Occludin_PA5-32524_Antibody_Immunohistochemistry

In addition to standard ELISA testing of custom monoclonal antibody cell lines, we offer a variety of additional screening and assay testing service options to validate monoclonal antibodies for use in specific applications.

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Screening options for monoclonal antibody development

Our standard monoclonal antibody development service includes ELISA testing to determine which mice (or rats) to use for fusion and ELISA screening of the resulting hybridomas to determine the best cell lines to deliver. Options for more specialized screening and testing are described here. The listed assay strategies demonstrate our broad capabilities to provide nearly any sort of screening and assay validation that customers require for antibody production and purification.

ELISA matched pair development

If a quantitative immunoassay is needed, we can develop suitable matched pairs for your ELISA assay. We utilize the expertise of our own internal ELISA development team which supports more than 50 standard cytokine and chemokine ELISA products. Projects can be initiated in conjunction with our custom hybridoma development services or antibodies can be supplied by customers for evaluation.
Supernatants from positive fusions are screened with the Biacore by injecting supernatant at a concentration of approximately 0.1 -0.25 mg/mL over an anti-mouse IgG Fc surface, followed by injection of the recombinant antigen.

Strong binding candidates are purified via Protein G or via 96-well melon gel depending on number of positive clones (see antibody purification kits and products).

Antibody testing

Plates are coated with purified antibody/s.
  1. At least 3 whole plates should be coated for each antibody to be tested.
  2. Plates are coated with 100 ul per well of a 10 ug/ml solution.

ELISA assay/s are run to determine best antibody pair.
  1. Each coating antibody is tested with each of the detection antibody candidates determined by the epitope mapping.
  2. A polyclonal ab for the antigen of interest (if available) can be used as a coating or detection option.
  3. A 5-7 point standard curve of the recombinant protein is typically used.
  4. A typical standard curve range is 5000 pg/ml - 6.8 pg/ml, plus a blank.
  5. Best pair/s is/are determined based on signal intensity, sensitivity, background and S/N.

ELISA assay/s is/are run to confirm activity with natural samples. Obtain natural samples from client.
  1. Antibodies that detect natural samples are reported to team for Screening Set and/or ELISA kit Development.
  2. If there is more than one clone available for ELISA, the "best" clones are saved for use in Screening sets and ELISA. Remaining clones, as well as clones used in kits can be evaluated for use as standalones.
  3. After we have confirmation that the antibodies detect natural samples, antibodies need to be scaled up for production.
  4. This information is reported on the Certificate of Analysis (CofA).
IHC and IF characterization and pathology services

The following antibody testing options are available for IHC/IF through our comprehensive antibody development platform. A final report that includes pathological scoring notes for specificity and intensity for tissues or fluorescent staining images and report for IF will be provided.

Qualification testing

Antibodies will be tested against researcher-supplied tissues or cell lines under standard tissue preparatory conditions for formulin-fixed, paraffin-embedded samples for tissues or with standard Thermo Scientific Cellomics Testing for IF.

Antibody testing

Confirm antibody reactivity from customer and expected expression patterns
  1. Expected tissue or cellular distribution
  2. Expression level intensity by qPCR or published protein expression reference
  3. Expected protein localization
  4. Protein ID (UniProt or Genbank¨ Databases)
  5. Protein orientation with the membrane (if applicable)

Obtain a minimum of two positive control and one negative control tissues / cell lines from customer
  1. Tissue in 10um or smaller sections or 2 vials of 10^6 cells.
  2. Inspected for penetration with formulin or grow cells to defined confluency
  3. Triplicate slides for each tissue or wells for each cell

Testing antibodies
  1. Antibodies will be tested at 5 working dilutions (1:100, 1:200, 1:500, 1:750 and 1:1,000) unless you specify other dilutions
  2. Antibodies will be screened using standard blocking conditions
  3. Pre-absorbing antigen may be supplied for control
  4. Appropriate secondary polymeric HRP will be used along with DAB staining for tissues and customer-selected fluorescent labels
  5. Appropriate hematoxylin and eosin (H&E) or organelle stains will be done with customer consultation for proper orientation

Data
  1. Final data report will be provided to customer showing scoring
  2. Images can be provided upon request for an additional fee
  3. Conditions for testing will be summarized with suggestions for any further testing optimization to be performed
Western blot characterization

The following western blotting testing options is available for any antibody developed. Standard conditions for gel gradient and testing samples will apply. Detection will be monitored by Pierce Supersignal chemiluminescence.

Qualification testing

Antibodies will be tested against researcher-supplied tissues or cell lines under standard preparatory conditions.

Simple western blot testing

Confirm antibody reactivity from customer and expected expression patterns by source and molecular weight.

  1. Expected tissue or cellular distribution, including overexpression construct and negative control if available (3 samples maximum)
  2. Electrophoresis by appropriate tris-glycine gradient gel to optimize for predicted or confirmed molecular weight.
  3. Antibodies will be tested at 3 working dilutions (1:100, 1:200, 1:500) unless otherwise directed.

Advanced western testing

Antibodies are tested in the same manner as described for "Simple Western blot testing" except samples are enriched for specific cellular locations prior to phoresis and up to 5 samples may be tested for each antibody. This is an important offering for customers who know their protein(s) of interest may be abundant at lower concentrations.
Confirm antibody reactivity from customer and expected expression patterns by source and molecular weight.

  1. Expected tissue or cellular distribution, including overexpression construct and negative control if available (5 samples maximum).
  2. Prepared subcellular fractions using appropriate subcellular fractionation kit.
  3. Electrophoresis by appropriate tris-glycine gradient gel to optimize for predicted or confirmed molecular weight.
  4. Antibodies will be tested at 3 working dilutions (1:100, 1:200, 1:500) unless otherwise directed.
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For Research Use Only. Not for use in diagnostic procedures.