Human Melanoma gp100 ELISA Kit (A2341)

Name: Human Melanoma gp100 ELISA Kit
Brand: Antibodies.com
SKU: A2341
Price: 475.0 GBP
Availability: InStock

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Name

Human Melanoma gp100 ELISA Kit

Description

Human Melanoma gp100 ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of human Melanoma gp100 in tissue homogenates, cell lysates or other biological fluids.

Assay Type

Sandwich (quantitative)

Principle of Assay

Human Melanoma gp100 ELISA Kit (A2341) employs the sandwich enzyme immunoassay technique for the quantitative measurement of human Melanoma gp100 in tissue homogenates, cell lysates or other biological fluids. An antibody specific for Melanoma gp100 has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the Melanoma gp100 present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-Melanoma gp100 Antibody, which binds the captured Melanoma gp100 present in each well. HRP-Avidin conjugate is then added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is proportional to the amount of Melanoma gp100 captured in each well. The concentration of Melanoma gp100 can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.

Platform

Pre-coated Microplate (12 x 8 well strips)

Detection Type

Colourmetric

Instrument

Colorimetric Microplate Reader

Sample Type

Tissue homogenates, cell lysates or other biological fluids.

Sensitivity

0.061 ng/ml

Product Range

0.156-10 ng/ml

Assay Time

4h 30m

Reactivity

Human

Recovery

Sample Type	n	Range	Average
Serum	5	83% - 100%	91%
EDTA Plasma	5	91% - 102%	96%
Heparin Plasma	5	89% - 102%	95%

Linearity

Sample Type	1:2	1:4	1:8	1:16
Serum (n=5)	75-104%	90-98%	82-104%	84-99%
EDTA Plasma (n=5)	95-102%	83-110%	85-94%	89-105%
Heparin Plasma (n=5)	72-102%	91-94%	92-105%	82-104%

Precision

Intra-assay: CV < 10%, Inter-assay: CV < 12%

General Notes

Information online is representative. Always refer to the Product Manual inside the kit.

Storage

Store kit components at +4°C or -20°C as instructed. Do not use past expiration date!

Synonyms

D12S53E, ME20-M, ME20M, Melanocyte protein PMEL, Melanocyte protein Pmel 17, Melanocytes lineage-specific antigen GP100, Melanoma-associated ME20 antigen, P1, PMEL, PMEL17, SILV

Product Note

Information online is representative. Always refer to the Product Manual inside the kit.

Disclaimer

This product is for research use only. It is not intended for diagnostic or therapeutic use.

Kit Components

Item	Quantity	Storage
Pre-Coated 96 Well Microplate	12 x 8 Well Strips	-20°C
Lyopholized Standard	2 Vials	-20°C
Detection Solution A	120μl	-20°C
Detection Solution B	120µl	-20°C
Wash Buffer (30X)	20ml	+4°C
Sample Dilution Buffer	45ml	-20°C
TMB Substrate	9ml	+4°C
Stop Solution	6ml	+4°C
Plate Sealers	5 Adhesive Strips	-

Required Materials

The following materials are not included in the kit, but are required to run this assay:

Microplate reader capable of measuring absorbance at 450nm ± 10nm.
Squirt bottle, multichannel pipette reservoir, or automated microplate washer.
Graph paper or computer software capable of generating logarithmic functions.
Test tubes and Eppendorf tubes to prepare standards and samples.
Multi- and single-channel pipettes and sterile pipette tips.
Deionized or distilled water.
Absorbent paper towels.
37°C incubator.

Important Notes

Sample concentrations should be estimated before being used in the assay and a proper dilution factor should be selected to make the diluted target protein concentration fall in the optimal detection range of this kit.
Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.
Samples should be brought to room temperature before starting the assay.
This ELISA kit is intended for the analysis of biological samples and has not been validated for purified/recombinant proteins, industrial, or commercial materials. Performance with such samples cannot be guaranteed, as differences in expression, sequence, tags, folding, post-translational modifications, purity, activity, or buffer composition may affect assay results. For further guidance, please contact our technical support team.

Bring all reagents and samples to room temperature before use.

We recommend that all standards and samples be assayed in duplicate.

Step 1

Prepare all reagents, samples, and working standards as directed in the previous sections.

Step 2

Determine the number of microplate strips required for the assay and insert them into the plate frame. Unused strips should be stored in a foil pouch at -20°C.

Step 3

Add 100μl of standard solutions to the standard wells. Note: A plate layout is provided to record standards and samples assayed.

Step 4

Add 100μl of samples to the sample wells.

Step 5

Cover with an adhesive plate sealer provided and incubate at 37°C for 2 hours.

Step 6

Remove the plate sealer and aspirate the liquid from the plate.

Step 7

Add 100μl of working Detection Solution A to each well.

Step 8

Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

Step 9

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate three times with Wash Buffer. Soak wells with 300μl of Wash Buffer for 1-2 minutes each time. Note: Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Blot the plate onto paper towels or other absorbent material. Do not let the wells dry completely at any time!

Step 10

Add 100μl of working Detection Solution B to each well.

Step 11

Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

Step 12

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate five times with Wash Buffer. Soak wells with 300μl of Wash Buffer for 1-2 minutes each time.

Step 13

Add 90μl of TMB Substrate to each well. Cover with a new plate sealer and incubate at 37°C in the dark for 15-25 minutes (do not exceed 30 minutes). Note: This incubation time is for reference only, the optimal reaction time should be determined by the end-user and can be shortened or extended according to the actual color change. The reaction may be terminated when a gradient is apparent in the standard wells.

Step 14

Add 50μl of Stop Solution to each well. The color in the wells should change from blue to yellow immediately. Note: If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

Step 15

Determine the optical density (O.D. value) of each well immediately using a microplate reader set to 450 nm.

Scientific Validation DataValidation Data (1)

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Standard Curve - Human Melanoma gp100 ELISA Kit (A2341)

Representative standard curve for Human Melanoma gp100 ELISA Kit (A2341).

Product Citations (1)

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(2)

Serum gp100 as an Independent Prognostic Biomarker in Early-Stage Uveal Melanoma

This publication cites Human Melanoma gp100 ELISA Kit (A2341).

Article Abstract

Purpose: To evaluate the prognostic significance of serum gp100 levels in patients with uveal melanoma (UM) without detectable metastases at diagnosis.

Methods: This prospective study included 37 patients with UM and no clinical evidence of metastasis at baseline. Serum gp100 concentration was quantified using a commercial ELISA kit. Tumors were molecularly profiled and categorized into high- or low-risk classes. Patients were stratified based on the median gp100 concentration (1.23 ng/mL). Survival analysis was performed using Kaplan-Meier estimates, and multivariate logistic regression was used to identify independent predictors of metastasis.

Results: During a mean follow-up of 24.6 months, 14 patients (37.8%) developed metastases. Patients with baseline gp100 >1.23 ng/mL had significantly shorter metastasis-free survival (P < 0.001). Receiver operating characteristic (ROC) analysis yielded an area under the curve of 0.89, with a cutoff of 1.387 ng/mL, achieving 85.7% sensitivity and 82.6% specificity. In multivariate analysis, serum gp100 remained an independent predictor of metastasis (odds ratio = 3849.9; 95% confidence interval, 9.66-1,534,206; P = 0.007), regardless of molecular classification. No significant correlations were found between gp100 and clinical or genetic parameters.

Conclusions: Elevated serum gp100 is an independent biomarker of early metastatic risk in UM, even in the absence of clinical or genetic high-risk features. ELISA-based measurement of gp100 may support noninvasive stratification and personalized surveillance strategies in clinical practice.

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