Description
LPS ELISA Kit(UNEB0072)
Lipopolysaccharides (LPS) are very large molecules that are composed of sugars and lipids. They are a major component of the outer membrane of Gram-negative bacteria and can trigger strong immune responses. LPS has been implicated in a variety of diseases, including sepsis, arthritis and atherosclerosis. LPS is often used as a biomarker for bacterial infection, with LPS ELISA Kits typically used in the research of immune responses. LPS ELISA kits can also be used to study pneumothroax (collapsed lung).
The Assay Genie LPS ELISA Kit allows for the quantitative determination of LPS in serum, blood, plasma, cell culture supernatant and other related supernatants and tissue samples.
| Pre-coated 96 well plate | |
| Kit includes all necessary reagents | |
| Reproducible and reliable results |
| Product Name: | LPS ELISA Kit |
| SKU: | UNEB0072 |
| Size: | 96 Assays |
| Reactivity: | General |
| Synonyms: | Lipopolysaccharide, LPS |
| Detection Method: | Sandwich |
| Range: | 7.8-500 pg/mL |
| Storage: | Please see kit components below for exact storage details |
| Note: | For Research Use Only |
|
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
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Protocol |
| 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. |
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. |
Lipopolysaccharides (LPS) are large, complex molecules primarily found in the outer membrane of gram-negative bacteria. These bacterial components play a crucial role in the integrity and functionality of the bacterial cell wall. In humans, they play a crucial role in the immune response and can have significant implications in disease pathology when present in excessive amounts.
Lipopolysaccharide (LPS) Structure
Lipopolysaccharides (LPS) are large molecules composed of three main regions: lipid A, core oligosaccharide, and O-specific polysaccharide. The lipid A region anchors LPS in the outer membrane of gram-negative bacteria and is responsible for its endotoxic properties. It consists of fatty acids and phosphate groups, and its structure can vary between bacterial species, influencing the potency of the endotoxin. The core oligosaccharide region connects lipid A to the O-specific polysaccharide. |
Lipopolysaccharide Function
The outer membrane, including the LPS component, serves several important purposes for gram-negative bacteria. It acts as a protective barrier, shielding the bacterium from harsh environments, including detergents, antibiotics, and host immune defenses. LPS also plays a role in adhesion to host cells, modulating immune responses, and promoting bacterial survival.
In hosts like humans, LPS functions as a potent activator of the immune system. It stimulates the release of various pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6), among others. LPS is recognized by immune cells, particularly macrophages, through pattern recognition receptors known as Toll-like receptors (TLRs), specifically TLR4. Upon binding to TLR4, LPS triggers a cascade of signaling events that promote the release of inflammatory mediators.
High LPS Levels in host - significance
LPS levels refer to the concentration or amount of lipopolysaccharides (LPS) present in a biological sample, such as blood, serum, or other body fluids. Measuring LPS levels can provide valuable information about the immune response, inflammatory conditions, and the presence of gram-negative bacterial infections.
In healthy individuals without ongoing infections or inflammatory disorders, LPS levels are typically very low or undetectable in the bloodstream. However, in the context of bacterial infections, sepsis, or certain chronic inflammatory conditions, elevated levels of LPS can be observed, indicating an abnormal immune response or the presence of gram-negative bacteria.
The measurement of LPS levels often involves specialized laboratory techniques, including the Limulus amebocyte lysate (LAL) assay or enzyme-linked immunosorbent assay (ELISA). These methods allow for the detection and quantification of LPS molecules in the sample.
It's important to note that diagnostic reference ranges for LPS can differ between laboratories and may require clinical correlation for accurate interpretation.
Q: What is the purpose of the LPS ELISA kit?
LPS ELISA is commonly used in research studies to investigate LPS levels in different biological samples, including serum, plasma, or cell culture supernatants. It can help in studying bacterial infections, sepsis, inflammation, immune responses, and gut permeability. The assay is also useful for evaluating the effects of drugs, therapies, or experimental interventions on LPS levels.
Q: What type of samples can be used with the LPS ELISA kit?
The Assay Genie LPS ELISA Kit allows for the quantitative determination of LPS in serum, blood, plasma, cell culture supernatant and other related supernatants and tissue samples.
Q: Can the LPS ELISA kit be used for diagnostic purposes?
While the LPS ELISA kit can provide valuable insights into LPS expression, it is primarily intended for research purposes. It can aid in evaluating the effects of drugs, therapies, or experimental interventions on LPS levels. For clinical diagnostic applications, regulatory-approved assays specifically designed for diagnostic use should be employed.
Q: Can the LPS ELISA kit cross-react with other molecules or contaminants?
The specificity of the LPS ELISA kit can vary depending on the antibodies and reagents used. Cross-reactivity with other molecules or contaminants may occur in some cases. Review the product information or contact the manufacturer for details regarding specificity and potential cross-reactivity.
Q: Where can I find additional technical support or assistance with the LPS ELISA kit?
For technical inquiries or assistance, reach out to our team.
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