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Endotoxin (ET) ELISA Kit

SKU:
UNEB0040
Product Type:
ELISA Kit
Size:
96 Assays
Range:
0.01-1 EU/mL
ELISA Type:
Competitive
Reactivity:
Universal
$719
Frequently bought together:

Description

Endotoxin (ET) 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:

Endotoxin (ET) ELISA Kit

Product Code:

UNEB0040

Alias:

Endotoxin, ET

Reactivity:

General

Range:

0.01-1 EU/mL

Detection Method:

Competitive

Size:

96 Assays

Storage:

Please see kit components below for exact storage details

Note:

For Research Use Only

Kit Components

Component Quantity Storage

ELISA Microplate (Dismountable)

8x12 strips

-20°C

Lyophilized Standard

2

-20°C

Sample Diluent

20ml

-20°C

Assay Diluent A

10mL

-20°C

Assay Diluent B

10ml

-20°C

Detection Reagent A

60µL

-20°C

Detection Reagent B

120µL

-20°C

Wash Buffer (25X)

30ml

4°C

Substrate

10mL

4°C

Stop Solution

10ml

4°C

Plate Sealer

5

-

Other materials required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Protocol

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

Step Procedure

1.

Add 50µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible.

2.

Immediately add 50µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.

3.

Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette, manifold dispenser or automated washer are needed) and let it sit in the well for 1-2 minutes. Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.

4.

Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 45 minutes at 37°C.

5.

Repeat the wash process for five times as conducted in step 3.

6.

Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminate the reaction.

7.

Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.

8.

Determine the optical density (OD value) of each well at once, using amicro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.

9.

After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

Endotoxin Background

Endotoxins, specifically lipopolysaccharide (LPS) molecules, are an integral part of the outer membrane of gram-negative bacteria. They are composed of lipid A, which anchors the endotoxin to the bacterial cell wall, a core oligosaccharide, and an O antigen. These endotoxins are released when gram-negative bacteria undergo cell lysis, destruction, or death, and the subsequent breakdown of the bacterial cell wall.

Immune Response and Effects

When endotoxins are released into the host's bloodstream, they activate the immune system, particularly macrophages. Macrophages recognize endotoxins through pattern recognition receptors, such as Toll-like receptor 4 (TLR4), initiating a cascade of immune responses. This activation leads to the release of inflammatory mediators, including cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). These inflammatory mediators cause systemic effects such as fever, hypotension, increased vascular permeability, and activation of the coagulation system.

Significance in Bacterial Infections

Endotoxins play a crucial role in the pathogenesis of infections caused by gram-negative bacteria. The presence of endotoxins can contribute to the severity and progression of bacterial infections by amplifying the inflammatory response and triggering cascades that can lead to tissue damage. In severe cases, the systemic effects of endotoxins, including the release of cytokines, can result in septic shock, a life-threatening condition characterized by widespread inflammation and organ dysfunction.

Distinction from Exotoxins

Endotoxins are distinct from exotoxins in several ways. While endotoxins are components of the bacterial cell wall and released upon cell breakdown, exotoxins are actively secreted by bacteria. Exotoxins often have specific targets and mechanisms of action, allowing them to exert precise effects on host cells or tissues. In contrast, endotoxins trigger a more generalized immune response without the same level of specificity. Understanding this distinction is essential for studying bacterial pathogenesis and designing therapeutic approaches targeting these toxins.

Endotoxin FAQs

What is the Endotoxin ELISA Kit?

The Endotoxin ELISA Kit is a diagnostic tool used to measure the levels of endotoxins in biological samples.

What are the advantages of using the Endotoxin ELISA Kit?

The Endotoxin ELISA Kit offers several advantages for endotoxin detection. It provides a sensitive and quantitative measurement of endotoxin levels in various samples. It provides reliable and reproducible results, streamlining the experimental workflow with its comprehensive package of components.

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

For more detailed information about the Endotoxin 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.

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