HUMAN ENDOCRINE LINCOplex

 By purchasing this product, which contains fluorescent-labeled microsphere beads authorized by Luminex Corporation ("Luminex"), you, the customer, acquire the right under Luminex’s patent rights, if any, to use this product or any portion of this product, including without limitation the microsphere beads contained herein, only with Luminex’s laser based fluorescent analytical test instrumentation marketed under the name of Luminex*.

 

*Luminex instrumentation refers to Luminex®100, Luminex 200 and other Luminex instruments from MiraiBio® (MasterPlex CT™), ACS® (STarStation™), Bio-Rad Laboratories, Inc.® (Bio-Plex™) and Qiagen® (LiquiChip™). For technical assistance on running LINCOplex Kits, contact our technical service department Toll Free U.S. at 866-441-8400 or 636-441-8400 or email us at [email protected]

 

I. INTENDED USE

This is a multiplex assay kit manufactured by Linco Research using methods for the simultaneous quantitative determination of the following human endocrine hormones in any combination: Leptin, GLP-1 (Active), C-Peptide, Insulin, Glucagon, Amylin (Active), and Amylin (Total). This kit may be used for analysis in human serum, plasma or tissue culture samples. This kit is for research purposes only.

II. REAGENTS SUPPLIED

A. Antibody-Immobilized Beads

The following beads are supplied as requested:

1 bottle containing #22-Human Leptin (Cat. #HE-LPTN)

1 bottle containing #53-GLP-1 (Active) (Cat. #HRE-GLP1)

1 bottle containing #73-Human C-Peptide (Cat. #HE-CP)

1 bottle containing #82-Human Insulin (Cat. #HE-INS)

1 bottle containing #34-Glucagon (Cat. #RME-GLU)

1 bottle containing #15-Amylin (Active) (Cat. #RME-AMLN)

1 bottle containing #55-Amylin (Total) (Cat. #HE-AMLN)

Mix beads (see Note) and dilute with HENDO-65K Bead Diluent as described in Section VIII. D.

Quantity: 0.2 ml antibody-immobilized beads/bottle

Note: Amylin (Total) Antibody-Immobilized Beads and Amylin (Active) Antibody-Immobilized Beads cannot be run together in the same assay.

B. Bead Diluent

1 vial containing diluent for bead preparation

Quantity: 3.5 ml/bottle

 

C. Human Endocrine Standard Cocktail

1 vial containing human endocrine standard cocktail, lyophilized

Quantity: 1 vial

D. Human Endocrine Controls

Control I – 1 vial containing mixed endocrine cocktail, lyophilized

Control II – 1 vial containing mixed endocrine cocktail, lyophilized

Quantity: 1 vial/Control

E. Serum Matrix , Lyophilized - Required for serum or plasma samples only.

Serum containing 0.08% Sodium Azide

Reconstitute with 1.0 mL of deionized water before use.

Quantity: 1 ml/vial

 

F. Human Endocrine Detection Antibodies

1 bottle containing a cocktail of all biotinylated detection antibodies in Assay Buffer

Quantity: 5.5 ml/bottle

G. Streptavidin-Phycoerythrin

1 bottle containing Streptavidin-Phycoerythrin prepared in Assay Buffer

Quantity: 5.5 ml/bottle

H. LINCOplex Assay Buffer

50 mM PBS with 0.08% Sodium Azide, 0.02% Tween 20, Protease Inhibitors and 1% BSA, pH 6.8

Quantity: 30 ml/bottle

I. LINCOplex 10X Wash Buffer

1:10 dilution required with deionized water to give 10 mM PBS with 0.05% Proclin, and 0.05% Tween 20, pH 7.4

Quantity: 30 ml/bottle

J. Mixing Bottle

Quantity: 1 Bottle

K. Microtiter Filter Plate

Quantity: 1- 96 Well Filtration Plate

L. Plate Sealers

Quantity: 2 Plate Sealers

III. STORAGE CONDITIONS UPON RECEIPT

Recommended storage for kit components is 2 - 8°C. See individual vials for long-term storage recommendations.

Once the standards, controls and serum matrix have been reconstituted, immediately transfer contents into polypropylene vials. DO NOT STORE RECONSTITUTED STANDARDS OR CONTROLS IN GLASS VIALS. For long-term storage, freeze reconstituted standards, controls, and serum matrix at £ -20°C. Avoid multiple (>2) freeze/thaw cycles.

DO NOT FREEZE Antibody-Immobilized Beads, Detection Antibody, and Streptavidin-Phycoerythrin.

IV. REAGENT PRECAUTIONS

Sodium Azide has been added to some reagents as a preservative. Although the concentrations are low, sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide build up.

V. MATERIALS REQUIRED BUT NOT PROVIDED

A. Reagents

Luminex Sheath Fluid (Luminex Catalogue #40-50000)

B. Instrumentation/Materials

1. Adjustable Pipettes with Tips capable of delivering 25 m l to 1000 m l

2. Multichannel Pipettes capable of delivering 25 m l to 200 m l

3. Reagent Reservoirs

4. Polypropylene Microfuge Tubes

5. Aluminum Foil

6. Absorbent Pads

7. Laboratory Vortex

8. Sonicator (Branson Ultrasonic Cleaner, Model # B200 or equivalent)

9. Titer Plate Shaker (Lab-Line Instruments, Model #4625, or equivalent)

10. Vacuum Filtration Unit (Millipore Vacuum Manifold Catalogue #MAVMO960R, or equivalent)

11. Luminex Instrument

VI. SPECIMEN COLLECTION AND STORAGE

A. A maximum of 25 m l per well of serum or plasma can be used. Tissue culture or other media may also be used.

B. Preparation of Tissue Culture Supernatant:

Centrifuge the samples to remove debris and assay immediately or aliquot and store samples at £ -20şC. Avoid multiple (>2) freeze/thaw cycles. Tissue Culture Supernatant may require a dilution with the appropriate medium prior to assay.

C. Preparation of Serum Samples:

For GLP-1 (Active) samples, after collecting blood samples, immediately add the appropriate amount of a DPPIV inhibitor according to the manufacturer’s instruction. Invert tube several times to mix (if using Linco Catalogue #DPP4, add 10 ul DPP4 per milliliter of blood).

For C-Peptide samples, after collecting blood samples, immediately add Trasylol (Aprotinin) at a concentration of 500 KIU per ml of serum to protect from proteolysis. Invert tube several times to mix.

For Amylin (Total and Active) samples, after collecting blood samples, immediately add the appropriate amount of a protease inhibitor according to the manufacturer’s instruction. Invert tube several times to mix.

Allow the blood to clot for at least 30 minutes before centrifugation for 10 minutes at 1000 xg. Remove serum and assay immediately or aliquot and store samples at £ -20şC. Avoid multiple (>2) freeze/thaw cycles. Use Serum Matrix as the diluent if certain samples require dilution prior to assay.

D. Preparation of Plasma Samples:

When collecting plasma, the use of EDTA as an anticoagulant is recommended.

For GLP-1 (Active) samples, after collecting blood samples, immediately add the appropriate amount of a DPPIV inhibitor according to manufacturer’s instruction. Invert tube several times to mix (if using Linco Catalogue #DPP4, add 10 ul DPP4 per milliliter of blood).

For C-Peptide samples, after collecting blood samples, immediately add Trasylol (Aprotinin) at a concentration of 500 KIU per ml of serum to protect from proteolysis. Invert tube several times to mix.

For Amylin (Total and Active) samples, after collecting blood samples, immediately add the appropriate amount of a protease inhibitor according to manufacturer’s instruction. Invert tube several times to mix.

Centrifuge for 10 minutes at 1000 xg within 30 minutes of blood collection. Remove plasma and assay immediately or aliquot and store samples at £ -20şC. Avoid multiple (>2) freeze/thaw cycles. It is recommended to centrifuge samples again prior to assay setup. Use Serum Matrix as the diluent if certain samples require dilution prior to assay.

E. Avoid using samples with gross hemolysis or lipemia.

F. Care must be taken when using heparin as an anticoagulant, since an excess will provide falsely high values. Use no more than 10 IU heparin per ml of blood collected.

VII. TECHNICAL GUIDELINES

To obtain reliable and reproducible results, the operator should carefully read this entire manual and fully understand all aspects of each assay step before attempting to run the assay. The following notes should be reviewed and understood before the assay is set-up.

A. The Antibody-Immobilized Beads are light sensitive and must be covered with aluminum foil at all times. Cover the assay plate containing beads with aluminum foil during all incubation steps.

B. It is critical to allow some of the reagents (Detection Antibody and Streptavidin-Phycoerythrin) to warm to room temperature (20-25° C) before use in the assay. (see Section IX).

C. The bottom of the Microtiter Filter Plate should not be in direct contact with any absorbent material during assay set-up or incubation times. The plate can be set on the non-flat side of the plate cover or any other plate holder to raise the plate from the surface.

D. After the wash steps, keep the bottom of the Microtiter Filter Plate clean by blotting on paper towels or absorbent pads to prevent any leakage due to capillary action.

E. Keep the vacuum suction on the plate as low as possible. It is recommended to have a vacuum setting that will remove 200 ml of buffer in > 5 seconds (equivalent to < 100 mmHg).

F. The standards prepared by serial dilution must be used within 1 hour of preparation. Discard any unused standards except the 10,000 pM stock standard which may be stored at £ -20° C for 1 month and at £ -80° C for greater than one month.

G. Mix only required amount of beads prior to assay setup. Discard any unused premixed beads.

H. During the preparation of the standard curve, make certain to vortex the higher concentration well before making the next dilution. Use a fresh tip with each dilution.

I. The plate should be read immediately after the assay is finished. If, however, the plate cannot be read immediately, do the final bead suspension in 100µl Sheath Fluid, seal the plate, cover with aluminum foil, and store at 2-8° C for up to 24 hours. Prior to reading, agitate the plate on the plate shaker at room temperature for 10 minutes. Delay in reading the plate may result in decreased signals and sensitivities for some analytes.

J.  The titer plate shaker should be set at a speed to provide maximum agitation without splashing of liquid outside the wells. For the recommended plate shaker, this would be a setting of 5-7, which is approximately 500-800 rpm in a 0.3 cm orbit.

K. Amylin (Total) Antibody-Immobilized Beads and Amylin (Active) Antibody-Immobilized Beads cannot be run together in the same assay.

L. Ensure the needle probe is clean. This may be achieved by sonication and/or Alcohol Flushes. Adjust the probe height to the Lincoplex filter plate prior to reading an assay.

M. For cell culture supernatants or tissue extraction, use the culture or extraction medium as matrix in blank, standard curve and controls. If samples are diluted in Assay Buffer, use the Assay Buffer as matrix.

VIII. PREPARATION OF REAGENTS FOR IMMUNOASSAY

A. Preparation of Human Endocrine Standard Cocktail

1). Before use, reconstitute the Human Endocrine Standard Cocktail with 250 ml Deionized Water to give a 10,000 pM concentration of standard. Invert the vial several times to mix. Vortex the vial for 10 seconds. Allow the vial to set for 5-10 minutes. Add 220 ml Assay Buffer into a polypropylene microfuge tube and take 180 ml standard (10,000 pM) to prepare the 4500 pM concentration of standard. This will be used as the 4500 pM standard; the unused portions may be stored at £ -20° for up to one month.

2). Preparation of Working Standards

Label six polypropylene microfuge tubes 1500, 500, 166.7, 55.6, 18.5 and 6.2 pM. Add 200 ml of Assay Buffer to each of the six tubes. Prepare serial dilutions by adding 100 ml of the 4500 pM reconstituted standard to the 1500 pM tube, mix well and transfer 100 ml of the 1500 pM standard to the 500 pM tube, mix well and transfer 100 ml of the 500 pM standard to the 166.7 pM tube, mix well and transfer 100 ml of the 166.7 pM standard to 55.6 pM tube, mix well and transfer 100 ml of the 55.6 pM pg/ml standard to the 18.5 pM tube and mix well and transfer 100 ml of the 18.5 pM standard to the 6.2 pM tube and mix well. The 0 pM standard (Background) will be Assay Buffer.