Custom Sandwich ELISA Antibody Pairs Services

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The enzyme-linked immunosorbent assay (ELISA) is a technique to detect the antigen-antibody reaction by the use of enzyme-linked antibodies. ELISA is used mainly to detect and quantify peptides, proteins, or antibodies in a sample. In ELISA, peptide or protein antigens are immobilized on a solid support, either coated directly or through specific capture antibodies. ELISA is based on the use of labelled antibodies so that the resulting conjugates have both immunological and enzymatic activity. The antigen-antibody reaction can be easily measured when adding the reaction substrate.

Types of ELISA Methods:

• Direct ELISA:
  

ELISA is a highly sensitive technique and has high specifity. So it is possible to perform population studies in a short time easily and economically. Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. This technique has good reproducibility and the results are easy to interpret.

• Indirect ELISA:
  

It is the most commonly used method for antibody detection. The coating of the ELISA plate with the antigen against the specific antibodies is needed. The next steps involve the detection with enzyme-conjugated secondary antibodies that recognize the primary antibodies. In this way, the primary antibodies have their maximum immunoreactivity, while the secondary antibodies are capable of signal amplification.

• Sandwich ELISA:
  

This is the most frequent ELISA for antigen detection. The plates are usually coated with an antibody (monoclonal or polyclonal antibody) against the unknown antigen. The antigen to be detected is between a layer of capture antibodies and a layer of detection antibodies. The method requires that antigens have at least two antibody binding sites to prevent cross-reactivity or competition of binding sites.

• Competitive ELISA:

This technique is also very common for the detection of specific antibodies. The antigen of interest from crude or impure samples and immobilized known antigen compete for binding to the capture antibody.

Custom Sandwich ELISA Antibody Pairs Services

The Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) measures the amount of antigen between two layers of antibodies. It is a sensitive and robust method to measure the antigen concentration in an unknown sample. The antigen to be measured must contain at least two antigenic sites capable of binding to two antibodies: the capture and the detection antibodies.

General Procedure Advantages Antigen preparation Immunization Hybridoma development and screening Subcloning Monoclonal antibody production Antibody pair testing High specificity: Two antibodies are used the antigen/analyte is specifically captured and detected Flexibility and sensitivity: Both direct and indirect detection methods can be used Suitable for complex samples: The antigen does not require purification prior to measurement with high signal to background Ratio.

Antibody Pair for Sandwich ELISA Service Phase I  Prepayment Non-refundable startup fee Antigen Preparation Customer provide proteins. We need at least 10-15 mg recombinant proteins as antigens for immunization and ELISA testing. To guarantee the success, we prefer to have the proteins expressed and purified by LifeTein. Phase II Immunization Day 0: 50ul pre-immune sera and 1st-immunization with CFA Day 21: 2nd-immunization with IFA Day 35-45: 3rd-immunizaiton with IFA and final serum collected for ELISA test Fusion & Screening Day 50-54: Further immunization and positive sera collection Day 64-68: ELISA testing for cell culture supernatant and select positive clones Day 69-84: Provide 200ul culture supernatants to customer for testing and confirmation. We will keep 10-20 clones of ELISA positive clones and keep all the positive clones for up to 2 weeks. Phase III Subcloning Day 61-71: Subcloning and ELISA testing Day 72-90: Further subcloning and choose subclone with the highest ELISA titer for each positive hydridoma Hybridoma expansion and monoclonal production Day 91-98: Cell culture expansion Day92: Hybridoma injection for ascites Day 100-106: Ascites collection Day 107-114: Antibody purification by protein A/G and ELISA testing Antibody pair testing Day 115-122: Antibody labeling with HRP Day 123-137: Sandwich ELISA testing Phase IV Delivery  1. 2 clones of hybridoma cells 2. 1-2 pairs of antibodies: 5mg capture antibody, 1mg HRP labelled detection antibody . ELISA sandwich testing data Final Payment Net 30 days     Total Lead time (8-10 Months)

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