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Frequently Asked Questions

Customer Service

Online:       www.immunochemistry.com/products
E-mail:       orders@immunochemistry.com 
Phone:       952-888-8788
Toll-Free:    800-829-3194
Fax:           952-888-8988

Online Ordering:

ICT is pleased to offer secure ordering on our website. You may create an account or order as a guest.  To order, select the size and quantity of your desired products, click the green "Add to Cart" button, and proceed with the checkout process.  We will confirm your order within 24 business hours.

Payment Methods:

Online: Domestic customers with approved accounts may order with a valid institutional purchase order or a credit card. Customers located outside of the U.S. may order online with a credit card.

Offline: ICT accepts orders placed with a credit card or a valid institutional purchase order (with an approved account). Acceptable payment methods after processing a purchase order are: check, EFT, credit card, or wire transfer. Foreign payment processing fees may apply.

International Sales:

To order from a country outside the U.S., please contact one of our distributors.  Please note that the prices on www.immunochemistry.com/products are U.S. prices only and do not include applicable shipping charges, duties, taxes, or handling fees.

To order directly from ImmunoChemistry Technologies, utilize the following methods:

Online: Orders for certain destinations may be placed and prepaid online with a credit card. Create an account or place your order as a guest. Please note: online ordering is not enabled for all countries.

Email: Send your order to orders@immunochemistry.com or fax 952-888-8988. An invoice will be generated for pre-payment via wire transfer or check. Foreign payment processing fees may apply.

Fax: Send your order form to 952-888-8988. An invoice will be generated for pre-payment via wire transfer or check. Foreign payment processing fees may apply.

Note on Shipping:

Please note that our e-commerce site only offers standard or priority overnight service.  If you are ordering a product that is shipped at room temperature (see product specs) and prefer 2-day or 3-day shipping for your order, please place your order offline to receive this option and appropriate pricing.  Online credit card orders are securely processed immediately for the approved amount, including the approved overnight shipping charges.

ICT accepts valid institutional purchase orders from approved US-based customers with payment due Net 30.  If you are a new customer, please contact us with your company and contact information (New Customer Account Form).  We are typically able to enter customers in our system and accept new purchase orders within 1 business day.  ICT also accepts orders prepaid with a credit card.

Acceptable payment methods for approved customers using a purchase order are: check; EFT; credit card; or wire transfer. Foreign payment processing fees may apply.

INTERNATIONAL CUSTOMERS: Orders may be placed and prepaid online with a credit card. If a credit card payment method is not available, please send your order to orders@immunochemistry.com or fax 952-888-8988. An invoice will be generated for pre-payment via wire transfer or check. Foreign payment processing fees may apply.

Product orders are shipped as soon as possible after verification of product inventory and payment terms. International sales require pre-payment.

Most product orders received by 1pm US CT are shipped on the same day they are received. US and Canadian orders are shipped Monday-Thursday via overnight delivery on ice. International orders, once confirmed and pre-paid, will take 2-10 days for delivery, depending on the country and type of product.

Online orders receive automatic email confirmations of an order's shipment. Shipment confirmations may also be requested for orders placed by phone, fax, or email; simply provide the email address to receive the notification.

Note on shipping:
Please note that our e-commerce site only offers domestic standard overnight or priority overnight service.  If you are ordering a product that is shipped at room temperature and prefer 2-day or 3-day domestic shipping for your order, please place your order offline to receive this option and appropriate pricing.  Online credit card orders are securely processed immediately for the approved amount, and whereas it is possible to issue a differential refund, this process will take 3-6 business days.

October 2013 Facebook Promotion

Sweepstakes instructions:

  1. Go to our New Products webpage:
    www.immunochemistry.com /products/new-products.html 
  2. Take and crop a screenshot of the new product that most interests you. 
    E.g., the image displayed next to the product name, fluorescent cell image, picture of bottle or kit, etc. Hint - images with dimensions greater than 200 px display best.
  3. Follow ICT on Facebook:
    www.facebook.com/ ImmunoChemistryTechnologies
  4. Post the image with a description* on ICT's Facebook Page using your Facebook account.
    *Indicate in the image description the name of the product and why this product interests you. You could also add how you would like to use the product credit if you win!
  5. Check that the image and your description are properly displayed; to be eligible, it should be obvious that it is a screenshot from the New Products webpage.
  6. Follow us! facebook.com/ImmunoChemistryTechnologiesVisit our Page on Friday 10/25 and Friday 11/1 for the winners! Two (2) winners will be selected each Friday through November 1, 2013: 10/25/13 and 11/01/13. If you win, reply when we contact you so you can receive your product credit! 

 

Terms:

Example screenshot of Data from FLICA 660 Caspase-3 Assay 

1. Eligibility requirements:

  • Sweepstakes is open to residents of the United States of America, Canada, Mexico, and the countries in the European Union.
  • One entry per participant per week will be considered eligible for each drawing. 
  • Each participant is only eligible to win once during the entire promotion.
  • Participants and winners must be 18 years of age or older.
  • The instructions above ("Sweepstakes instructions") must be executed to create a qualifying entry.
  • Winner must be able to accept contact from ImmunoChemistry Technologies to be notified via Facebook Messenger. A response with the winner's contact information is required for ImmunoChemistry Technologies to provide the product credit, which will be sent via email (not Facebook Messenger) in the form of a quote. If no response is received within 15 business days, the prize will not be awarded and all claims to such shall be made void. 
  • It is up to the discretion of ImmunoChemistry Technologies to make a determination of how to proceed in the case of fraudulent entries.

2. Prizes:

  • Up to four (4) Product Credit Vouchers for qualifying products from ImmunoChemistry Technologies will be awarded. Each Product Credit Voucher will have the product credit value of $100 USD.
  • Product credit has no value except when used under these terms and conditions.
  • Product credit may be used on qualifying product purchases from ImmunoChemistry Technologies that meet or exceed a total value of $150 before applicable shipping, handling, and product credits are applied.
  • Product credit will be issued in the form of a quote, which will expire one (1) year after issuance. 
  • Product credit may be used by winner or a colleague of the winner who has been authorized by winner to use it. Product credit may not be sold.
  • Qualifying product purchases include any standard catalog item available for purchase on the ICT e-commerce website, www.immunochemistry.com/products. Credit is not valid on reserve material, service work, or custom products.
  • Product credit must be used on a single purchase. Credit has no residual value. 
  • Credit is not valid with any other discounts or quotations. 
  • Credit is valid for purchases made by direct customers (end-users) and will not apply to distributor or reseller purchases. 
  • Standard shipping and handling fees will apply to product purchases.

3. Selection of Winning Entries

  • Winners will be selected randomly at 11 am US-CDT from the qualifying entries submitted that week. 
  • The instructions above ("Sweepstakes instructions") must be executed to create a qualifying entry.
  • Up to two (2) unique winners will be selected per week during the sweepstakes. In the case of less than two (2) qualifying entries being submitted per week, ImmunoChemistry Technologies may award no prize or one (1) prize during that promotional sub-period.
  • Participants may enter during both weeks, but each participant is only eligible to win once.
  • Winners will first be notified via Facebook Messenger and asked to make arrangements for the product credit to be provided in the form of a quote. If no response is received within 15 business days, the prize will not be awarded and all claims to such shall be made void.
  • It is up to the discretion of ImmunoChemistry Technologies to make a determination of how to proceed in the case of fraudulent entries.

4. Disclaimers and Releases:

  • This promotion does not have a connection
    with Facebook in any way. It is in no way sponsored, endorsed or administered by, or associated with, Facebook.
  • Each entrant or participant of this sweepstakes consents to a complete release of Facebook from any liability pertaining to this offer.
  • The recipient of the information provided by participants is ImmunoChemistry Technologies, LLC, not Facebook. Facebook is not collecting information about participants in this offer.
  • By posting an image(s) via his/her Facebook account on the ImmunoChemistry Technologies Facebook Page, entrant consents to have the uploaded image(s) and its (their) source(s) shared on the ImmunoChemistry Technologies Facebook Page. As part of the sharing mechanism, the entrant's name and/or profile photo may be displayed with the shared image. Entrant also consents to having his/her name posted by ImmunoChemistry Technologies in the event that his/her entry is chosen to win. It is the sole responsibility of the entrant to manage their profile security settings within Facebook.

 

ELISA SOLUTIONS

APOPTOSIS ASSAYS

CELLULAR IMAGING DYES

IN VIVO APOPTOSIS TRACERS

 

Place an Order:

Online: www.immunochemistry.com/products

E-mail: orders@immunochemistry.com

Tel: 952-888-8788

Toll-Free: 800-829-3194

Fax: 952-888-8988

 

Stay informed of new products, promotions, and events with the following communication options:

Join our mailing list to receive a brief, monthly email from ICT

 

Join our mailing list - we will only send one email per month, promise!

 

Immunochemistry Technologies, LLC on LinkedIn

 

Tweet @Immunochemistry

Twitter @ImmunoChemistry - adding our tweets to the chorus of science chirps and buzzes

 

Get Social with Us

Facebook - Get Social with Us

 

 

 

 

Adding us to your email system's Safe Senders List will help ensure that you receive our email communications, such as a response to a quote request or our monthly e-newsletter. To do this, please follow the appropriate steps below:

Microsoft Outlook

With an email from ICT selected in Outlook, click on the "Actions" menu. Select "Junk E-mail", and click "Add Sender's Domain to Safe Sender's List". Without an ICT email selected, click on the "Actions" menu, select "Junk E-mail" and add e-mail@immunochemistry.com to the list.

Gmail

From the inbox screen, click on "Contacts" in the Gmail menu. Click on the "Add to My Contacts" button. Enter e-mail@immunochemistry.com and click the "Add" button.

Windows Live Hotmail

Click "Options," then "Safe and blocked senders" under the "Junk e-mail" heading. Next, click "Safe Senders" and then add e-mail@immunochemistry.com to the list.

Yahoo! Mail

Click on "Contacts," then click on the "New Contact" button. Enter e-mail@immunochemistry.com into the "Email" area and click the "Save" button.

AOL

Click on the "Mail Options" arrow, and then "Address Book." Next, click the plus sign (+) with "Add" beneath it. Enter e-mail@immunochemistry.com in the screen name section.

 

ICT utilizes FedEx Express shipping services.  Our shipping and handling charges vary by destination, package size and weight, service type, and incoterm (if applicable). 

The majority of our products ship on blue ice with FedEx standard overnight; shipments of a single kit or a few small bottles typically cost between $40 and $48 within the continental United States. International shipments vary by destination, size, and incoterm (e.g., 1 kit, DDU to France, is approx $105 USD).

If you need a shipping estimate before placing your order, please add your desired products to the cart and use the store's checkout tool to calculate the charge.

Online Orders:
When ordering products from our e-commerce site, the prepaid FedEx overnight shipping charge will be automatically calculated and added to the order total. Online credit card orders are securely processed immediately for the approved amount, including the overnight shipping charge.

Online Shipping Options: Domestic standard, priority overnight or International Priority

Non-Overnight Options: Offline Only
If you are ordering a product that is shipped at room temperature (see product specs) and prefer domestic FedEx Express 2-day or 3-day shipping for your order, please place your order offline to request this option. FedEx Ground services (best for heavy shipments) are available for an additional $10 pickup fee. 

Collect Shipping
You may also request the use of your collect shipping account for product delivery. ICT will assess a $10 handling charge per package (under 24"x20"x20") for collect shipping with FedEx or a $17 handling charge per package (under 24"x20"x20") for collect shipping with UPS or another major carrier.

To request this option, please clearly provide the account number with your order by fax, phone, or email. When ordering online, enter your FedEx number in the designated field and select "FedEx Collect." Orders that do not explicitly instruct the use of a valid account number will be shipped FedEx prepaid, and freight charges will be assessed on the invoice.

Ordering
Phone: 952-888-8788 / 800-829-3194
Fax: 952-888-8988
Email: orders {at} immunochemistry.com

ELISA Solutions

Stabilizers, buffers, and diluents to build a better assay:
www.immunochemistry.com/products/elisa-solutions.html

The first step in making a reliable microtiter plate immunoassay is proper coating of the antibody or antigen onto the plate. ICT has specifically formulated two plate coating buffers for coating antibodies and antigens onto polystyrene microtiter ELISA plates. Both coating buffers stabilize coated proteins by maintaining their tertiary three-dimensional structure, allowing for greater binding reactivity with the detection molecule, thereby enhancing the specific signal. 


For antibody-sandwich ELISAs, use ICT's Antibody Plate Coating Buffer (5x). Antibody Coating Buffer is a unique buffer to coat antibodies onto polystyrene microtiter ELISA plates. It can also be used for antigen-down ELISAs as well. However, for most antigen-down ELISAs, we recommend ICT's Antigen Coating Buffer (5x). This coating buffer is formulated for antigen-down ELISAs.

Simply dilute the coat buffer 1:5 (for example, add 100mL 5x coat buffer to 400mL diH20 for a final volume of 500mL), add your protein antigen or antibody, let the solution stir for 15 minutes, and pipette onto the plate. As both coat buffers are concentrated 5x, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm the buffer until all crystals are dissolved before diluting. Do not let it boil.

Antibody Coating Buffer, 5x:

The pH and ionic strength of this buffer have been specifically formulated to create an optimal coating environment which maximizes the adsorption efficiency of most antibodies and some protein antigens onto polystyrene plate surfaces.

This buffer also stabilizes coated antibodies by maintaining their tertiary three-dimensional structure during the adsorption process. By stabilizing the adsorbed antibody, antigenic regions are preserved, allowing for greater binding reactivity with the detection molecule, thereby enhancing the specific signal.


In antibody-sandwich assays, there is a finite quantity of antibody that may be coated effectively onto microtiter plates.  The use of antibody concentrations in excess of the optimal range will result in surface saturation and clumping on the plate surface. This produces lower specific signals, and higher nonspecific signals.

By generating a higher specific signal with a stabilizing plate coating buffer from ICT, a lower concentration of coating antibody may be needed when this buffer is used, thereby saving valuable reagents.

Antigen Coating Buffer, 5x:

This buffer is well suited to coat all antigens onto polystyrene plates. Antigen Coating Buffer enhances the coating adsorption of antigens onto polystyrene plates by using an alkaline pH binding environment so that this buffer can be used for all antigen-down ELISAs. During the adsorption process, Antigen Coating Buffer stabilizes and hydrates coated antigens without compromising their structure. This allows for greater accessibility to detector antibody that are present in sample solutions, thereby enhancing the specific signal. By generating a higher specific signal, the assay may require a lower concentration of coat antigen when this buffer is used, saving valuable reagents.

ICT’s proprietary coating buffers contain an antimicrobial agent to retard bacterial growth during the plate coating process, allowing the procedure to be performed at room temperature. Depending on the activity of the coated antibody or antigen, plates may be stored at 2°-8°C (or even at room temperature for hardy proteins) for several months, or even years. 


Both of ICT's coating buffers are supplied as a 5x concentrate to conserve valuable storage space. Simply add 4 parts deionized water to 1 part buffer, mix, and it’s ready to use. No pH adjustment is required. After dilution, the 1x buffer will suppress bacterial growth for up to 1 month at room temperature and 6 months at 2°-8°C. The concentrated 5x buffer will suppress bacterial growth up to 18 months at 2°-8°C. During refrigeration, some salts in the buffer may precipitate. Simply warm the bottle in a water bath (do not boil) and mix. The crystals should go into solution.

Which plates should I use?


Be sure to use a 96-well microtiter plate specific for ELISA applications, as 96-well tissue culture plates will cause background problems. For antibody sandwich ELISAs, try Costar EIA/RIA Stripwell plates, and secondarily Immulon II HB plates. For antigen-down ELISAs, we have found that Immulon II plates tend to work best.

How much coating buffer do I need?

Plates can be coated at many different volumes depending on the protein you are coating with. For example, if you coat with 100 uL/well (in a 96-well plate), you will need 9.6 mL of 1x buffer per plate. The 25 mL trial size is enough for 125 mL of 1x buffer = 12-13 plates. You can coat 51 plates with 100mL of 5x size, and 520 plates with the 1L bottles. If you are coating 1000s of plates, we offer each coat buffer in 10L carboys = 5,200 plates.
 If you coat with 200 uL/well, you will need 19.2 mL of 1x coat buffer per 96-well microtiter plate. Therefore, 6 plates can be coated with the 25 mL trial size; 25 plates can be coated with 100 mL of 5x coat buffer; 125 plates can be coated with 500 mL 5x, and 250 plates can be coated with the 1L size. If you are coating 1000s of plates, we offer each coat buffer in 10L carboys = 2,600 plates.

How do I coat plates?

  1. Dilute the 5x coating buffer by adding 1 part buffer to 4 parts deionized water (100 mL coat buffer to 400 mL diH2O, yielding a total volume of 500 mL) and mix for 15 minutes.

  2. Dilute your antigen or antibody into the coating buffer; coating concentration varies significantly from less than 0.1 ug/mL to over 10 ug/mL.
  3. Let the solution stir (10 - 15 minutes) and pipette onto the plate (coating volume generally ranges between 50-300 uL per well).

  4. Once added to the plate, incubate the coating solution from 3 - 24 hours at room temperature (RT) protected from light. Minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a covered, humidified storage box.
  5. After incubation, dump or aspirate the coating solution out of the wells. 

  6. Wash the plate 2 - 4 times with ICT’s ELISA Wash Buffer.

  7. Aspirate and pipette one of ICT’s blocking buffers onto the plate at a higher volume than the coating solution (300-400 uL per well). 

  8. Once added to the plate, incubate the blocking buffer from 3 - 24 hours at RT protected from light. Minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a covered, humidified storage box. 

  9. Aspirate the blocking buffer. 

  10. The assay can be run at this point, or the plate can be dried and packaged for later use.

  11. Dry the plate by letting it sit on the bench top from 2 - 24 hours (but protected from light – loosely cover with aluminum foil), or dry in a drying oven from 2 - 24 hours at RT or warmer.

  12. When dry, seal the plate in an air-tight foil pouch with a desiccant and store at RT or 2°-8°C protected from light.

For more information on these and other ELISA buffers, please browse our ELISA Solutions selection or call us: 1-800-829-3194.

Related:

ELISA Solutions
ELISA Blocking Buffers

Typical Immunoassay Protocol

Preparation

In this example, 1 blank (which is simply the sample diluent), 7 standards, 3 controls, and 37 unknown samples are being tested in duplicate in 1 microtiter plate MoAb-PoAb sandwich immunoassay.

Standards

As each kit typically comes with only 1 vial of a high-concentrate standard which is lyophilized (freeze dried into a powder), it must be reconstituted, and then further diluted with sample diluent. If the lyophilized standard was at 5000pg, when reconstituted with 5mL, it would have a concentration of 5000pg/5mL or 1000pg/mL. To generate a standard curve, it should be serially diluted 1:2 (500uL standard with 500uL sample diluent) to create standards at 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, and 15.13pg/mL.

Controls

The controls may or may not come with a kit, but are often lyophilized and must also be reconstituted, typically with 2mL sample diluent. Controls are usually not diluted further.

Unknown Samples

Any samples thought to have a value higher than the reconstituted standard (at 1000pg/mL) should be diluted with the sample diluent so that it falls between the high standard (at 1000pg/mL) and the low standard (at 15.13 pg/mL). If they do not need to be diluted, samples often can be run neat. However, if the samples have some sort of interfering substance, such as rheumatoid factors, or complement, they may need to be treated prior to running in the assay. If you do not know about your samples, simply run them in the assay once and see where they fall (neat and diluted samples can be run at the same time). If the values are too high, just dilute, and/or treat the samples and run the assay again. Each investigator must determine their own protocols for sample dilutions and pre-assay treatments (see Consultation for assistance from ICT).

Immunoassay Procedure

  • Step 1: Add 50 uL per well of assay diluent into every well of the plate.
  • Step 2: Add 200 uL per well of your blanks, standards, controls, or samples onto the plate. Each item should be tested in duplicate (in 2 wells).
  • Step 3: Cover with plate sealer and incubate 2 hours at room temperature.
  • Step 4: Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4 cycles. Blot on paper towels.
  • Step 5: Add 200uL per well of PoAb-HRP conjugate solution into every well of the plate.
  • Step 6: Cover with plate sealer and incubate 2 hours at room temperature.
  • Step 7: Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4 cycles. Blot on paper towels.
  • Step 8: Add 200uL per well of TMB substrate solution into every well of the plate.
  • Step 9: Incubate 20 minutes at room temperature.
  • Step 10: Add 50 uL per well 2N HCl stop solution into every well of the plate.
  • Step 11: Read plate at 450nm while subtracting a reference wavelength of 540nm.
  • Step 12: Calculate data based on OD values of the unknown samples compared to the known values of the standard curve.

Typical Plate Map

  1 2 3 4 5 6 7 8 9 10 11 12
A Blank Blank Lo C Lo C Sp 6 Sp 6 Sp 14 Sp 14 Sp 22 Sp 22 Sp 30 Sp 30
B Std 1 Std 1 Mid C Mid C Sp 7 Sp 7 Sp 15 Sp 15 Sp 23 Sp 23 Sp 31 Sp 31
C Std 2 Std 2 Hi C Hi C Sp 8 Sp 8 Sp 16 Sp 16 Sp 24 Sp 24 Sp 32 Sp 32
D Std 3 Std 3 Sp 1 Sp 1 Sp 9 Sp 9 Sp 17 Sp 17 Sp 25 Sp 25 Sp 33 Sp 33
E Std 4 Std 4 Sp 2 Sp 2 Sp 10 Sp 10 Sp 18 Sp 18 Sp 26 Sp 26 Sp 34 Sp 34
F Std 5 Std 5 Sp 3 Sp 3 Sp 11 Sp 11 Sp 19 Sp 19 Sp 27 Sp 27 Sp 35 Sp 35
G Std 6 Std 6 Sp 4 Sp 4 Sp 12 Sp 12 Sp 20 Sp 20 Sp 28 Sp 28 Sp 36 Sp 36
H Std 7 Std 7 Sp 5 Sp 5 Sp 13 Sp 13 Sp 21 Sp 21 Sp 29 Sp 29 Sp 37 Sp 37

 

Coating Plates

The first step in making a reliable ELISA is proper coating of the antibody or antigen onto the plate. ICT has specifically formulated two plate coating buffers for coating antibodies and antigens onto polystyrene microtiter ELISA plates. Both coating buffers stabilize coated proteins by maintaining their tertiary three-dimensional structure, allowing for greater binding reactivity with the detection molecule, thereby enhancing the specific signal. 


ICT has developed two coating buffers based on the type of ELISA being developed. For antibody-sandwich ELISAs, use ICT's Antibody Plate Coating Buffer (5x, CB1). CB1 is a unique buffer to coat antibodies onto polystyrene microtiter ELISA plates. It can also be used for antigen-down ELISAs as well. However, for most antigen-down ELISAs, we recommend ICT's Antigen Coating Buffer (5x, CB2). This buffer is formulated for antigen-down ELISAs.

Simply dilute the coat buffer 1:5 (for example, add 100mL 5x coat buffer to 400mL diH20 for a final volume of 500mL), add your protein antigen or antibody, let the solution stir for 15 minutes, and pipette onto the plate. As both coat buffers are concentrated 5x, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm the buffer until all crystals are dissolved. Do not let it boil.

Antibody Coating Buffer CB1, 5x:

The pH and ionic strength of this buffer have been specifically formulated to create an optimal coating environment which maximizes the adsorption efficiency of most antibodies and some protein antigens onto polystyrene plate surfaces.

This buffer also stabilizes coated antibodies by maintaining their tertiary three-dimensional structure during the adsorption process. By stabilizing the adsorbed antibody, antigenic regions are preserved, allowing for greater binding reactivity with the detection molecule, thereby enhancing the specific signal.


By generating a higher specific signal, a lower concentration of coating antibody may be needed when this buffer is used, thereby saving valuable reagents. There is a finite quantity of antibody that may be coated effectively onto microtiter plates. Use of antibody concentrations in excess of the optimal range will result in surface saturation and clumping on the plate surface. This produces lower specific signals, and higher nonspecific signals.

5x Antigen Coating Buffer CB2:

This buffer is well suited to coat all antigens onto polystyrene plates. CB2 enhances the coating adsorption of antigens onto polystyrene plates by using an alkaline pH binding environment so that this buffer can be used for all antigen-down ELISAs. During the adsorption process, CB2 stabilizes and hydrates coated antigens without compromising their three-dimensional structure. This allows for greater accessibility to detector antibody that are present in sample solutions, thereby enhancing the specific signal. By generating a higher specific signal, a lower concentration of coat antigen may be needed when this buffer is used, thereby saving valuable reagents.

ICT’s proprietary antibody coating buffers contain an antimicrobial agent to retard bacterial growth during the plate coating process, allowing the procedure to be performed at room temperature. Depending on the activity of the coated antibody or antigen, plates may be stored at 2°-8°C (or even at room temperature for hardy proteins) for several months, or even years. 


Both of ICT's coating buffers are supplied as a 5x concentrate to conserve valuable storage space. Simply add 4 parts deionized water to 1 part buffer, mix, and it’s ready to use. No pH adjustment is required. After dilution, the 1x buffer will suppress bacterial growth for up to 1 month at room temperature and 6 months at 2°-8°C. The concentrated 5x buffer will suppress bacterial growth up to 18 months at 2°-8°C. During refrigeration, some salts in the buffer may precipitate. Simply warm the bottle in a water bath (do not boil) and mix. The crystals should go into solution. Which plates should I use?
 Be sure to use a 96-well plate specific for ELISA applications, as 96-well tissue culture plates will cause background problems. For antibody sandwich ELISAs, try Costar EIA/RIA Stripwell plates, and secondarily Immulon II HB plates. For antigen-down ELISAs, we have found that Immulon II plates tend to work best.

How much coating buffer do I need?

Plates can be coated at many different volumes depending on the protein you are coating with. For example, if you coat with 100 uL/well (in a 96-well plate), you will need 9.6 mL of 1x buffer per plate. The 25 mL trial size is enough for 125 mL of 1x buffer = 12-13 plates. You can coat 51 plates with 100mL of 5x size, and 520 plates with the 1L bottles. If you are coating 1000s of plates, we offer each coat buffer in 10L carboys = 5,200 plates.
 If you coat with 200 uL/well, you will need 19.2 mL of 1x coat buffer per 96-well microtiter plate. Therefore, 6 plates can be coated with the 25 mL trial size; 25 plates can be coated with 100 mL of 5x coat buffer; 125 plates can be coated with 500 mL 5x, and 250 plates can be coated with the 1L size. If you are coating 1000s of plates, we offer each coat buffer in 10L carboys = 2,600 plates.

How do I coat plates?

  1. Dilute the 5x coat buffer by adding 1 part buffer to 4 parts deionized water (100 mL coat buffer to 400 mL diH2O, yielding a total volume of 500 mL) and mix for 15 minutes.

  2. Dilute your antigen or antibody into the coating buffer; coating concentration varies significantly from less than 0.1 ug/mL to over 10 ug/mL.
  3. Let the solution stir (10 - 15 minutes) and pipette onto the plate (coating volume generally ranges between 50-300 uL per well).

  4. Once added to the plate, incubate the coating solution from 3 - 24 hours at room temperature (RT) protected from light. Minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a covered, humidified storage box.
  5. After incubation, dump or aspirate the coating solution out of the wells. 

  6. Wash the plate 2 - 4 times with ICT’s ELISA wash buffer.

  7. Aspirate and pipette one of ICT’s blocking buffers onto the plate at a higher volume than the coating solution (300-400 uL per well). 

  8. Once added to the plate, incubate the blocking buffer from 3 - 24 hours at RT protected from light. Minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a covered, humidified storage box. 

  9. Aspirate the block buffer. 

  10. The assay can be run at this point, or the plate can be dried and packaged for later use.

  11. Dry the plate by letting it sit on the bench top from 2 - 24 hours (but protected from light – loosely cover with aluminum foil), or dry in a drying oven from 2 - 24 hours at RT or warmer.

  12. When dry, seal the plate in an air-tight foil pouch with a desiccant and store at RT or 2°-8°C protected from light.

Help! I can't get my assay to work!


Just give us a call at 1-800-829-3194. To help you develop your assay, ICT has provided some background information on ELISA technology online and offers consultation services in assay development.

Fluorescent Detection Reagents

Fluorescent probes and assay kits for cellular analysis and imaging, including apoptosis and protease detection: www.immunochemistry.com/products/fluorescent-detection-reagents.html

Some of our whole cell assays, including MitoPT™ Mitochondrial Permeability Transition Kits, FLICA™ Caspase Assays, and FLISP™ serine protease assays require a short wash step to remove unbound background reagent.

After incubating with the reagent, remove the media and spin with fresh media or ICT’s wash buffer. Adherent cells can be spun in a plate. If that is not feasible, or if your cells cannot withstand centrifugation, replace the labeled media with fresh media or our wash buffer, and incubate 1 hour to allow any unbound reagent to diffuse back out of the cell. Remove this solution and analyze the cells. Perform aspiration steps carefully to avoid removal of floating apoptotic cells.

Our Magic Red caspase and cathepsin detection assays are substrate-based and do not require a wash step, so they may work best with sensitive cells and/or frozen cells.

The FLICA™ kits may work with some frozen cells and thin frozen sections. The reagents require the cell membrane to remain intact, and freezing often creates pores in the membranes, allowing the positive fluorescent signal to leave the cell. The Magic Red™ kits may work best with frozen cells when measuring total fluorescence with a plate reader.

No, our fluorescent protease detection reagents will not work with fixed cells, nor will they work with paraffin-embedded cells. The reagents' detection mechanisms require the enzymes to be active; fixation and paraffin inactivate the enzymes.  It is possible, however, to fix the cells after labeling and washing.  Our FLICA and FLISP kits include fixative.

The green cholinesterase, FLICA™, and FLISP™ reagents excite at 490nm and emit at 520nm. The red FLICA™ and FLISP™ reagents excite at 560nm and emit at 600nm. MR reagents excite at 540-590 and emit at >610nm. MitoPT™ excites at 488nm and emits at >590nm. Other filter pairings can be used which closely approximate these ranges.

GREEN: Our detection reagents that utilize the green carboxyfluorescein fluorophore (FAM) or fluorescein (FITC) excite at 490nm and emit at 520nm. Green apoptosis detection reagents:

FAM-FLICA™ Caspase and Apoptosis Assays
Annexin V-FITC Apoptosis Detection Kit
FAM-FLISP™ Serine Protease Assays
FAM-FLIVO™ in vivo Apoptosis Assay

ORANGE-RED: Our reagents labeled with sulforhodamine-B or sulforhodamine-101 excite at 560nm and emit at 600nm. Orange-red apoptosis detection reagents:

SR-FLICA™ Caspase and Apoptosis Assays
Total Cytotoxicity and Apoptosis Assay
SR101-FLISP™ Serine Protease Assays
SR-FLIVO™ in vivo Apoptosis Assay

FAR-RED: FLICA 660 Caspase and Apoptosis Assays, our newest FLICA assays, utilize a far-red 660 fluorophore that excites at 660 nm with an optimal emission at 690 nm. Far-red caspase assays:

FLICA™ 660 Poly Caspase Assay
FLICA™ 660 Caspase-1 Assay
FLICA™ 660 Caspase-3/7 Assay (coming soon!)

NEAR-INFRARED: NIR-FLIVO in vivo apoptosis tracers utilize the near-infrared fluorescent labels DyLight® 690 and DyLight® 747 for noninvasive apoptosis detection with whole animal imaging systems. DyLight® 690 excites at 690 nm and emits at 709 nm. DyLight® 747 excites at 747 nm and emits at 776 nm.

NIR-FLIVO™ in vivo Apoptosis Tracers

 

OTHER PROBES: Magic Red Cathepsin and Caspase substrate reagents excite at 540-590 and emit at >610nm. MitoPT™-JC1 cationic mitochondrial permeability transition dye excites at 488nm and emits at >590nm. Other filter pairings can be used which closely approximate these ranges.

The reagents are provided as stabilized lyophilized powders. Just reconstitute the vial with ~50uL DMSO, dilute with ~300uL PBS, and then add ~10uL of the diluted reagent to your cells. Your particular cell line may require more or less reagent. ICT’s kits can be customized by adapting the amount of reagent used and the length of the incubation periods.

 As a general guide, “1 test” is a 300uL aliquot of your cell culture for testing on a fluorescence plate reader. Your cell culture may be quite different; the kits work with just a few cells for viewing under a fluorescence microscope, or up to 1x106 cells/mL for flow cytometry.

The reagents start to react within 15 minutes of addition to the cells, and can be incubated up to 72 hours without killing the cells. Typical incubation periods are 1-4 hours so you can start and finish your experiment in one day. The exact incubation time should be determined by your experimental protocol. 

Our kits are very sensitive. They even detect natural apoptosis occurring in the control cells, generally 2-6% of the population. Positive apoptotic cells aften generate a signal 2-15x greater than the control population.

Services

Contract Laboratory Services, including Assay/ELISA Development, Consultation, Protein Lyophilization, and more: http://www.immunochemistry.com/services

ImmunoChemistry Technologies is a custom service laboratory specializing in immunoassay development and manufacturing. Once developed, we will ship the finished test to you so it can be used on-site by your staff, or in the field by your customers. We will then manufacture the components of the test whenever you need more.

There are several different formats of an immunoassay. A typical 96-well microtiter plate sandwich immunoassay kit may include the following components ():

Typical Components of an Sandwich Immunoassay and Typical Immunoassay Protocol

  • Pre-coated, stabilized 96-well microtiter plate
  • Sample diluent
  • Assay diluent
  • Calibrator diluent
  • Standards and controls
  • Conjugated detection antibody (in a working dilution)
  • 10X wash solution
  • Single component substrate
  • Stop solution

Pre-Coated, Stabilized 96-well Microtiter Plate:

For microtiter plate assays, the plates are provided ready-to-use. They are pre-coated with the capture antibody, blocked, stabilized, and packaged in a resealable foil pouch with a desiccant packet. Using our methods, the plates may have a real time stability of 1 year when stored at 2o – 8o C. With a standard curve (1 blank and 7 standards) and 3 controls, a 96-well microtiter plate format can test 21 samples in triplicate and 37 samples in duplicate (also see Plate Coating Buffers and Blocking Buffers for more information).

Sample Diluent:

The sample diluent is used to ensure that the analyte in the sample matrix is measured accurately. It is used to dilute the sample within the target range of the assay (also see Sample Diluents for more information).

Assay Diluent:

In addition to a specimen diluent, certain matrixes require the use of a special assay diluent that is applied to the plate just prior to adding the samples, standards, and controls. Assay diluents are often paired with a specific type of sample (such as serum, or cell culture media) to eliminate interference and non-specific binding generated from the matrix of the sample itself. These interferences are especially noticeable when running neat samples. An assay diluent may not be required for every assay. See our Assay Diluent products for more information.

Calibrator Diluent:

A calibrator diluent is used to ensure that the standards and controls will be measured accurately. This diluent must compliment the target analyte, capture antibody, and resemble the matrix of the sample. See our Sample Diluent products for more information.

Standards and controls:

A known amount of the analyte (such as 1 ng) is included with a kit and run on every plate. The standard is often lyophilized. Once reconstituted, it is diluted several times to prepare a range of known values with which to compare and properly quantitate the unknown amount of analyte in a sample. Controls are used to confirm the readings of the standard and to compare readings from different plates. Because they contain a known amount of analyte, they should always read within a certain optical density (OD) value based on the standard.

Conjugated Detection Antibody:

For some assays, the detection or ‘top’ antibody is often an affinity purified polyclonal antibody conjugated to HRP. The enzyme-antibody conjugate can often be supplied ready-to-use in its working concentration in a special conjugate diluent. The conjugate diluent stabilizes the conjugate and minimizes nonspecific binding of the conjugate onto the blocked plate or matrix residue. The working conjugate will often have a real-time stability of 1 year when stored at 2o–8o C.  See our Conjugate Stabilizer products for more information.

10X Wash Solution:

This specially formulated buffer is used to rinse the plate after the sample and conjugate incubation periods, just prior to the addition of the next reagent. It minimizes matrix residue and non-specific binding interferences of the samples and conjugate (also see Wash Buffer for more information).

Single Component Substrate:

This reagent reacts with HRP to generate a colored signal product. It can come in many formulations (powdered, 2-component liquid, etc.). ICT recommends a low-background, high-signal-generating ABTS or TMB that needs no preparation prior to use.

Stop Solution:

An appropriate stop solution is added to the plate with the ABTS or TMB substrate and stops its reaction with HRP. By stopping this reaction after 20 minutes, the plates can equilibrate before reading, which increases the accuracy and sensitivity of the assay.

Evolution of diagnostic tests began in the 1940s with colorimetric measurements of the enzymes and metabolites found in biological fluids using classical chemistry methods and agglutination reactions. In the 1950s, the radio-immunoassay (RIA) was developed by Rosalyn Yalow and Solomon Berson.  Tthis group was later awarded the Nobel prize in 1977 for developing an RIA to detect and measure blood glucose levels in diabetic patients. In the 1960s, immunoassay technology was enhanced by replacing radio-isotopes with enzymes for color generation. The use of enzymes eliminates the use of radioactive materials - and laboratory regulation by the Nuclear Regulatory Commission (NRC). EIAs also have faster reaction times, higher specificity to the target molecule, and longer shelf-lives compared to RIAs.  

Although immunoassay techniques were first described in the 1950s, they were not readily applied outside of clinical laboratories until the advent of economical automated plate-reading systems and personal computers to analyze data. Within the past 10 years, EIAs have become increasingly popular. Scientists in fields outside of medicine are now finding EIAs to be a convenient tool that can be used on a daily basis to detect and monitor specific molecules during the processing of materials. Immunoassays make it possible for scientists to run tests in-house with a minimal investment of time and expense.


Although unskilled technicians can run immunoassays, the development of these tests requires knowledge in many areas of immunology (such as antibody specificity) and protein chemistry (such as binding interactions). As the typical scientist is a specialist in a very narrow field of study, he or she typically may not have the ability to make his or her own rugged tests.

Because Immunochemistry Technologies develops assays with the end-user in mind, we try to eliminate as many dilution, mixing, and measuring steps as possible.  Most of our reagents come ready-to-use and typically have a shelf life of 12 months.  The scientists at ImmunoChemistry Technologies have the knowledge to develop reliable, sensitive, and specific immunoassays. For more information, call ICT at 1-800-829-3194.

Introduction

For over 40 years, immunoassays have been used in hospitals, laboratory medicine, and research to improve the health and well-being of humans and animals. Information gained by clinical immunoassay testing has shortened hospital stays and decreased the severity of illness by identifying and assessing the progression of disease, thereby leading to improved therapeutic choices. In life science research, immunoassays are used in the study of biological systems by tracking different proteins, hormones, and antibodies. In industry, immunoassays are used to detect contaminants in food and water, and in quality control to monitor specific molecules used during product processing.

What is an Immunoassay or ELISA?


Immunoassays are quick and accurate tests that can be used on-site and in the laboratory to detect specific molecules. Immunoassays rely on the inherent ability of an antibody to bind to the specific structure of a molecule. Antibodies are proteins generated by animals in response to the invasion of a foreign molecule (antigen) into the body. Antibodies are found in blood and tissue fluids and will bind to the antigen whenever it is encountered. Because antibodies are developed to the specific three-dimensional structure of an antigen, or analyte, they are highly specific and will bind only to that structure. Once purified from the blood, monoclonal and polyclonal antibodies are ideal assay reagents to detect and monitor specific target molecules with limited interferences from other substances. Four typical ELISA formats are: monoclonal-polyclonal sandwich assays, competitive inhibition assays, antigen-down immunoassays, and rapid assays.

Monoclonal-Polyclonal Sandwich Immunoassay

In a typical microtiter plate sandwich immunoassay, a monoclonal antibody is adsorbed onto a plastic microtiter plate. Build an Antibody Sandwich ELISA with Immunochemistry Technologies' ELISA Solutions When the test sample is added to the plate, the antibody on the plate will bind the target antigen from the sample, and retain it in the plate. When a polyclonal antibody is added in the next step, it also binds to the target antigen (already bound to the monoclonal antibody on the plate), thereby forming an antigen ‘sandwich’ between the two different antibodies. 


This binding reaction can then be measured by radio-isotopes, as in a radio-immunoassay format (RIA), or by enzymes, as in a enzyme immunoassay format (EIA or ELISA) attached to the polyclonal antibody. The radio-isotope or enzyme generates a color signal proportional to the amount of target antigen present in the original sample added to the plate. Depending on the immunoassay format, the degree of color can be detected and measured with the naked eye (as with a home pregnancy test), a scintillation counter (for an RIA), or with a spectrophotometric plate reader (for an EIA) (for a price quote, see Sandwich ELISA Development Sample Proposals).

  • Step 1: Monoclonal antibodies adsorbed onto the well of a plastic microtiter plate with coating buffer (no sample added). 

  • Step 2: Addition of a sample (such as human blood, diluted appropriately) to the well of the microtiter plate. The target antigen binds to the antibody adsorbed on the plate, retaining the antigen in the well. 

  • Step 3: Binding of an enzyme-conjugated polyclonal antibody to the target antigen (bound to the monoclonal antibody on the plate), thereby forming an antigen ‘sandwich’ between the two different antibodies. 
  • Step 4: Addition of a colorimetric substrate for detection of the enzyme-conjugated polyclonal antibodies will generate a color signal proportional to the amount of target antigen present in the original sample added to the plate.

Antigen-Down Immunoassay

In an antigen-down immunoassay, the analyte is coated onto a 96-well microtiter plate (rather than an antibody) and used to bind antibodies found in a sample. When the sample is added (such as human serum), the antigen on the plate is bound by antibodies (IgE for example) from the sample, which are then retained in the well. A species-specific antibody (anti-human IgE for example) labeled with HRP is added next, which, binds to the antibody bound to the antigen on the plate. The higher the signal, the more antibodies there are in the sample. 
 Antigen-down assays can be configured as rapid tests and are often used to diagnose allergy conditions - routinely a patient's blood is tested against different allergens to see if the person has antibodies to that allergen (for a price quote, see Antigen-Down ELISA Development Sample Proposal).

Competitive Inhibition Immunoassay


In addition to the Monoclonal-Polyclonal (Mo-Po) Antibody Sandwich format, many immunoassays are structured in a competitive inhibition format. Competitive inhibition assays are often used to measure small analytes because competitive inhibition assays only require the binding of one antibody rather than two, as in standard ELISA formats. 
 Because of the high probability for steric hindrance occurring when two antibodies attempt to bind to a small molecule at the same time, a sandwich assay format may not be feasible. Therefore a competitive inhibition assay would be preferable.

In a sequential competitive inhibition assay, the sample and conjugated analyte are added in steps like a sandwich assay, while in a classic competitive inhibition assay, these reagents are incubated together at the same time. 
 In a sequential competitive inhibition assay format, a monoclonal antibody is coated onto a 96-well microtiter plate. When the sample is added, the MoAb captures free analyte out of the sample. In the next step, a known amount of analyte labeled with either biotin or HRP is added. The labeled analyte will then also attempt to bind to the MoAb adsorbed onto the plate, however, the labeled analyte is inhibited from binding to the MoAb by the presence of previously bound analyte from the sample. This means that the labeled analyte will not be bound by the monoclonal on the plate if the monoclonal has already bound unlabeled analyte from the sample. 
 The amount of unlabeled analyte in the sample is inversely proportional to the signal generated by the labeled analyte. The lower the signal, the more unlabeled analyte there is in the sample. A standard curve can be constructed using serial dilutions of an unlabeled analyte standard. Subsequent sample values can then be read off the standard curve as is done in the sandwich ELISA formats. 
 The classic competitive inhibition assay format requires the simultaneous addition of labeled (conjugated analyte) and unlabeled analyte (from the sample). Both labeled and unlabeled analyte then compete simultaneously for the binding site on the monoclonal capture antibody on the plate. Like the sequential competitive inhibition format, the colored signal is inversely proportional to the concentration of unlabeled target analyte in the sample. 
 Detection of labeled analyte may be made by using a peroxidase substrate such as TMB, which can be read on a microtiter plate reader (for price quotes, see Competitive Inhibition ELISA Sample Proposal).

Rapid Immunoassay

In addition to microtiter plates, immunoassays are also configured as rapid tests, such as a home pregnancy test. Like microtiter plate assays, rapid tests use antibodies to react with antigens and can be developed as MoAb-PoAb sandwich formats, competitive inhibition formats, and antigen-down formats. With a rapid test, the antibody and antigen reagents are bound to porous membranes, which react with positive samples while channeling excess fluids to a non-reactive part of the membrane. 
 Rapid immunoassays commonly come in 2 configurations: a lateral flow test where the sample is simply placed in a well and the results are read immediately; and a flow through system, which requires placing the sample in a well, washing the well, and then finally adding an analyte-colloidal gold conjugate and the result is read after a few minutes. One sample is tested per strip or cassette. 
 Because rapid tests are faster than microtiter plate assays, require little sample processing, are often cheaper, and generate yes/no answers without using an instrument, they often used in the field by non-laboratory people testing whole samples. However, rapid immunoassays are not as sensitive nor can they be used to accurately quantitate an analyte. (Self-monitoring of blood glucose levels by diabetics is considered quantitative rapid testing, however, immunoassay technology is not used for these tests.) All rapid immunoassay tests can be converted to a microtiter plate assay, but not all microtiter plate assays can be converted to a rapid test (for price quotes, see Rapid Test ELISA Development Sample Proposals).

ICT offers a commercial rapid test immunoassay for isotyping mouse monoclonal antibodies.

Typical Immunoassay Protocol

Preparation

In this example, 1 blank (which is simply the sample diluent), 7 standards, 3 controls, and 37 unknown samples are being tested in duplicate in 1 microtiter plate MoAb-PoAb sandwich immunoassay.

Standards

As each kit typically comes with only 1 vial of a high-concentrate standard which is lyophilized (freeze dried into a powder), it must be reconstituted, and then further diluted with sample diluent. If the lyophilized standard was at 5000pg, when reconstituted with 5mL, it would have a concentration of 5000pg/5mL or 1000pg/mL. To generate a standard curve, it should be serially diluted 1:2 (500uL standard with 500uL sample diluent) to create standards at 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, and 15.13pg/mL.

Controls

The controls may or may not come with a kit, but are often lyophilized and must also be reconstituted, typically with 2mL sample diluent. Controls are usually not diluted further.

Unknown Samples

Any samples thought to have a value higher than the reconstituted standard (at 1000pg/mL) should be diluted with the sample diluent so that it falls between the high standard (at 1000pg/mL) and the low standard (at 15.13 pg/mL). If they do not need to be diluted, samples often can be run neat. However, if the samples have some sort of interfering substance, such as rheumatoid factors, or complement, they may need to be treated prior to running in the assay. If you do not know about your samples, simply run them in the assay once and see where they fall (neat and diluted samples can be run at the same time). If the values are too high, just dilute, and/or treat the samples and run the assay again. Each investigator must determine their own protocols for sample dilutions and pre-assay treatments (see Consultation for assistance from ICT).

Immunoassay Procedure

  • Step 1: Add 50 uL per well of assay diluent into every well of the plate.
  • Step 2: Add 200 uL per well of your blanks, standards, controls, or samples onto the plate. Each item should be tested in duplicate (in 2 wells).
  • Step 3: Cover with plate sealer and incubate 2 hours at room temperature.
  • Step 4: Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4 cycles. Blot on paper towels.
  • Step 5: Add 200uL per well of PoAb-HRP conjugate solution into every well of the plate.
  • Step 6: Cover with plate sealer and incubate 2 hours at room temperature.
  • Step 7: Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4 cycles. Blot on paper towels.
  • Step 8: Add 200uL per well of TMB substrate solution into every well of the plate.
  • Step 9: Incubate 20 minutes at room temperature.
  • Step 10: Add 50 uL per well 2N HCl stop solution into every well of the plate.
  • Step 11: Read plate at 450nm while subtracting a reference wavelength of 540nm.
  • Step 12: Calculate data based on OD values of the unknown samples compared to the known values of the standard curve.

Typical Plate Map

  1 2 3 4 5 6 7 8 9 10 11 12
A Blank Blank Lo C Lo C Sp 6 Sp 6 Sp 14 Sp 14 Sp 22 Sp 22 Sp 30 Sp 30
B Std 1 Std 1 Mid C Mid C Sp 7 Sp 7 Sp 15 Sp 15 Sp 23 Sp 23 Sp 31 Sp 31
C Std 2 Std 2 Hi C Hi C Sp 8 Sp 8 Sp 16 Sp 16 Sp 24 Sp 24 Sp 32 Sp 32
D Std 3 Std 3 Sp 1 Sp 1 Sp 9 Sp 9 Sp 17 Sp 17 Sp 25 Sp 25 Sp 33 Sp 33
E Std 4 Std 4 Sp 2 Sp 2 Sp 10 Sp 10 Sp 18 Sp 18 Sp 26 Sp 26 Sp 34 Sp 34
F Std 5 Std 5 Sp 3 Sp 3 Sp 11 Sp 11 Sp 19 Sp 19 Sp 27 Sp 27 Sp 35 Sp 35
G Std 6 Std 6 Sp 4 Sp 4 Sp 12 Sp 12 Sp 20 Sp 20 Sp 28 Sp 28 Sp 36 Sp 36
H Std 7 Std 7 Sp 5 Sp 5 Sp 13 Sp 13 Sp 21 Sp 21 Sp 29 Sp 29 Sp 37 Sp 37

 

Coating Plates

The first step in making a reliable ELISA is proper coating of the antibody or antigen onto the plate. ICT has specifically formulated two plate coating buffers for coating antibodies and antigens onto polystyrene microtiter ELISA plates. Both coating buffers stabilize coated proteins by maintaining their tertiary three-dimensional structure, allowing for greater binding reactivity with the detection molecule, thereby enhancing the specific signal. 


ICT has developed two coating buffers based on the type of ELISA being developed. For antibody-sandwich ELISAs, use ICT's 5x Universal Antibody Plate Coating Buffer (CB1). CB1 is a unique buffer to coat antibodies onto polystyrene microtiter ELISA plates. It can also be used for antigen-down ELISAs as well. However, for most antigen-down ELISAs, we recommend ICT's 5x Antigen Coating Buffer (CB2). This buffer is targeted for antigen-down ELISAs.

Simply dilute the coat buffer 1:5 (for example, add 100mL 5x coat buffer to 400mL diH20 for a final volume of 500mL), add your protein antigen or antibody, let the solution stir for 15 minutes, and pipette onto the plate. As both coat buffers are concentrated 5x, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm the buffer until all crystals are dissolved. Do not let it boil.

5x Universal Antibody Coating Buffer CB1:

The pH and ionic strength of this buffer have been specifically formulated to create an optimal coating environment which maximizes the adsorption efficiency of most antibodies and some protein antigens onto polystyrene plate surfaces.

This buffer also stabilizes coated antibodies by maintaining their tertiary three-dimensional structure during the adsorption process. By stabilizing the adsorbed antibody, antigenic regions are preserved, allowing for greater binding reactivity with the detection molecule, thereby enhancing the specific signal.


By generating a higher specific signal, a lower concentration of coating antibody may be needed when this buffer is used, thereby saving valuable reagents. There is a finite quantity of antibody that may be coated effectively onto microtiter plates. Use of antibody concentrations in excess of the optimal range will result in surface saturation and clumping on the plate surface. This produces lower specific signals, and higher nonspecific signals.

5x Antigen Coating Buffer CB2:

This buffer is well suited to coat all antigens onto polystyrene plates. CB2 enhances the coating adsorption of antigens onto polystyrene plates by using an alkaline pH binding environment so that this buffer can be used for all antigen-down ELISAs. During the adsorption process, CB2 stabilizes and hydrates coated antigens without compromising their three-dimensional structure. This allows for greater accessibility to detector antibody that are present in sample solutions, thereby enhancing the specific signal. By generating a higher specific signal, a lower concentration of coat antigen may be needed when this buffer is used, thereby saving valuable reagents.

ICT’s proprietary antibody coating buffers contain an antimicrobial agent to retard bacterial growth during the plate coating process, allowing the procedure to be performed at room temperature. Depending on the activity of the coated antibody or antigen, plates may be stored at 2°-8°C (or even at room temperature for hardy proteins) for several months, or even years. 


Both of ICT's coating buffers are supplied as a 5x concentrate to conserve valuable storage space. Simply add 4 parts deionized water to 1 part buffer, mix, and it’s ready to use. No pH adjustment is required. After dilution, the 1x buffer will suppress bacterial growth for up to 1 month at room temperature and 6 months at 2°-8°C. The concentrated 5x buffer will suppress bacterial growth up to 18 months at 2°-8°C. During refrigeration, some salts in the buffer may precipitate. Simply warm the bottle in a water bath (do not boil) and mix. The crystals should go into solution. Which plates should I use?
 Be sure to use a 96-well plate specific for ELISA applications, as 96-well tissue culture plates will cause background problems. For antibody sandwich ELISAs, try Costar EIA/RIA Stripwell plates, and secondarily Immulon II HB plates. For antigen-down ELISAs, we have found that Immulon II plates tend to work best.

How much coating buffer do I need?

Plates can be coated at many different volumes depending on the protein you are coating with. For example, if you coat with 100 uL/well (in a 96-well plate), you will need 9.6 mL of 1x buffer per plate. The 25 mL trial size is enough for 125 mL of 1x buffer = 12-13 plates. You can coat 51 plates with 100mL of 5x size, and 520 plates with the 1L bottles. If you are coating 1000s of plates, we offer each coat buffer in 10L carboys = 5,200 plates.
 If you coat with 200 uL/well, you will need 19.2 mL of 1x coat buffer per 96-well microtiter plate. Therefore, 6 plates can be coated with the 25 mL trial size; 25 plates can be coated with 100 mL of 5x coat buffer; 125 plates can be coated with 500 mL 5x, and 250 plates can be coated with the 1L size. If you are coating 1000s of plates, we offer each coat buffer in 10L carboys = 2,600 plates.

How do I coat plates?

  1. Dilute the 5x coat buffer by adding 1 part buffer to 4 parts deionized water (100 mL coat buffer to 400 mL diH2O, yielding a total volume of 500 mL) and mix for 15 minutes.

  2. Dilute your antigen or antibody into the coating buffer; coating concentration varies significantly from less than 0.1 ug/mL to over 10 ug/mL.
  3. Let the solution stir (10 - 15 minutes) and pipette onto the plate (coating volume generally ranges between 50-300 uL per well).

  4. Once added to the plate, incubate the coating solution from 3 - 24 hours at room temperature (RT) protected from light. Minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a covered, humidified storage box.
  5. After incubation, dump or aspirate the coating solution out of the wells. 

  6. Wash the plate 2 - 4 times with ICT’s ELISA wash buffer.

  7. Aspirate and pipette one of ICT’s blocking buffers onto the plate at a higher volume than the coating solution (300-400 uL per well). 

  8. Once added to the plate, incubate the blocking buffer from 3 - 24 hours at RT protected from light. Minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a covered, humidified storage box. 

  9. Aspirate the block buffer. 

  10. The assay can be run at this point, or the plate can be dried and packaged for later use.

  11. Dry the plate by letting it sit on the bench top from 2 - 24 hours (but protected from light – loosely cover with aluminum foil), or dry in a drying oven from 2 - 24 hours at RT or warmer.

  12. When dry, seal the plate in an air-tight foil pouch with a desiccant and store at RT or 2°-8°C protected from light.

Help! I can't get my assay to work!


Just give us a call at 1-800-829-3194. To help you develop your assay, ICT has provided some background information on ELISA technology online and offers consultation services in assay development.