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HOME > Protocols > Immunology > Antibody Labeling > Protocol for Chemiluminescence Labeling

Protocol for Chemiluminescence Labeling

1. INTRODUCTION

Assay Designs' Sub-Attomole Labeling Kit, combined with Turner BioSystems TD-20/20 Luminometer, gives rise to rapid attachment and analysis of chemiluminescent Acridinium Esters to lysine groups on most antibodies, proteins, nucleic acids and some peptides. As an alternative to 125I labels, the Acridinium Esters can be used to label antigens for competitive immunoassays, or signal antibodies for immunometric assays. In addition, chemiluminescent-labeled nucleic acids may be used as reporter molecules in DNA probe based assay systems. Labeling, purifying, and then detecting the labels on the TD-20/20 Luminometer can be achieved in less than 60 minutes.

2. MATERIALS REQUIRED

From Turner BioSystems:

  • TD-20/20 Luminometer with Dual Injectors (P/N 2020-050)
  • 12 mm Test Tube Holder and
  • 12 mm x 50 mm Polypropylene Test Tubes (P/N 2020-911)

From Assay Designs:

  • Sub-Attomole Labeling Kit (Cat. No. 90701) contains enough material to perform up to 5 labeling experiments equivalent to 1 mg of IgG each and includes the following materials:
    • Acridinium Ester with a N-hydroxysuccinimide Ester group, 100 mg
    • Dry Dimethyl Formamide, 0.5 ml (for dissolving the Acridinium Ester)
    • Bicarbonate Labeling Buffer(for diluting the antibody, protein or nucleic acid)
    • 10% Solution of Lysine (to stop the labeling reaction)
    • Gel Filtration Column (for separation of the labeled product from excess Acridinium Ester)
    • Column Buffer 10x Concentrate, 100 ml
    • Set of Trigger Solutions, 30 ml each (for detection of the labeled antibody, protein or nucleic acid)

NOTE: The Acridinium Ester supplied in this kit has an N-hydroxysuccinimidyl (NHS) ester-labeling group attached to a 2-carbon spacer arm. The NHS ester-labeling group will covalently attach to any primary amino group on the protein, peptide or nucleic acid to be labeled. At pH's above neutral, the NHS ester labeling group is subject to nucleophilic attack by the amine group on the protein, peptide or nucleic acid. As a result, the NHS group is displaced from the Acridinium Ester to form a stable amide bond between the Acridinium Ester and the protein, peptide or nucleic acid.

In addition, the sample to be labeled must be free of all aliphatic primary amines and free of all preservatives. Samples should not contain Tris based buffers, sodium azide, or other nucleophilic materials. Salt free lyophilized Preparations or concentrated solutions are ideal. These can be diluted in the Bicarbonate Labeling Buffer to achieve optimal pH for labeling.

3. INSTRUMENT SET-UP

3.1 Turn on TD-20/20 Luminometer and warm up for 5 minutes.

3.2 Place sample adaptor and holder into sample chamber.

3.3 Prime (at least 4 times) injector 1 with Trigger Solution 1 and injector 2 with Trigger Solution 2. Set injection volumes to 100 l on each injector. Refer to your operating manual for further details on priming injectors and setting injection volumes.

3.4 Set parameters of instrument: Delay=0, Integrate Time=4 seconds, Reps=1. Refer to your operating manual for further details on how to set parameters.

3.5
Set Mode to DLR by pressing [SETUP][2], and then Toggle to select DLR. Press [ESC] when done.

4. LABELING PROCEDURE

4.1 Allow the diluted Peptide, Protein or Nucleic Acid to come to room temperature. If the material to be labeled is lyophilized, add sufficient Labeling Buffer to make a 5 mg/ml solution.

4.2 Add the calculated volume of the Acridinium Ester solution in DMF to the peptide, protein or nucleic acid solution and vortex. Stir at room temperature for 30 minutes.

4.3 After 30 minutes, add 10 l of the 10% lysine solution to stop any further reaction and vortex. Stir at room temperature for 15 minutes.

4.4 Apply the labeled sample to the top of the washed, drained column. Let the sample enter the column bed. With a clean pipet tip, add
800 l of column buffer to the reaction tube and vortex to mix. Apply this to the column. With a clean pipet tip, follow with 1 ml aliquots of the column buffer. Collect 1 ml fractions. The column will stop draining automatically after each 1 ml aliquot is added. Collect 12-14 fractions.

5. ANALYZING SAMPLES

5.1 Pipet 1 l of each fraction into numbered duplicate 12 mm x 50 mm test tubes.

5.2 Insert test tube in the luminometer and press [GO]. The luminometer will inject Trigger Solution 1, take a reading, inject Trigger Solution 2, and then take another reading. It will also calculate the ratio between the two readings. You will only be concerned with the second reading, when both Trigger Solutions have been added to the sample.

5.3 Pool the first fraction(s) that contain light activity. The pooled sample(s) should be aliquoted and stored at -20C.

6. EXAMPLE OF TYPICAL RESULTS

Graph 1 below is an example of the Acridinium Ester labeling of mouse IgG. The graph shows the total Relative Light Units (RLU) obtained from the TD-20/20 Luminometer for 1 m l of each fraction. The total RLU includes any dilutions that were performed.

Graph 1

Graph 1: Acridinium Ester Labeling of mouse IgG using Turner BioSystems TD-20/20 Luminometer.


7. REFERENCE INFORMATION

7.1 The uses of Acridinium Ester labeled antibodies, antigens and DNA have been described in a number of publications. These references are available upon request from Assay Designs by phones at (734) 668-6113, by fax (734)668-2793, or by Email at TechServ@assaydesigns.com.

8. GENERAL USES

8.1 Streptavidin, progesterone, thyroxin and free thyroxin, alpha-fetoprotein, thyroid stimulating hormone, human chorionic gonadotropin, human complement component C9, staphylococcal peptidoglycan, carcinoembryonic antigen, Chlamydia trachomatis and Neisseria gonorrhea RNA have all been detected using this molecule or analogs. Invariably the assays developed using this material show greater sensitivity, speed of analysis, dynamic range and/or decreased sample volume.

9. SPECIAL THANKS

Turner BioSystems would like to express their appreciation to Assay Designs for their help in providing much of the information in this applications note.

Turner BioSystems would also like to express their appreciation to Teresa Yang of Sentinel Biosciences for providing the graph in Section 6.

10. ABOUT ASSAY DESIGNS, INC.

Orders for Assay Designs' products may be placed by:

Phone: (800) 833-8651 (U.S. or Canada)
(734) 668-6113 (local or international)
Fax: (734) 668-2793
E-mail: info@assaydesigns.com
Internet: http://www.assaydesigns.com

Mailing Address (HQ):
Assay Designs, Inc.
1327 Jones Drive
Ann Arbor, MI 48105

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