Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3030 Procedure for Human LONG®R³IGF-I Double monoclonal ELISA (BK / MAN1 / MAO1)

ASSAY SHOULD BE VALIDATED FOR SPECIFIC PURPOSE IN THE USER LABORATORY

PRINCIPLE OF THE ASSAY

The assay employs a quantitative ‘sandwich’ enzyme linked immunoassay technique (ELISA) to measure LONG®R³IGF-I. A monoclonal antibody against LONG®R³IGF-I is coated into wells of an ELISA plate. Standards and samples are pipetted into the wells and LONG®R³IGF-I is bound by the immobilized antibody. A biotin-labeled monoclonal antibody against LONG®R³IGF-I is added to the wells to form a complex with the immobilized antibody and LONG®R³IGF-I. Avidin-labeled horseradish peroxidase is added to the wells followed by a chromogenic substrate for HRP. Color develops in proportion to the amount of LONG®R³IGF-I bound to the immobilized monoclonal antibody. The color development is stopped and color intensity is measured by absorbance on a microplate reader.
A calibration curve is prepared, plotting the absorbance versus the concentration of LONG®R³IGF-I. The concentration of LONG®R³IGF-I in the unknown samples is then determined by comparing the absorbance of the samples to the calibration curve.

LONG is a trade mark owned by Novozymes GroPep Limited

LONG®R³IGF-I is covered by the following patents assigned to Novozymes GroPep:
US patent 5,330,971; European patent 429,586; Japanese patent 2,682,738; Australian patent 633,099; Canadian patent 2,033,176;

REAGENTS SUPPLIED
Store reagents refrigerated at 2 - 8°C.

Coating Antibody (Product Code: MAN1)
Lyophilized mouse monoclonal anti LONG®R³IGF-I antibody. The vial contains sufficient reagent to coat 1 x 96 well microtiter plate.

Detection Antibody (Product Code: MAO1)
Biotin-labeled mouse monoclonal anti-LONG®R³IGF-I antibody. The vial contains sufficient reagent to detect samples in 1 x 96 well microtiter plate.

LONG®R³IGF-I Standard (Product Code: BK01)
Only use the LONG®R³IGF-I standard supplied with this kit.

REAGENTS AND EQUIPMENT REQUIRED (NOT SUPPLIED)

Results presented below were obtained using the reagents and equipment indicated. Each laboratory should obtain their own supplementary reagents and validate the assay in their laboratory

96 well ELISA plates (Greiner Microlon 600, Greiner Bio-One, Frickenhausen, Germany. Catalog Code: 705071).
www.greinerbioone.com

Streptavidin-labeled Horseradish peroxidase (Streptavidin-HRP - R&D Systems, Minneapolis, MN, USA. Catalog Code DY998).
www.rndsystems.com

Tetramethylbenzidine substrate solution (R&D Systems, Minneapolis, MN, USA. Catalog Code: DY999).
www.rndsystems.com


Coating Buffer, PBS - Phosphate-buffered saline, pH7.4
NaCl 80g
KH₂PO₄ 2g
Na₂HPO₄ 11.5g
KCl 2g
Add Milli-Q (or equivalent standard) water to 10 litres. pH should be 7.4 without adjustment.

PBS-Tween (for blocking, all dilutions, reagents and washing steps)
Phosphate buffered saline containing 0.5 ml Tween 20 per litre PBS. Store at 4°C for a maximum of 14 days.

Stop Solution
Sulphuric Acid (1 M)

Plate reader
Configured for ELISA plates.

Plate shaker
A shaking platform capable of holding ELISA plates and operating at approximately 500 oscillations per minute.

Pipettes
Adjustable pipettes able to deliver 10 µl to 1 ml.
5 and 10 ml disposable pipettes.
Multichannel pipettes to deliver 50, 100 and 250 µl.

Plate washer
Manifold dispenser, multi-channel pipette, squirt bottle or automated ELISA plate washer.

Water
Milli-Q or equivalent standard water is required.

Laboratory items
Tubes, racks, reagent reservoirs and other general laboratory items are required for preparation steps in the assay.

SAMPLE COLLECTION and STORAGE
The reagents have been developed as a support product for companies using LONG®R³IGF-I in serum-free media for cell culture. It is likely to be used to measure LONG®R³IGF-I in media and during downstream processing of media following a production cycle and is not intended for other use.
Normal precautions should be taken for sample collection and storage. Cell culture supernatants should be centrifuged and stored frozen at -20°C. Avoid freeze-thaw cycles.
Samples at extremes of pH should be buffered to neutral pH before adding to the assay wells.
Since LONG®R³IGF-I has a low affinity for insulin-like growth factor binding proteins, there is no requirement for an extraction step.

PREPARATION OF REAGENTS

Note: Assay plates should be coated the day before the assay is performed; therefore not all reagents are prepared at the same time.

ASSAY PROCEDURE

DAY 1

1. Reconstitute the vial of lyophilized Coating Antibody in 1 ml of PBS (not PBS-Tween). Add the entire contents of the vial to a further 10 ml of PBS (not PBS-Tween). Pipette 100 µl of the solution into each well of one 96 well ELISA plate. Cover plate with parafilm, place in ziplock plastic bag and incubate overnight at 4°C.

DAY 2

Efficient removal of the liquid at each step is essential to good performance.

1. Aspirate or decant contents of the 'coated' plate. Blot against paper toweling.
2. Fill all wells with 250 µl PBS-Tween. Incubate for 30 - 60 minutes at room temperature.
3. Prepare the standards as follows:
Reconstitute 1 vial of LONG®R³IGF-I ELISA standard with 1 ml 100 mM acetic acid (Solution A).

Solution B
Take 50 µl of Solution A and add 4.95 ml PBS-Tween. Mix thoroughly.


Solution C
Take 500 µl of Solution B and add 9.5 ml PBS-Tween. Mix thoroughly.

Follow the Table below to prepare the Assay Standards:

Note that standard solutions should not be stored. The assay must be performed on the same day the standards are prepared.

4. Samples of culture media or from chromatography steps in a downstream processing protocol may be tested undiluted or a dilution series may be prepared in PBS-Tween or in the appropriate culture media to bring samples into the assay range.
5. Aspirate or decant the contents of the 'blocked' plate. Blot against paper toweling and wash once with PBS-Tween. Blot plate dry.
6. Pipette 100 µl of standards and samples in at least duplicate (or other replicate set) into the wells. Cover and incubate plates at room temperature on a plate shaker for 60 minutes. Make sure that the position of each standard and sample is recorded accurately.
7. Reconstitute the vial of lyophilized Detection Antibody in 1 ml of PBS. Gently mix at room temperature to allow complete solution of freeze-dried product. Add the entire contents of the vial to a further 10 ml of PBS-Tween.
8. Aspirate or decant the contents of the plate and wash 3 times with PBS-Tween. Blot against paper toweling after each wash.
9. Pipette 100 µl of Detection Antibody solution into each well of the assay plate. Cover plate and incubate on a plate shaker for 60 minutes at room temperature.
10. Prepare the streptavidin-HRP conjugate by diluting it to the manufacturer's recommended working concentration in PBS-Tween. Prepare 11 ml for one 96 well plate.
11. Aspirate or decant the contents of the plate and wash 3 times with PBS-Tween. Blot against paper toweling after each wash.
12. Pipette 100 µl streptavidin-HRP conjugate solution into each well of the assay plate. Cover plate and incubate on a plate shaker for 30 minutes at room temperature.
13. Dilute the substrate solution as recommended by the manufacturer. Prepare 11 ml for one 96 well plate. This reagent should be prepared no more than 30 minutes before use and protected from light.
14. Aspirate or decant the contents of the plate and wash 3 times with PBS-Tween. Blot against paper toweling after each wash.
15. Pipette 100 µl substrate solution into each well. Cover plate and incubate on a plate shaker in the dark (e.g. under aluminum foil) for 20 minutes at room temperature.
16. Stop the enzyme reaction by adding 50 µl of 1M H₂SO₄ to each well.
17. Determine the absorbance of each well within 30 minutes of stopping the reaction, using a microplate reader set to 450 nm. If possible, use dual wavelength readings at 450 nm and 650 nm. Subtract the absorbance values obtained at 650 nm from those obtained at 450 nm.

CALCULATION OF RESULTS
For computer aided data analysis, an appropriate equation producing a dose-reponse fit (or similar) is recommended. Plot the concentration of LONG®R³IGF-I standard on the X axis and the absorbance units on the Y axis. Unknown values can be read from this curve and the LONG®R³IGF-I concentration in samples calculated by multiplying by the dilution factor.

TYPICAL DATA
Any variation in assay diluent, operator, pipetting and washing technique, incubation time and temperature can cause variation in the binding and affect the final results. The following example is data obtained using the ELISA plates, buffers, reagents, incubation times and temperatures described above.

Standard curves, assay parameters and quality control data should be established for each individual laboratory.

ASSAY CHARACTERISTICS

SPECIFICITY
The assay is intended for quantification of LONG®R³IGF-I. Cross reaction with human IGF-I is 32 %. Cross reaction with human IGF-II is less than 0.01 %.

INTERFERING SUBSTANCES
No interference was seen in culture media (DMEM) or DMEM containing 1% or 10% fetal calf serum. Recovery of LONG®R³IGF-I spiked into these media was 100 %.

Each laboratory should determine whether the matrix in which LONG®R³IGF-I is present interferes with this assay. It may be necessary to dilute standards and test samples in the same matrix.

TECHNICAL HINTS

• No difference was found using PBS containing bovine serum albumin for blocking or dilution of samples or reagents. PBS-Tween alone is an effective “blocking” solution.
• No 'soak' is required between washes.
• Substrate solution should remain colorless until added to the plate.
• Substrate solution incubated in the wells should change from colorless to gradations of blue. After stopping the reaction with acid, the substrate will turn yellow.
• Add the stop solution to the plate in the same order as the substrate solution was added.
• Avoid excessive foaming of solutions during mixing.
• To avoid cross-contamination, change pipette tips between addition of each level of standard, sample additions and reagents.
• Use unique reservoirs for each reagent.

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