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Comparative data

Lightning-Link® RPE - flow cytometry data 

A mouse monoclonal antibody, clone RPA-T4, specific for human CD4 was purchased from a commercial source in both unconjugated and RPE conjugated formats. The unconjugated antibody was linked to RPE using a Lightning Link kit, and the two conjugates were compared in flow cytometry staining human peripheral blood lymphocytes as shown below. Lightning-Link® conjugated antibody is shown on the left hand side, and commercially conjugated antibody on the right hand side. The blue curve shows unstained cells.

flow cytometry    flow cytometry

A mouse monoclonal antibody, clone HCD54, specific for human CD54 was purchased from a commercial source in both unconjugated and RPE conjugated formats.  The unconjugated antibody was linked to RPE using a Lightning Link kit, and the two conjugates were compared in flow cytometry staining human peripheral blood lymphocytes as shown below. Lightning-Link® conjugated antibody is shown on the left hand side, and commercially conjugated antibody on the right hand side.  The blue curve shows unstained cells.

flow cytometryflow cytometry

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Lightning-Link® Fluorescein - flow cytometry data

A mouse monoclonal antibody, clone RPA-T4, specific for human CD4 was purchased from a commercial source in both unconjugated and FITC conjugated formats.  The unconjugated antibody was linked to fluorescein using a Lightning Link kit, and the two conjugates were compared in flow cytometry staining human peripheral blood lymphocytes as shown below. Lightning Link® conjugated antibody is shown on the left hand side, and commercially conjugated antibody on the right hand side.  The blue curve shows unstained cells.

flow cytometryflow cytometry

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Lightning-Link® RPE-Cy5 - flow cytometry data

A mouse monoclonal antibody, clone RPA-T4, specific for human CD4 was purchased from a commercial source in both unconjugated and RPE-Cy5 conjugated formats.  The unconjugated antibody was linked to RPE-Cy5 using a Lightning Link kit, and the two conjugates were compared in flow cytometry staining human peripheral blood lymphocytes as shown below. Lightning Link conjugated antibody is shown on the left hand side, and commercially conjugated antibody on the right hand side.  The blue curve shows unstained cells.

flow cytometryflow cytometry

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Lightning-Link® Alkaline phosphatase - ELISA data

A mouse monoclonal antibody specific for human IgG was purchased from a commercial source in both unconjugated and alkaline phosphatase conjugated formats.  The unconjugated antibody was linked to Alkaline Phosphatase using a Lightning Link kit, and the two conjugates were compared in ELISA as shown. The Lightning-Link® conjugated antibody demonstrates increased sensitivity and may be used at higher dilutions than the commercial conjugate.

alkaline phosphatase

Protein A was conjugated to alkaline phosphatase using Lightning-Link®, and compared to commercially available Protein A:Alk Phos available from two commercial suppliers. Species IgG was coated to an ELISA plate at 100ng/well, and Protein A:Alk Phos used at 50ng/ml as detection reagent. Note the significantly increased sensitivity demonstrated for detection of the various species IgG's in comparison to both commercial sources, being especially significant in detection of mouse.

alkaline phosphatase

 

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Lightning-Link® HRP - ELISA data

A mouse monoclonal antibody specific for human IgG was purchased from a commercial source in both unconjugated and HRP conjugated formats.  The unconjugated antibody was linked to HRP using a Lightning Link kit, and the two conjugates were compared in ELISA as shown.

The Lightning-Link® conjugated antibody demonstrates increased sensitivity and may be used at higher dilutions than the commercial conjugate.

horseradish peroxidase

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Scalability of Lightning-Link®

The simplicity of the Lightning-Link® approach means that it is very easy to scale up (or scale down) the amount of antibody to be labeled. Whether you are labeling small or bulk amounts of antibody, the hands on time remains the same – just 30 seconds. Because there are no new technical issues created by scaling up, the performance of trial conjugate prepared at small scale is essentially identical to that prepared at bulk scale (see Figure below). For any new antibody, or in situations where the antibody is extremely valuable and in limited supply, Lightning-Link kits allow labeling reactions to be carried out with as little as 10µg of antibody. Other regular pack sizes include 100ug, 1mg and 5mg. 

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Phosphate detection

Competitor assays are beset with several problems including reagent precipitation. At the heart of Innova Biosciences assay kit is PiColorLock™ Gold a non-radioactive, superior phosphate detection reagent which ensures high stability of the colored dye-phosphate complexes (green color- see below).

 

phosphate detection

 

PiColorLock™ Gold has also been designed to:

Prevent background signals arising from non-enzymatic (i.e. acid) hydrolysis of the substrate. The figure below shows ATP incubated in three detection reagents. A steadily rising background signal is seen with competitor reagents, whereas PiColorLock™ gives baseline readings.

phosphate detection

Have a large linear range, thus reducing the need for sample dilution.

phosphate detection

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InnovaCoat® GOLD - lateral flow data

A comparison of antibody conjugation using InnovaCoat® GOLD to that using traditional passive conjugation techniques with naked gold, showing both enhanced signal intensity and improved specificity. 40nm gold particles were labelled with anti-IgA antibody, and used to measure IgA concentration in a lateral flow inhibition assay, with IgA bound to a lateral flow strip.  

The data in the left hand graph shows the enhanced signal seen using InnovaCoat® in comparison to passive gold conjugation. Increasing amounts of IgA in the buffer matrix result in a reduction in signal that can be used to measure IgA concentration.  The enhanced curve seen with InnovaCoat® GOLD makes it easier to accurately measure the IgA concentration.

The right hand graph shows that InnovaCoat® GOLD also demonstrates improved specificity over the passive gold conjugation.  Even at high levels of soluble IgA the passive method still shows 50% of the maximum signal, whilst this is reduced to only 10% with InnovaCoat® GOLD.  This highlights the enhanced specificity and reduction of non-specific binding seen with InnovaCoat® GOLD.

gold conjugation

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LifeXtend HRP Conjugate Stabilizer - ELISA data

HRP conjugated goat anti-rabbit IgG (1mg/ml) was diluted 1/10,000 in each of four stabilizers and incubated for 12 days at 37°C. Am undiluted control sample was incubated at 4°C for the same period and diluted at 1/10,000 prior to ELISA testing. Conjugates were incubated on a rabbit IgG-coated plate for 1 hour at room temperature, followed by washing and determination of HRP activity using ABTS substrate.

fig1

As can be seen above, even some of the best known stabilisers perform either poorly or only modestly in standard accelerated stability trials at 37°C. LifeXtend HRP stabiliser/diluent offers the greatest protection and can substantially extend the shelf-life of HRP conjugates stored at 4°C or at room temperature.

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