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Create your own Alpha Assay


Overview


The information on these pages offers guidance for the design and development of any Alpha assay. Please note that there are two sections that link to individual pages containing detailed information: one for those who will be developing an immunoassay specifically, and the other for those who will be working with tagged reagents as one of their assay components. Further below, you will find a section that discusses how to conjugate your own reagent to a bead.

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Guides


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Immunoassays


Detailed information on immunoassay design and development using the Alpha technology. Many assays (analyte detection, antibody quantitation/characterization, post-translational modifications, kinase assays, protease assays, etc.) can be designed as immunoassays. Existing ELISAs can be converted to a no-wash AlphaLISA® assay. Assays can be used for screening or quantitation of an analyte (by running samples against a standard curve created with a recombinant analyte). There are two major formats: a sandwiching antibody assay and a competition (displacement) assay. Each format can be performed using directly-conjugated beads (one antibody directly conjugated to an AlphaLISA Acceptor bead), or via an indirect method of antibody-bead association (Figure 1).

Alphaformats1 
Figure 1. Sandwiching and competition assay formats in Alpha, and direct vs. indirect antibody association methods.

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Assays That Use Tagged Reagents


Detailed information on how to design and develop Alpha assays that use tagged reagents. The use of tagged proteins, peptides, and other reagents (6xHis, GST, c-myc, HA, digoxigenin, etc.) can be particularly useful when studying biomolecular interactions or enzymatic activity with recombinant or synthetic substrates (kinase assays, protease assays, helicase assays, etc.). A tagged reagent with an appropriate anti-tag bead or affinity bead can be used in place of a target-specific antibody in your assay design (Figure 2).

p53MDM2Fig 
Figure 2. Alpha assay that uses tagged reagents (His-p53, GST-MDM2) in a protein-protein interaction assay. The His-tagged p53 associates with a nickel chelate bead, and the GST-tagged protein associates with a glutathione-coated bead.

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Bead Conjugation


Detailed information, tips, and FAQs for bead conjugation. You can use our unconjugated beads to directly conjugate a peptide, protein, or antibody of interest directly to an Alpha Donor, AlphaScreen Acceptor, or AlphaLISA Acceptor bead. Our unconjugated beads can be used in a reductive amination reaction to link your reagent via free amines (lysines, N-termini) as shown in Figure 3.

conjugationchem 
Figure 3. Reductive amination conjugating reaction.

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Working Range of Binding Affinities


AlphaLISA assays have a very broad working range in terms of binding affinity, as demonstrated in Figure 4. This makes the assays particularly well-suited for antibody-based assays and for protein-protein interaction assays, where binding affinities are typically much lower.

AlphaaffinityFig3 
Figure 4. Binding affinities for various biomolecular interactions. Key: 1 = highest affinity biological interaction, 2 = biotin-streptavidin interaction, 3 = very high affinity antibody or receptor ligands, 4 = most antibodies of good quality, 5 = most protein-protein interactions, 6 = lectins.

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Available Choices of Donor and Acceptor Beads


Bead selection is an important aspect of assay design. Bead selection can impact assay performance, and in some cases, can mean the difference between a successful assay and an unworkable assay. An understanding of the various ways bead choice can affect your assay may be helpful when you are planning your experiments.

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Theoretical Bead Binding Capacities


Relative capacities of various beads (beads used at 20 μg/mL final concentration in assay).

Bead CoatingUsed to Bind/CaptureMolecule and Partner Bead Used to Determine Theoretical Binding CapacityTheoretical Binding Capacity1(Provided for Relative Comparison)
StreptavidinBiotinylated peptides, proteins, oligos, sugars, small molecules, etc.Biotinylated peptide30 nM
Strep-Tactin®Strep-Tag® II and One-STrEP tagged proteins/peptides
Biotinylated peptides, proteins, oligos, sugars, small molecules, etc.
One STrEP-azurin-6X His with nickel chelate beads (100 nM)
Strep-Tag II-6X His with nickel chelate beads (300 nM)
Biotinylated rabbit IgG with Protein A beads (10 nM)
Anti-GST antibodyGST-fusion proteins and peptidesBiotinylated GST with streptavidin bead3 nM
Anti-6X His antibodyHis-tagged proteins and peptides6X His-GST with glutathione bead100 nM
Anti-FLAG antibodyFLAG-tagged proteins and peptidesBiotinylated FLAG with streptavidin bead100 nM
Anti-maltose binding protein (MBP) antibodyMBP-tagged proteinsBiotinylated MBP with streptavidin bead1 nM
Anti-HA antibodyHemagluttinin-tagged proteins and peptidesBiotinylated-PEG-HA with streptavidin bead10 nM
Anti-c-myc antibodyc-myc-tagged proteins and peptidesBiotinylated c-myc with streptavidin bead100 nM
Anti-DIG antibodyDigoxigenin labeled proteins, peptides oligos, etc.Biotinylated digoxigenin with streptavidin bead1 nM
Anti-FITC antibodyFITC or fluorescein-labeled proteins, peptides, oligos, sugars, small molecules, etc.Biotinylated-ERE-FITC with streptavidin bead>1 nM
Anti-V5 antibodyV5-tagged proteins/targetsBiotin-Chromalink V5 (14 aa) with streptavidin bead~3 nM
Anti-GFP antibodyGFP-tagged (green fluorescent protein-tagged) proteins and peptidesBiotinylated GFP with streptavidin beads3 nM
Glutathione (GSH)GST-fusion proteins and peptides6X His-tagged GST with nickel chelate bead300 nM -1 μM
Nickel chelate (Ni2+)His-tagged proteins and peptides6X His-tagged GST with glutathione bead300 nM -1 μM
Protein AAntibodies2Biotinylated rabbit IgG with streptavidin bead3 nM (antibody3)
Protein GAntibodies4Biotinylated rabbit IgG with streptavidin bead1 nM (antibody3)
Protein LAntibodies5Biotinylated human IgG (kappa) with streptavidin bead1 nM (antibody3)
Anti-human IgGFc portion of human IgG antibodiesBiotinylated human IgG with streptavidin bead3 nM (antibody3)
Anti-rabbit IgGFc portion of rabbit IgG antibodiesBiotinylated rabbit IgG with streptavidin bead1 nM (antibody3)
Anti-mouse IgGFc portion of mouse IgG antibodiesBiotinylated mouse IgG with streptavidin bead3 nM (antibody3)
Anti-mouse IgMMouse IgM immunoglobulinsBiotin-mouse IgM with streptavidin bead0.3 nM (antibody3)
Anti-rat IgGFc portion of rat IgG antibodiesBiotinylated rat IgG with streptavidin bead1 nM (antibody3)
Anti-sheep IgGFc portion of sheep IgG antibodiesBiotinylated sheep IgG with streptavidin beads1 nM (antibody3)
Anti-goat IgGFc portion of goat IgG antibodiesBiotinylated goat IgG with streptavidin bead3 nM (antibody3)
Anti-chicken IgYFc fragment of chicken IgY immunoglobulinsBiotinylated chicken IgY with streptavidin beads0.3 nM (antibody3)

1The numbers provided are examples given to compare relative bead capacities, and are derived from probe titration experiments, as shown in the QC data on the product tech data sheets. The actual bead capacity for your bead system should be determined empirically for a given assay. The bead capacities given in this table are influenced by partner bead and the size of the probe used in these assays. The actual bead capacity will be influenced by size of the protein associating with the bead, as well as affinity of the protein (or antibody) for the bead.

2Protein A interacts strongly with particular subclasses of antibodies, including human IgG1, IgG2, and IgG4; mouse IgG2A and IgG2B; and rabbit, human and mouse total IgG. Protein A also has weaker affinity for other antibody subclasses. The affinity depends on the isotype and species of the antibody associating with the bead. Pierce has a nice chart that may be helpful.

3The bead capacity for these products refers to the capacity for antibody. The amount of protein you will then be able to bind to the antibody on the bead will be dependent on the affinity of your antibody for your protein and the size of your protein, and may range anywhere from 1 pM to 1 μM.

4Protein G binds to all subclasses of human IgG and mouse IgG. In addition it binds to rat, goat, sheep, guinea pig, rabbit, cow, pig and horse antibodies. Pierce has a nice chart that may be helpful.

5Protein L binds efficiently total human IgG, IgM, IgA, IgE, IgD, mouse IgG and rat IgG. It binds only poorly mouse IgM and rabbit IgM and it does not bind human IgG (lambda light chain), rabbit, sheep, goat and bovine IgG and rat IgM. Pierce has a nice chart that may be helpful.

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Buffer Selection


Visit Buffer selection for Alpha assays for help in choosing a suitable buffer for your assay. This page describes the various Alpha immunoassay buffers supplied by PerkinElmer and will help you avoid interfering substances in buffers of your own design.

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Working With Serum Samples


When analyzing samples in serum, it is best to prepare the standard curve in a sample matrix as similar as possible to the actual samples. This will help ensure that the assay accuracy is as high as possible.

Analyte-Depleted Serum Standard Curve

For serum samples, the best option is to prepare the standard curve in analyte-depleted serum.

FBS Standard Curve

When analyte-depleted serum is not conveniently available, the analyte standard curve can be prepared in 100% FBS (fetal bovine serum). The same lot of FBS should be used for all asays in a given study. To ensure high accuracy (percent recovery), it may be necessary to dilute the serum sample somewhat (perhaps 2- or 4-fold) in FBS.

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Custom Bead Conjugation and Custom Assay Development at PerkinElmer


PerkinElmer offers custom bead conjugation services as well as custom assay development. If you are interested in having your biomolecule custom-conjugated to a bead, or in custom assay development, please contact our custom teams:

Custom Bead Conjugation Services

Custom Assay Development Services

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