FAQ's

Can reagents be purchased separately?

All reagents are available for purchase separately. Although a particular lot may not be available, all IFA Product Reagents are Universal within their respective test systems.

Are reagents universal (within a test system, between different test systems, etc)?

All IFA Reagents are Universal within their test system and can be used independent of lot as long as the following are adhered to: The Product Number for the new reagent lot matches the proper Product Number for that test system, the reagent must have been stored in its required storage conditions and the new reagent is still within its expiration dating.

How can a cutoff be determined for pos/neg results?

The cut off to all IFA Test Systems is dependent on the initial dilution required by that specific test system Package Insert. Positivity at and above that initial starting dilution means that the patient sample is considered positive. A lack of fluorescence at the starting dilution means that the patient sample is negative. The cutoff is determined by a fluorescence grading system of 1-4+ (4+ being the strongest intensity of fluorescence while the 1+ is the weakest fluorescent staining that is considered significant). All positive control vials provide a manufacturer’s suggested end point titer, or 1+ dilution, that can be used as a reference to judge minimally reactive, 1+ end point fluorescence. Anything equal to or above this fluorescence is positive while anything less intense is negative. Note: Due to laboratory environment, instrument variations & lab tech subjectivity, this 1+ dilution can only be considered a recommended dilution. It is fully acceptable for laboratories to show variance within +/- 1 serial dilution from the recommended.

Will negative results display a complete lack of fluorescence?

Negative results rely on the 1 – 4+ fluorescence grading system. 1+ will be the weakest fluorescence intensity that is considered reactive. While a sample may exhibit some fluorescence, positivity is dependent on its comparison to the 1+ fluorescence grade. Therefore, if the fluorescence is below the 1+ intensity the result would be considered negative.

How long can reagents be left out of their required temperature requirements?

Reagents should be allowed to come to room temperature for testing and then returned to their required storage conditions immediately after use.

What is the best way for slides to come to room temperature?

IFA Kits will be shipped in a specific configuration so that all reagents are grouped together. The amount of slides required for a particular day’s testing should be pulled and laid out on the lab bench, spread out from each other. If slides are allowed to remain grouped together, they will take longer to come to room temperature. Therefore, all slides should be laid out and allowed to come to room temperature evenly. Please note that slides should remain in their pouches until ready to add the diluted sample and controls.

What are the ZORBA® Reagents and are they required to perform testing or are they optional?

ZORBA® IgG Removal Reagent should be used for pre-treatment when running IgM IFA Test Systems. ZORBA® NS Sample Diluent is not required for use on any particular test system, yet it should be used to reduce any possibility of conflicting sample related effects. Utilization of the ZORBA® Reagents will ensure that samples have been pretreated to the best of their ability and this will reduce both cost & product waste by minimizing the need to retest samples that contain the interfering substances.

Can PBS/Mounting Media from another manufacturer be utilized for this test system? All test systems have been optimized utilizing the reagents provided in each test system. ZORBA® manufactured by ZEUS Scientific and optimized for our test systems only. Other manufacturer’s reagents may not consist of the same reagents and formulation.

What is the proper technique for washing slides?

In order to properly wash slides; one needs a Squeeze Bottle, a Coplin Jar and a Slide Holder. While slides are close to the end of their incubation period, the Slide Holder should be placed in the Coplin Jar and then filled with fresh PBS. The Squeeze Bottle should then also be filled with PBS. The slides should be removed from the Moist Chamber, one at a time, and then the slides should be gently rinsed with the Squeeze Bottle PBS. The optimal way to perform this would be to direct the stream of PBS on the slide mask and not on the wells themselves. The slides should be tilted forward, gently rinsed and then tilted backwards and gently rinsed again. After this, the slides should have any excess liquid expelled from them by “snapping” the slide with a quick snap motion. Performing the gentle wash step in this format will reduce the possibility of well to well cross contamination. It is important to avoid aiming the PBS Stream directly on the wells as this can dislodge substrate and interfere with results. Once this has been completed, the slide should be placed in the Slide Holder, in the Coplin Jar and then the next consecutive slide should be processed in the same manner. Package Insert Specifications should be followed for each test system’s respective wash requirement. Most IFA Assays require a change of PBS in between wash incubations. This should be performed by removing the slide holder from the Coplin Jar and then the old PBS should be removed and fresh PBS should be added to the Coplin Jar. The Slide Holder should then be immediately replaced in the Coplin Jar for their second wash incubation. Unless otherwise specified to dry slides after the wash procedure, the slides should remain in the Coplin Jar filled with PBS and processed for Conjugation or Cover Slipping individually, while remaining slides are still in PBS as to avoid drying. Slides that do not require drying after washing will contain a Blotter in the Slide Packaging. After the required Wash Steps have been performed, the blotter should be laid on the lab bench (Note: avoid placing them on another absorptive surface) and then the slide should be lined up with the Blotter so that the wells match up with the holes in the Blotter. The actual well surface should never come in contact with the Blotter or any other absorptive surface. Once the slide has been aligned, a paper towel should be used to dry the full glass, back portion of the slide. After this has been completed, the slide mask should be inspected for any additional moisture. If the slide mask appears dry, then the slides are ready to be Conjugated or Cover Slipped depending on the next consecutive step in the Package Insert.

What is meant by a recommended endpoint titer?

A recommended endpoint titer refers to the last dilution that shows a measurable degree of reactivity. It is considered recommended because it was derived during the production of the Positive Control & it was the dilution that yielded a 1+, Minimally Reactive Result. This is the weakest fluorescence grade that is considered significant. Due to the subjectivity of the IFA Test Systems from differences in laboratory environments, instrumentation and laboratory technologist subjectivity, this endpoint titer may vary between laboratories. Therefore ZEUS Scientific recommends that each laboratory establish its own endpoint titer using the provided recommended endpoint titer as a reference. It has been found that most variation is still within 1+/- titer from the recommended endpoint titer, if it does not exactly match up with the recommended endpoint titer for that specific lab.

How do I utilize the IgG Removal Reagent for IgM assays and is this dilution my starting for titrations?

The ZORBA® IgG Removal Reagent should be allowed to warm to room temperature before testing has been initiated. Prior to testing, the ZORBA® IgG Removal Reagent should be shaken well. Then a 1:10 screening dilution should be created by diluting 5uL of patient sera with 45uL of ZORBA IgG Removal Reagent. The sample is now ready for screen testing. If the sample is to be further tittered, the serial dilutions should be made in PBS with the initial dilution being made in the ZORBA® IgG Removal Reagent as the starting dilution. Flocculent material may form due to immune complex formations. This material will not interfere with the test system yet can be removed by centrifugation prior to testing if desired.

What are the benefits of screening with Zorba® Reagent as the diluents?

ZORBA® Reagents are specifically geared toward removing interfering substances with patient sera to better prepare patient sera for optimal IFA testing conditions. The ZORBA® NS Reagent was developed and optimized to reduce nonspecific fluorescence, resulting from the non-immunological attachment of immunoglobulins to a substrate. This effect may vary from sample to sample but if nonspecific interference is high within a sample,  it can mask a potential pattern and would have to be reported as uninterpretable.  Proactive use of this reagent will minimize retesting requirements for samples with high nonspecific concentrations.

The ZORBA® IgG Removal Reagent was designed to functionally remove potentially interfering immunoglobulin G (IgG) antibodies from human sera prior to testing for IgM. Specific IgG in a patient specimen may compete with specific IgM for antigenic sites on the substrate. Depending on the ratio of IgG to IgM, false negative IgM results may be obtained. In addition, RF IgM  (IgM class anti-IgG) if present in conjunction with specific IgG, may interact with the antigen bound IgG resulting in a false positive IgM test. Therefore accurate measurement of pathogen-specific IgM is highly dependent upon the effective removal or neutralization of specific IgG. Proactive use of this reagent will minimize this effect and provide more accurate testing results, meaning that further repeat and reference testing would be minimized as well.