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HCY (Homocysteine) ELISA Kit

SKU:
UNFI0065
Product Type:
ELISA Kit
Size:
96 Assays
Sensitivity:
4.688pmol/ml
Range:
7.813-500pmol/ml
ELISA Type:
Competitive
Synonyms:
Homocysteine
Reactivity:
Universal
€599
Frequently bought together:

Description

HCY (Homocysteine) ELISA Kit

system_update_alt Datasheet system_update_alt MSDS

Key Features

Save Time Pre-coated 96 well plate
Quick Start Kit includes all necessary reagents
Publication Ready Reproducible and reliable results

Overview

Product Name:

HCY (Homocysteine) ELISA Kit

Product Code:

UNFI0065

Size:

96 Assays

Target:

HCY

Alias:

Homocysteine

Reactivity:

Universal

Detection Method:

Competitive ELISA, Coated with Antibody

Sensitivity:

4.688pmol/ml

Range:

7.813-500pmol/ml

Storage:

4°C for 6 months

Note:

For Research Use Only

Additional Information

Recovery:

Matrices listed below were spiked with certain level of HCY and the recovery rates were calculated by comparing the measured value to the expected amount of HCY in samples. Please contact us for more information.

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of HCY and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Please get in contact for more information.

Intra-Assay

CV <8%

Inter-Assay:

CV <10%

Protocol

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step Procedure

1.

Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!

2.

Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).

3.

Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.

4.

HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.

5.

Wash: Repeat the aspiration/wash process for five times.

6.

TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.

7.

Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.

8.

OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testing parameters.

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

HCY Background

Homocysteine (HCY) is a non-protein amino acid that is produced during the metabolism of methionine, an essential amino acid obtained from the diet. It is a byproduct of various biochemical reactions in the body. Homocysteine can exist in two forms: free homocysteine and bound homocysteine. Free homocysteine is the form that circulates in the bloodstream and can be measured to assess the levels of HCY.

Homocysteine Function

Homocysteine has been associated with various functions in the body. It plays a role in methylation reactions by serving as a methyl group donor for important biochemical processes, including DNA and RNA synthesis, protein synthesis, and neurotransmitter metabolism. Additionally, homocysteine is involved in the synthesis of cysteine, an amino acid essential for protein formation and the production of antioxidants like glutathione. It has also been implicated in antioxidant defense, acting as a free radical scavenger to protect cells from oxidative damage. However, further research is needed to fully understand the precise functions and significance of homocysteine in the body.

Factors Affecting Homocysteine Levels

Various factors can influence homocysteine levels in the body. Nutritional factors play a significant role, as deficiencies in certain vitamins, such as folate (vitamin B9), vitamin B12, and vitamin B6, can lead to elevated homocysteine levels. Genetic factors are another important consideration.

Hyperhomocysteinemia

Hyperhomocysteinemia can be caused by two main factors. Firstly, nutritional deficiencies can result in elevated homocysteine levels. Inadequate intake or impaired absorption of vitamins involved in homocysteine metabolism, such as folate (vitamin B9), vitamin B12, and vitamin B6, can contribute to this condition. Secondly, genetic factors can play a role. Genetic mutations, such as the MTHFR gene mutation, can interfere with the enzymes responsible for homocysteine metabolism, leading to increased homocysteine levels.

HCY FAQs

What is the HCY ELISA Kit?

The HCY ELISA Kit is a specialized assay kit used for quantitatively measuring HCY levels in biological samples.

What are the advantages of using the HCY ELISA Kit?

The HCY ELISA Kit provides precise and quantitative measurements of HCY levels, allowing accurate assessment and monitoring. It provides reliable and reproducible results, streamlining the experimental workflow with its comprehensive package of components.

Where can I find more information about the HCY ELISA Kit?

For more detailed information about the HCY ELISA Kit, including technical specifications, performance characteristics, and ordering details, please refer to the product brochure or contact our customer support team. We are here to assist you with any inquiries you may have.