Section11:Reagents

From Assay Guidance Wiki

Contents 1 Reagents 2 Example Plate Types 3 Coating Buffers 4 Blocking Buffers 5 Wash Buffers 6 Antibody Diluents Buffers 7 Matrix Diluent 8 Enzymes and Substrates 9 Stop Solutions 10 Absorbance Readout 11 Specific Antibodies 12 Standards or Antigen (Analyte) 13 Control Samples

Reagents

Reagents are a critical piece of any assay development process. This refers to all of the reagents that will be used in the assay. There are certain items that need to be considered when obtaining reagents:

1. Quality of standards and antibodies.
2. Quantity of standards and antibodies.
3. Purity of standards and antibodies (when possible antibodies are affinity purified).
4. Selectivity and specificity of antibodies.

Example Plate Types

1. Greiner high binding plates
2. Costar EIA/RIA high-low binding plates
3. Immunotech
4. Falcon

Note: Other plate types can also be used based the experience of the investigators and appropriate quality control to demonstrate acceptability.

Coating Buffers

1. 0.05 M sodium bicarbonate, pH 9.6
2. 0.2 M sodium bicarbonate, pH 9.4
3. PBS-50 mM Phosphate, pH 8.0, 0.15 M NaCl
4. Carbonate-bicarbonate
5. Phosphate Buffer: 1.7 mM NaH2PO4, 98 mM Na2HPO4.7H2O, 0.1% NaN3, pH 8.5
6. TBS - 50 mM TRIS, pH 8.0, 0.15 M NaCl

Blocking Buffers

1. 1% BSA or 10% host serum in TBS, or TBS with 0.05% Tween-20
2. Phosphate Buffer: 73 mM Sucrose, 1.7 mM NaH2PO4, 98 mM Na2HPO4.7H2Om 0.1% NaN3, pH 8.5
3. 1% HSA in PBS
4. Casein Buffer: Pierce Blocker cat # 37528
5. Pierce has many blocking buffers that are available in their catalog

Wash Buffers

1. PBS, 0.05% Tween-20, Chimeras
2. TBS, 0.05% Tween-20, Chimeras

Antibody Diluents Buffers

1. 1% BSA or 10% host serum in TBS, or TBS with 0.05% Tween-20
2. 1% BSA or 10% host serum in PBS, or PBS with 0.05% Tween-20
3. 50 mM HEPES, 0.1 M NaCl, 1%BSA, pH 7.4

Matrix Diluent

1. Serum from the host animal-mouse serum, human serum, etc
2. 0.1 M HEPES, 0.1 M NaCl, 1% BSA, 0.1 % Tween-20
3. Tissue culture medium for samples.
4. Cell lysates will contain SDS or other denaturing reagents that may interfere with the assay.

Enzymes and Substrates

1. HRP: horseradish peroxidase
2. TMB: 3,3', 5,5'-tetramethyl benzidine
3. OPD: o-phenylene diamine
4. ABTS: 2, 2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)

Stop Solutions

1. HRP/TMB: 2M H2SO4 solution (at a 1:1 volume with the HRP/TMB substrate/enzyme solution)
2. OPD: 3M H2SO4 solution, (at a 1:1 volume with the OPD substrate/enzyme solution)
3. ABTS: 1%SDS

Absorbance Readout

1. HRP TMB: 450 nm
2. OPD: 490 nm
3. ABTS: 405 nm

Specific Antibodies

1. Sandwich Immunoassay: matched pair of antibodies, one for analyte capture on a solid surface and one for detection that binds to the antigen/hapten/analyte. Antibodies need to be affinity purified for optimal results.
2. Competitive Immunoassay; a single antibody specific for the hapten/analyte. For optimal results affinity purified reagents are preferred.

Standards or Antigen (Analyte)

1. The analyte to be measured is typically a recombinant form of the natural analyte or peptide.
2. Enough standard should be obtained for use in the development phase, validation phase and the continued support of the method to avoid changing lots and or running out of standard.
3. Standard quality: Can vary from vendor to vendor and from lot to lot from a vendor.
4. Standard stability: Information on the stability of a standard can be obtained from the vendor and their recommendations should be followed in storing the standards.

Control Samples

1. Spiked controls are created by adding a known concentration of the standard analyte into the matrix (for example: tissue culture, serum, cell lysates). Spiked controls can be used to determine assay performance based on calculating the percent recovery.
2. Control samples are real samples where the antigen analyte level has been determined by another validated method. Samples are aliquoted, frozen and used as control samples in each experiment to track assay performance.