rev:July 11, 1997 

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Immunoasaay Enhancers

for Researchers and Manufacturers


Alkaline Phosphatase CONJUGATE STABILIZERS


ALKALINE PHOSPHATASE (AP) -CONJUGATE STABILIZER

The major advantage of a liquid AP-conjugate is it eliminates the chance for human error when dissolving or diluting the lyophilized or concentrated conjugate. Adding too much or not enough buffer results in assay to assay variation. Until now, the disadvantage of this format was the inherent instability of the liquid AP-conjugates. This problem has now been addressed!


The AP-CONJUGATE STABILIZER is formulated to allow you to provide pre-diluted, READY-TO-USE CONJUGATES.

This product is prepared in a proprietary buffer system and contains a non mercury containing preservative (no azide, no thimerosal).

cat#AP-Stab READY-TO-USE : This formulation was designed to reconstitute lyophilized or concentrated AP-conjugates. Conjugates are tittered and diluted to working concentrations with the AP-CONJUGATE STABILIZER.


Use at full strength or dilute 1:1 with 10mM MES Buffer, pH 6.0 (or 0.85% NACL. The use of more than 10% or 1.5mM Phosphate will cause precipitation.

For additional protection, add 10% glycerol to the conjugate stabilizer solution (if compatible with your system). Store at 4 DEG C and protect diluted material from light.

Conjugates prepared in this manner will maintain AP activity for up to 2 years at 4 DEG C. (actual time must be determined with your particular conjugate and assay characteristics).


CAT# RDI-APSTAB-1X =$375.00/Liter

                                           $344.00/Liter for 5-9 liters

                                           $325.00/liter 10-20 Liters

Please allow 2 weeks for delivery on multi-liter quantities (to ensure product from same production lot!).

You should titer your conjugate with each batch. Our testing shows you may need slightly more of your conjugate but the long term stability of the "working conjugate" makes for a much more reproducible product. The addition of glycerol is generally only needed for very unstable conjugates.Minimize the number of times you open and close the bulk stabilizer bottle. Try to maintain aseptic techniques. Ideally, sterile filter your conjugate prior to addition to the stabilizer (or after dilution for maximum stabilizing effect).

FOR RESEARCH OR FURTHER MANUFACTURING USE ONLY.


Technical Recommendations for Stabilizing Solutions:

You should titer your conjugate with each batch. Our testing shows you may need slightly more of your conjugate but the long term stability of the "working conjugate" makes for a much more reproducible product.

Minimize the number of times you open and close the bulk stabilizer bottle. Try to maintain aseptic techniques. Ideally, sterile filter your conjugate prior to addition to the stabilizer (or after dilution for maximum stabilizing effect). Package diluted conjugates in oxygen barrier bottles (PET or brown glass lyophilization vials with stoppers and tight caps). NOTE: Stability of HRP conjugates is prolonged by keeping conjugates cool and reducing exposure to light and oxygen. Thorough vacuum degassing with nitrogen flushing of all buffers, conjugates and diluents will extend diluted conjugate stability under severe conditions as will addition of metal free glycerol.


Our primary formulation of  Alk-Phos Stabilizer is a proprietary formulation made with certified reagents under a standardized GMP production protocol. I regret that I cannot give you any additional details on its preparation or formulation.

As for validating an extended stability with such a product, this is also too dependent on the product to give you much information. However, the following is a "SAMPLE" protocol of process that must be investigated for using such a product.

1) You should first titer your conjugate with the stabilizer (full strength ) to obtain a working dilution. Generally, you will need approx 10% more conjugate initially than if used with a standard buffer diluent.


2) Then , you should sterile filter the conjugate (if possible) and add it to the stabilizer. If you have the capabilities and you have a very unstable conjugate, you may also wish to add  glycerol.

-if you conjugate is susceptible to bacterial decay, you can add additional thimerosal or other non azide preservatives as desired (as our microcide I).

3) Bottle the cold, diluted material with nitrogen pressure or gravity fill into pre-chilled low oxygen transfer bottles. Ideally, brown borosilicate glass with stopper and screw cap or tight crimp seals. Also good is PETE type cell culture bottles (which have low O2 and CO2 transfer).

4) Stability testing: The only truly applicable method is real time studies in various packages at your intended storage conditions 4-8 DEG C. One generally would perform 24 hours at 37 DEG C and then 24 hours at 20 DEG C and then regular store at 4-8 DEG C (this simulates generally shipping conditions of one stressful day in a delivery truck, one day waiting for receiving to put kit in refrigerator and then the remaining time for storage as needed).


5) Some initial accelerated stability testing can be performed to stress the conjugate and see if any changes in packaging methods will have an effect. These include the following: a) make up 16 bottles each of your conjugate ( 8 in the stabilizer, and 8 in your regular assay buffer or  diluent).

Before bottling, remove enough to run your assay (to set baseline characteristics).

Additional bottles may be made if your decide to test different packages (ie, glass versus plastic etc). Each bottle MUST be made up to the volume intended for final use so that head space/void space is at actual dimensions.


b) Place Two sets in 37 DEG C bath, two at room temp, two at 4-8 DEG C and two at -20 DEG C (to study frozen effects long term).

c) Each 24 hours, open one designated set, remove only enough to run a standard curve . Close vial and place back in designated temp. Repeat every 48 hours until such time that the 37 DEG C conjugate looses enough activity to make it unsuitable for your assay. Then continue testing once per week observing the room temp studies. At conclusion of study, check the unopened vials and compare that activity with activity at beginning and from the last of your sampled vials (this checks to see if some of the additional loss of activity was due to opening and closing the vials themselves).


As you can see, this requires a comprehensive evaluation. Since there are too many factors involved, we cannot say what effect any one item might have on the result. The varied stability/purity of the  conjugate itself will have a great effect on the stability of any stabilizer to be of help.


For IN Vitro Research or Further Manufacturing Use Only.


RDI Division of Fitzgerald Industries Intl

34 Junction Square Drive

Concord MA 01742-3049

USA

phone (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

Email:antibodies@fitzgerald-fii.com

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