay. The data from the literature demonstrated that Anti-Drug Antibody positivity was significantly associated with low Golimumab levels and poor therapeutic response. This ImmunoGuide Golimumab ELISA (mAb-based) is developed for the specific measurement of Golimumab in sera, plasma and other biological fluids by the advantage of using a site-directed DWZ-1E4 mouse monoclonal antibody (mAb) specific for Golimumab only. Enzyme immunoassay (ELISA) for the determination of free Golimumab (Simponi®) (mAb-based) in serum and plasma samples. For research use only, not for use in diagnostic procedures.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01350
Species Human
Design Enzyme immunoassay (ELISA) technique.
Standards 5 standards, ready to use.
Controls None provided.
Sample Types Serum and plasma.
Sample Volume 10 µL
Assay Desc. 60 min. incubation (RT) + 30 min. (RT) + 10 min. (RT) = 1 hour, 40 min. total incubation time.
Standard Range 0 / 6 - 200 ng/mL
Sensitivity 5 ng/mL
Radioactivity
Other Names
Storage 2 - 8°C
Postscript For research use only, not for use in diagnostic procedures.

Intended use

This ELISA kit applies to the in vitro quantitative determination of Golimumab (Simponi®) (mAb-based) concentrations in serum, plasma and other biological fluids.

Test principle

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to Golimumab (Simponi®) (mAb-based). Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Golimumab (Simponi®) (mAb-based) and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Golimumab (Simponi®) (mAb-based), biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Golimumab (Simponi®) (mAb-based). You can calculate the concentration of Golimumab (Simponi®) (mAb-based) in the samples by comparing the OD of the samples to the standard curve.

Typical data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Precision

Intra-assay Precision (Precision within an assay):
3 samples with low, middle,high concentration of Golimumab (Simponi®) (mAb-based). And the sample of Golimumab (Simponi®) (mAb-based) were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Golimumab (Simponi®) (mAb-based). And the sample of Golimumab (Simponi®) (mAb-based) were tested on 3 different plates, 30 replicates in each plate.

Recovery

Matrices listed below were spiked with certain level of Golimumab (Simponi®) (mAb-based) and the recovery rates were calculated by comparing the measured value to the expected amount of Golimumab (Simponi®) (mAb-based) in samples.

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 89-103 98
EDTA plasma (n=5) 90-103 92
heparin plasma (n=5) 82-102 95

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Golimumab (Simponi®) (mAb-based) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

    Serum (n=5) EDTA plasma (n=5) heparin plasma (n=5)
1:2 Range (%) 87-101 92-107 88-108
Average (%) 93 95 94
1:4 Range (%) 89-106 93-108 93-102
Average (%) 95 94 93
1:8 Range (%) 90-103 93-100 93-101
Average (%) 93 94 93
1:16 Range (%) 88-105 87-100 93-102
Average (%) 95 94 94

Stability

The stability of Golimumab (Simponi®) (mAb-based) is determined by the loss rate of activity. The loss rate of Golimumab (Simponi®) (mAb-based) is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Item Specifications Storage
Micro ELISA Plate(Dismountable) 8 wells ×12 strips -20℃, 6 months
Reference Standard 2 vials
Concentrated Biotinylated Detection Ab (100×) 1 vial, 120 μL
Concentrated HRP Conjugate (100×) 1 vial, 120 μL -20℃(shading light), 6 months
Reference Standard & Sample Diluent 1 vial, 20 mL 4℃, 6 months
Biotinylated Detection Ab Diluent 1 vial, 14 mL
HRP Conjugate Diluent 1 vial, 14 mL
Concentrated Wash Buffer (25×) 1 vial, 30 mL
Substrate Reagent 1 vial, 10 mL 4℃(shading light)
Stop Solution 1 vial, 10 mL 4℃
Plate Sealer 5 pieces
Product Description 1 copy
Certificate of Analysis 1 copy

Other supplies required

Microplate reader with 450 nm wavelength filter
High-precision transfer pipette, EP tubes and disposable pipette tips
Incubator capable of maintaining 37℃
Deionized or distilled water
Absorbent paper
Loading slot for Wash Buffer

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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