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What is the composition of a standard diluent buffer in a leptin ELISA kit?

What is the composition of a standard diluent buffer in a leptin ELISA kit?


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I am trying to find the composition of the diluent buffer used for the dilution of a human leptin stock to be used in an ELISA assay. The manufacturer of the kit (https://www.thermofisher.com/elisa/product/Leptin-Human-ELISA-Kit/KAC2281) only indicates that the buffer contains 8mM of sodium azide, but I cannot find the precise composition of the buffer anywhere on the internet.


Myeloma and hybridoma culture medium (serum-containing)

Hybridoma growth medium with hypoxanthine and thymidine (serum-containing)


What is the composition of a standard diluent buffer in a leptin ELISA kit? - Biology

Spiking & Recovery Results

Linearity Results

Application Notes

  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Stop Solution
  • Assay Diluent(s)
  • Lyophilized Standard
  • Biotinylated Detection Antibody
  • Streptavidin-Conjugated HRP
  • TMB One-Step Substrate
  • Distilled or deionized water
  • Precision pipettes to deliver 2 µl to 1 µl volumes
  • Adjustable 1-25 µl pipettes for reagent preparation
  • 100 µl and 1 liter graduated cylinders
  • Tubes to prepare standard and sample dilutions
  • Absorbent paper
  • Microplate reader capable of measuring absorbance at 450nm
  • Log-log graph paper or computer and software for ELISA data analysis
  1. Prepare all reagents, samples and standards as instructed in the manual.
  2. Add 100 µl of standard or sample to each well.
  3. Incubate 2.5 h at RT or O/N at 4°C.
  4. Add 100 µl of prepared biotin antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared Streptavidin solution to each well.
  7. Incubate 45 min at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

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ELISA Kit for Leptin (LEP)

  • Product No. SEA084Eq
  • Organism Species Equus caballus Equine (Horse) Same name, Different species.

Specificity

This assay has high sensitivity and excellent specificity for detection of Leptin (LEP).
No significant cross-reactivity or interference between Leptin (LEP) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Leptin (LEP) and the recovery rates were calculated by comparing the measured value to the expected amount of Leptin (LEP) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 88-105 95
EDTA plasma(n=5) 86-95 92
heparin plasma(n=5) 78-101 95

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Leptin (LEP) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Leptin (LEP) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV

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  • Single-component Reagents of Assay Kit
  • Lysis Buffer Specific for ELISA / CLIA
  • Quality Control of ELISA / CLIA
  • ELISA Kit Customized Service
  • Disease Model Customized Service
  • Serums Customized Service
  • TGFB1 Activation Reagent
  • Real Time PCR Experimental Service
  • Streptavidin

Blocking Buffers

Blocking is a critical step for ELISAs and there are a number of options available. Choosing the right blocking buffer increases sensitivity by reducing non-specific binding and background noise.

Block Ace (BUF029)

Superior to 1% BSA, Block Ace is a high-performance blocking buffer which can also be used as a wash buffer, and for diluting antibodies. In comparison to BSA, Block Ace provides reduced backgrounds and sharper standard curves.

BSA Block (BUF032)

Minimize non-specific binding in sandwich and antigen-down ELISAs using BSA Block. Based on BSA, this buffer has a proprietary protein stabilizer that creates a long-term stable environment for coating antigens or capture antibodies.

Ultrablock (BUF033)

A buffer containing proprietary non-mammalian proteins from fish designed to reduce non-specific binding. With extra blocking strength, it is suitable for use in antigen-down and sandwich ELISAs with high background and when working with human, bovine and porcine serum samples. Ultrablock can coat non-specific binding sites that are sterically inaccessible to traditional blockers.

Synblock (BUF034)

For maximum blocking strength use Synblock, a novel non-protein blocking formulation for antibody-coated and sandwich ELISAs. This synthetic, inert blocking agent provides maximum reduction of interference and eliminates non-specific background noise.


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We typically use a stabilizer for pre-coated plates. The additional washing step is designed to remove these components before you start the assay. If you do not perform the washing, the effect on assay performance is negligible.

What antibody clones are used in your ELISA Kits?

Information regarding the antibody clones used in our kits is proprietary. However, the clonality (polyclonal or monoclonal) and host species of the antibodies may be provided upon request.

Should I use serum or plasma samples for my ELISA experiment?

This is dependent on what targets you are analyzing and the specifics of your study. Ideally, we advise determining the difference between serum and plasma for the targets of interest and deciding on the sample type to be used for quantification. Depending on the targets, there may be a difference in concentrations of the targets between serum and plasma. The most important factor in preparing plasma or serum samples is consistency in preparation to ensure precise measurements. In general, plasma/serum samples should be free of particulate matters, contain no excess lipids, and have no hemolysis.

These types of contaminants will contribute to background, and adversely affect the precision of the assay. The key is to prepare the sample the same way each time. That is, centrifuging samples at the same speed, for the same time, removing the serum or plasma immediately after centrifugation and aliquoting and freezing the samples at the same time. Avoid repeated freezing and thawing cycles.

My sample may have very low levels of analyte. What methods can I use to improve detection?

For our LEGEND MAX&trade and ELISA MAX&trade Deluxe formats we recommend following the provided protocol. We cannot guarantee kit performance if there are deviations from the protocol. Below are general suggestions if you are using our ELISA MAX&trade Standard format:

  • Control background noise by increasing the number of washings and soaking time between washes.
  • Control assay precision. For example, be more careful and more consistent in your pipetting, use fresh paper towels for tapping plates to avoid contamination of avidin-HRP and increase washing volumes.
  • Increase incubation times. Be aware that this typically also increases background so the assay sensitivity may not necessarily increase.
  • Concentrate your sample, if possible.
  • Use a five-parameter logistic curve fitting method to more accurately calculate sample concentration at the lower end of the standard curve.

In many cases, the sample may simply contain very low to non-detectable levels of the analyte. No matter how you manipulate the assay, you may still not be able to obtain detectable concentrations.

We ran out of capture/detection antibody in our ELISA kit. Can we use a standalone/single antibody to replace it?

No, we do not recommend using standalone reagents to replace kit components and cannot guarantee the performance of the kit if you replace or combine reagents from different kits/manufacturers.

How many samples can I run with your kit?

This will depend on whether your samples are analyzed in duplicate or triplicate. For example, after adding the standards, you can run 80 samples with no replicates or 40 samples in duplicate and so on.

How should the plates be stored after coating?

If coated plates cannot be used immediately, they should be sealed and stored overnight at 4°C.

I have multiple LEGEND MAX™ ELISA kits that I want to run simultaneously. Can I use the same wash buffer for all the kits?

The wash buffer provided in all our LEGEND MAX&trade kits is the same and the part numbers on the wash buffer bottles in these kits should identical. For ELISA MAX&trade Deluxe and ELISA MAX&trade Standard Sets, we provide a recipe for the wash buffer on each kit&rsquos technical data sheet. This recipe is the same for all ELISA MAX&trade sets.

I am using the biotin and purified formats of the same antibody clone to try to set up my own ELISA, but I am having no success.

If you are using the same clone for both the detection and capture antibodies, the epitope may already be occupied by one of the antibodies and prevent binding of the other. We recommend choosing different clones for your capture and detection antibodies.

Where can I find troubleshooting tips for ELISA assays?

When should I use LEGEND MAX™ ELISA kits with pre-coated plates?

LEGEND MAX&trade Kits are ready-to-go, designed for ease-of-use, and minimize requirements for coating the ELISA plates and preparing buffer dilutions. We recommend LEGEND MAX&trade Kits for users who are new to ELISAs.

What kind of coating buffer should I use for my ELISA?

In general, we recommend using PBS (pH 7.4) or carbonate buffer (pH 9.5). The exact coating buffer you choose may be dependent on the cytokine being detected.

Why do we recommend not using azide in ELISA buffers?

Azide is an irreversible inhibitor of HRP.

What is the sensitivity of your ELISA kits?

If you are using LEGEND MAX&trade or ELISA MAX&trade Deluxe format, please consult the individual kit&rsquos manual for more information on sensitivity. We do not provide this information for the ELISA MAX&trade Standard Sets but in theory, it should match that of the ELISA MAX&trade Deluxe format if you use all of BioLegend&rsquos reagents.

What is the shelf-life of BioLegend ELISA products?

BioLegend's LEGEND MAX&trade Kits are guaranteed for 3 months from the date of receipt. ELISA MAX&trade Sets are guaranteed for 12 months from the date of receipt. For a lot-specific expiration date, refer to the box label on each product.

Can I use the recombinant standard provided with the kit for bioassay?

No, we do not recommend using the recombinant protein standard for any other applications. The protein standards included in the kit are calibrated for use as an ELISA standard and have been not tested for bioactivity. Additionally, ELISA protein standards may contain other carrier proteins and have not undergone the same sterility testing as our bioactive recombinant proteins.

Can I use tissue culture grade sterile plates for ELISA?

No. Tissue culture plates are designed for cell culture purposes and do not typically have high protein-binding capacity. For ELISAs, we recommend using high protein-binding plates such as Nunc Maxisorp&trade plates (Cat. No. 423501).

Can I use the capture and detection antibody provided as part of an ELISA Kit for other applications such as flow cytometry staining?

The antibodies used in our ELISA Kits have been validated for their use in ELISA assays and we cannot guarantee that they will work in other applications, like flow cytometry. Instead, we recommend you use flow cytometry validated antibodies for these experiments.

Can I use your ELISA kits for my tissue samples?

In general, BioLegend&rsquos ELISA products can be used for tissue or cell extracts/homogenates as long as the samples are prepared in such a way that they are compatible with immunoreactivity. For this, we recommend using the following guidelines:

  • Tissues/cells should be lysed or homogenized in a neutral pH buffer that contains no denaturing chemicals (such as urea, thiourea, SDS).
  • No or minimal levels of detergent (such as SDS, Triton&trade X-100).
  • No excessive ionic strength (salt concentration greater than physiological ionic strength).
  • The buffers should contain sufficient protease inhibitor cocktails to preserve the target proteins from proteolytic degradation by enzymes released from cells.

We do not recommend reusing samples. For accurate results, samples should be aliquoted and stored for one-time use only. If you decide to reuse a sample, you would need to perform additional validation experiments to ensure you are able to get accurate readings. This would be influenced by a number of factors including sample stability.

Can I use your matched ELISA antibody pairs with a protein standard from another company?

If the antibodies were provided as part of a kit, we do not recommend combining reagents from different ELISA Kits. We cannot guarantee the performance of the kit if you combine reagents from multiple kits or manufacturers. If you have purchased individual antibodies and are developing your own ELISA, it may be possible but would need to be tested. Antibodies may not recognize or show poor binding toward the protein standard if the immunogen used to generate those antibodies is different from the protein standard in terms of sequence or structure.

At what step can I delay my ELISA procedure for a few days? Can I freeze my plates for later use?

It is best to follow the recommended protocol without any additional delays. However, if you wish to pause the experiment, we would recommend delaying the procedure at the blocking step (after coating with the capture antibody). You can add 200 &muL of blocking buffer in the wells and keep the plates overnight at 4°C (minimal or no loss of signal) or frozen at -20°C for few days (it may have some impact on the signal). Delaying the procedure at the first step (coating with capture antibody beyond 16-20 hrs. at 4°C) is not advisable because it may lead to high background.

I ran out of some ELISA kit components, can I them buy separately?

It may be possible to purchase some kit components individually. Please contact our technical service group and provide lot information of the kit and its components to determine whether this is feasible.

Can I mix reagents from different ELISA MAX™ Sets or use them for other applications?

This is not recommended. The ELISA MAX&trade reagents are optimized for a particular lot, and they have not been quality tested for applications other than ELISA.

Can coating with capture antibody be carried out for longer than overnight?

Generally, coating for 16-20 hours at 4°C is recommended. Longer incubation time may increase the amount of capture antibody bound to the plates and this may increase the background noise.

Can I add an extra standard at the higher end of the standard curve in order to calculate higher concentration of the analyte?

We don&rsquot recommend this practice. Sensitivity of a kit depends on the individual components and their collective validation as a kit and it will not change by adding extra points to the standard curve.

Can I add an extra standard at the lower end of the standard curve to enhance the sensitivity of my assay?

While this may be possible, you may end up with a signal plateau at higher concentrations of the standard. It is generally recommended to use the concentration range recommended in the user manual.

Does phenol red in the medium interfere with an ELISA assay?

No, phenol red in cell culture media will not interfere with the readings from an ELISA assay.

For some of your ELISA kits, why do my serum samples require dilution with assay buffer?

In some cases, dilution with assay buffer is required to minimize the matrix difference between the samples and the standards to achieve better accuracy.

How can I obtain better signal and sensitivity for my ELISA assay?

&bull Increase incubation times (with samples, detection antibodies, or avidin-HRP or TMB substrate). Please note, this may also increase the background in your assay and may not increase sensitivity.
&bull Shake plates during incubation steps.
&bull Ensure that the standard is completely reconstituted before use.
&bull Increased washing and soaking in between washings to further decrease background.
&bull If possible, read the plate at 450-570 (or closest) nm for background subtraction.
&bull Improve duplicate CV% by controlling pipetting error or washing with larger volume of washing buffer.
&bull Use a 5-PL or 4-PL curve-fitting method, rather than a linear curve fitting. This is usually performed with a curve fitting software and provides better calculation at the lower end of the curve.

What is the difference between your ELISA Kits and Sets?

The primary differences between these kits are the reagents included within the kit or set.

LEGEND MAX&trade: Fully validated and ready-to-use kits. They contain all required reagents, including a 96-well strip plate pre-coated with capture antibody.

RAPID MAX&trade: cut assay time down to less than 90 minutes. Each kit is fully validated and contains a 96-well strip plate that has been pre-coated to immobilize the capture antibody.

ELISA MAX&trade Deluxe: A more cost-effective option that includes most buffers, pre-titrated antibodies, recombinant standard and substrate. Plates are not included in this kit and must be purchased separately.

ELISA MAX&trade Standard: Provides the basic components to complete an ELISA including a recombinant protein standard, capture and detection antibodies, and Avidin-HRP. This set requires some optimization and is perfect for those that want to stretch their budget by providing their own buffers and plates.

Do the ELISA MAX™ Deluxe Sets come with plates?

No, ELISA MAX&trade Deluxe Sets do not come with plates. Coating Buffer and Assay Diluent (for blocking and dilutions) are included in these sets. Plates can be ordered separately (Cat No. 423501), and instructions for coating the plates are in the sets&rsquo product manuals.

Can I use different components from different companies for my ELISA?

Different manufacturers often use different antibody clones in their kits. The specificity of the antibodies partially dictate how much signal is being detected.

Additionally, different kits may use recombinant protein standards that were expressed and purified using different methods. For example, recombinant proteins expressed in E. coli can show different immunoreactivities primarily due to refolding inconsistences. Kit standards are produced and calibrated against different references between manufacturers, making it difficult to compare components from different kits. Each BioLegend ELISA Kit was developed and validated with reagent concentrations and protocols optimized for analytical robustness. Any changes to the reagents (standards, antibodies, matching matrices) and protocols will affect the final assay performance.

Why are my sample’s cytokine concentrations different using LEGEND MAX™ kits vs other ELISA kits?

It is very difficult to obtain exactly the same sample concentrations at pg/ml levels when comparing different cytokine kits from different vendors, in part, because suppliers may use different reagents (including antibody clones) that have different immunoreactivity toward the target protein. It is not uncommon that the cytokine concentration may vary a few-fold between kits.

In general, particularly for those cytokines in pg/mL ranges, it is more important to have matching biological trends instead of matching absolutely concentrations. This is to say, the same cytokine measured by different kits from different vendors should show the same pattern of biological changes for relevant treatments.

What is the expected concentration of a particular analyte in biological samples (e.g. in serum samples of naÏve animals or normal humans)?

Since every sample is unique, it is difficult to predict as this may depend on the sample preparation and the nature of the analyte. If you are using a LEGEND MAX&trade Kit, please refer to the respective manual for more information.


What is the composition of a standard diluent buffer in a leptin ELISA kit? - Biology

The western blotting is a well-established technique used in molecular biology to detect specific proteins from a complex mixture of proteins extracted from tissue or cells. Synthetic or animal-derived antibodies are created that react with a specific target protein. The sample material undergoes protein denaturation, followed by gel electrophoresis. Next, the electrophoresis membrane is washed in a solution containing the specific antibody. The excess antibody is then washed off, and a secondary antibody that reacts with the first antibody is added. Through various methods such as staining, immunofluorescence, and radioactivity, the secondary antibody can then allow visualisation of the protein.

A. Sample preparation

1. Protein extraction

Improved on the basi c RIPA buffer , containing protease inhibitors and phosphatase inhibitors .

Classic formulation , i t is suitable for most immunological experiments of protein-protein interaction

The protein from the lysate is active, and retains the properties and functions , properly . It is suitable for co-IP. The protein cleavage under non-denaturation condition, the product retains the protein characteristics and functions to the maximum extent it is suitable for co-IP .

Note: Th e reagent of protein extraction containing detergent is not suitable for Bradford As s ay Kit (PC0010) . P lease select BCA Protein Assay Kit (PC0020) or Lowry Protein As s ay Kit (PC0030) .

2. Measurement of protein concentration

The protein content will be determined by comparing the absorbance of sample to the absorption coefficient determined from a standard curve. A standard curve is prepared by plotting the absorbance of samples containing known concentrations of the standard protein.

The choice of method of protein quantification is supposed to take into account the influence of factors such as protein structure (standard protein) and interfering substances in protein solution.

M easured at 595nm. Low interference factor s, t he determination is not disturbed by Tris , carbohydrate , glycerol , β-Mercaptoethanol , ammonia , EDTA , etc.

M easured at 562nm. The determination is not disturbed by detergent .

M easured at 650nm. This kit is s uitable for samples with high lipid content . I n certain range, it is not affected by detergent

B. WB Experiment Procedure

1. Gel P reparation

a. Prepare separation gel according to the protein molecular weight. Shake slowly to mix completely, avoid oxygen in when shaking hard. Pour the mixed gel in between two glass panes, inject anhydrous ethyl alcohol above it to avoid oxygen affect polymerization.

Concentration of separation gel ( % )

b. Prepare gel solution ( Choose either of the following two options )

Gel P reparation T able 1

4 × separating buffer ( PH8.8 )

30% Acrylamide/Bis Solution, 29:1

Gel P reparation T able 2

30% Acrylamide/Bis Solution, 29:1

For your convenience, our SDS-PAGE gel preparing kit ( P1200 ) and Tris-Tricine-SDS-PAGE gel preparing kit ( P1320 ) including all reagent for gel preparation .

To save you time, we provide Precast-Gel to skip the gel preparation step and run electrophoresis directly.

1) Our Precast-Gel is suitable for both denaturing gel electrophoresis and non denaturing polyacrylamide gel electrophoresis .

2) C ompare d to Tris-Gly Precast-Gel, the Tris-Hepes Precast-Gel can run more clear bands and higher resolution.

3) P lease prepare the electrophoretic buffer use the p rovide d power, do not suggest use the Tris-Gly buffer.

The loading buffer is used for both Native-PAGE (P1017, P 1027) and SDS-PAGE (P1015, P1016, P1017, P1018) which will fit all you needs .

W e also have Protein markers of different specifications and uses for your needs.

1) All of these three methods are suitable for gel staining.

2) Silver Staining method ( G7210 ) is more sensitive than Coomassie Brilliant Blue method(P1300 & P1305) , i t's suitable for samples with low protein concentration.

3) P1300 has shorter staining time than P1305

The transfer of proteins or nucleic acids to microporous membranes is referred to as " blotting " and this term encompasses both " spotting " (manual sample deposition) and transfer from planar gels. Proteins that are resolved on SDS-PAGE are typically transferred to adsorbent membrane supports under the influence of an electric current in a procedure that is known as western blotting (WB) or protein blotting.

The Western blot detection system includes HRP-conjugated secondary antibodies and chromogenic agent as well. Sola r bio pro vides reagents and kits for DAB and ECL.

P0012 Ponceau S Solution,10× can be used to detect the membrane transfer efficiency, especially for the NC membrane.

W e provide a Stripping Buffer ( SW3020 ) which can totally make membrane clean and be able to test another antibody in the same membrane.

Stripping Buffer can wipe off the antibody without affecting antigen in the membrane. In fact, membrane pattern, type and concentration of antibody, and properties of antigens matter to elution efficiency.

N ote: Suitable for Western detection of ECL and similar chemiluminescent reagents. Not suitable for Western detection with non-chemiluminescent reagents such as DAB, NBT/BCIP.


ELISA Kit for Aspartate Aminotransferase (AST)

  • Product No. SEB214Ra
  • Organism Species Rattus norvegicus (Rat) Same name, Different species.

Specificity

This assay has high sensitivity and excellent specificity for detection of Aspartate Aminotransferase (AST).
No significant cross-reactivity or interference between Aspartate Aminotransferase (AST) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Aspartate Aminotransferase (AST) and the recovery rates were calculated by comparing the measured value to the expected amount of Aspartate Aminotransferase (AST) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 98-105 101
EDTA plasma(n=5) 91-99 96
heparin plasma(n=5) 84-92 88

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Aspartate Aminotransferase (AST) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Aspartate Aminotransferase (AST) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV

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  • Single-component Reagents of Assay Kit
  • Lysis Buffer Specific for ELISA / CLIA
  • Quality Control of ELISA / CLIA
  • ELISA Kit Customized Service
  • Disease Model Customized Service
  • Serums Customized Service
  • TGFB1 Activation Reagent
  • Real Time PCR Experimental Service
  • Streptavidin

ELISA: Glossary of Terms

The ELISA Basics Guide has the right amount of detail to help you plan your experiment and achieve a successful ELISA.

Adsorption - the passive attachment of a liquid to a solid surface creating a thin film.

Antigen - substance, protein, chemical compound, or virus that is able to elicit an immune response against which antibodies are raised.

AP (Alkaline Phosphatase) - phosphatase enzyme that removes a phosphate group from a substrate. In ELISAs the substrate is p-NitroPhenyl Phosphate (pNPP).

Assay diluent (see also buffer) - buffer solution in which the sample to be analyzed is diluted in.

Assay sensitivity - a measure of the ability of the ELISA to distinguish between small changes in concentration.

Background - the signal readout attributable to all reagents excluding the analyte. Should be low.

Blocking - application of reagents, generally buffers, to lower background by binding to the potential non-specific binding sites of antibodies and enzyme conjugates.

Buffer - solutions containing compounds, generally proteins, to reduce the non-specific binding of antibodies used in blocking to reduce background.

Cross-reactivity - an antibody binding to a target that is very similar to but not the intended target analyte, i.e. a closely related molecule with structural similarities to the target antigen.

Detection limit - the smallest quantity of analyte that can be reliably measured by the ELISA assay it is often set to 2 standard deviations (2 SD) above background level.

Dilution - addition of a buffer to a protein solution to make it less concentrated, used in optimizing antibody concentration but also applied to samples to obtain readings within the dynamic range of the assay.

Dynamic range - range in the ELISA over which the absorbance reading increases in a linear mode and the analyte can be reliably measured.

Edge effect - the result of inconsistencies in the production of ELISA multiwell plates or when assay conditions, such as stacking plates, cause the outer wells to behave differently. As a result, unexpected values can appear in the outer wells which may be out of line with neighboring well. This can best be controlled for by using duplicates or triplicates for all samples, and noting any large variations in the results for a given sample.

Heterophilic interference - arises from antibodies found in the sample analyzed by the ELISA it is binding by these antibodies to the detection antibody used in the assay. The best known example of heterophilic interference is HAMA (human anti-mouse antibody) found in some patients where it interferes with the accurate analyte determination, showing false positive readings.

HRP (Horseradish Peroxidase) - an enzyme that breaks down hydrogen peroxide to water &ndash a peroxidase. Chromogenic substrates such as TMB serve as indicators of that enzyme activity.

Hook effect - caused by very high levels of antigen in the sample. As a result specific binding of the antigen by the antibody is insufficient to match analyte levels and signal is lower than expected. The best way to avoid this issue is to test several dilutions of each sample.

Immunoassay - any type of assay that uses antibodies to measure the concentration of an analyte in a sample.

Interference - effects on the immunoassay that interfere with the accurate measurement of the analyte (e.g. matrix effect, heterophilic interference).

Matrix effect - the effect compounds in the sample have on the measurement of the analyte.

pNPP (para-Nitrophenylphosphate) - colorimetric substrate for alkaline phosphatase, it precipitates as a yellow substance.

Protein stabilizers - reagents that promote maintenance of the native structure of proteins during adsorbtion to the assay surface.

Substrate - compound such as pNPP and TMB that is used to measure the analyte in an immunoassay.

TMB - a colorimetric substrate for horseradish peroxidase, turning blue upon completion of the enzymatic reaction.


ELISA Kit for 25-Hydroxyvitamin D3 (HVD3)

  • Product No. CEA915Ge
  • Organism Species Pan-species (General) Same name, Different species.

Specificity

This assay has high sensitivity and excellent specificity for detection of 25-Hydroxyvitamin D3 (HVD3).
No significant cross-reactivity or interference between 25-Hydroxyvitamin D3 (HVD3) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of 25-Hydroxyvitamin D3 (HVD3) and the recovery rates were calculated by comparing the measured value to the expected amount of 25-Hydroxyvitamin D3 (HVD3) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 99-105 102
EDTA plasma(n=5) 78-89 85
heparin plasma(n=5) 93-105 98

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level 25-Hydroxyvitamin D3 (HVD3) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level 25-Hydroxyvitamin D3 (HVD3) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV

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  • Single-component Reagents of Assay Kit
  • ELISA Kit Customized Service

SARS-CoV-2 Nucleoprotein ELISA Kit

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for SARS-CoV-2 Nucleoprotein has been pre-coated onto a microplate. Standards and samples are pipetted into the wells, and then a horseradish peroxidase-conjugated detection antibody specific for SARS-CoV-2 Nucleoprotein is added to the wells, producing an antibody-antigen-antibody "sandwich complex". Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of SARS-CoV-2 Nucleoprotein present in the sample. The reaction is terminated by the addition of acid and absorbance is measured at 450nm. A standard curve is prepared from six SARS-CoV-2 Nucleoprotein standard dilutions and SARS-CoV-2 Nucleoprotein sample concentration determined.

TARGET INFORMATION

Coronaviruses are enveloped viruses with a positive-sense RNA genome and with a nucleocapsid of helical symmetry. Coronavirus nucleoproteins localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. Coronavirus N protein is required for coronavirus RNA synthesis and has RNA chaperone activity that may be involved in template switch. Nucleocapsid protein is the most abundant protein of coronavirus. During virion assembly, N protein binds to viral RNA and leads to the formation of the helical nucleocapsid. Nucleocapsid protein is a highly immunogenic phosphoprotein also implicated in viral genome replication and in modulating cell signaling pathways. Because of the conservation of the N protein sequence and its strong immunogenicity, the N protein of coronavirus is chosen as a diagnostic tool.