Troubleshooting Immunoassays

Troubleshooting normally proceeds along a logical investigation path:

Sandwich assay troubleshooting guide:

Inconsistent absorbances across the plate, or otherwise atypically spurious results

Plates stacked during incubations

Stacking of plates does not allow even distribution of temperature across the wells of the plates. Avoid stacking.

Pipetting inconsistent

Ensure pipettes are working correctly and are calibrated. Ensure pipette tips are pushed on far enough to create a good seal. Take particular care when diluting down the plate and watch to make sure the pipette tips are all picking up and releasing the correct amount of liquid. This will greatly affect consistency of results between duplicates

Antibody dilutions/reagents not well mixed

To ensure a consistent concentration across all wells, ensure all reagents and samples are mixed well and allowed to equilibrate at room temperature before pipetting onto the plate.

Wells allowed to dry out

Do not allow plates to become dry by leaving unattended for a prolonged period following washing steps.

Inadequate washing

Some wells may not be washed as well as others, leaving different amounts of unbound antibody behind giving inconsistent results.

Bottom of the plate is dirty affecting absorbance readings

Clean the bottom of the plate carefully, and then read the plate again.

For Ultra-Sensitive AMH ELISA and picoAMH ELISA Customers outside the United States

Ansh Labs has two main assays for human AMH, the original Ultra-Sensitive (U.S.) AMH kit and an assay designed with even more sensitivity for measuring the very low AMH levels in peri-menopausal women, the picoAMH ELISA, AL-124-i. The U.S. AMH assay can be used to measure AMH levels between 0.06 and 23 ng/mL without dilution. The picoAMH assay is designed to measure AMH levels between 0.006 and 1.0 ng/mL without dilution, but specimens with AMH levels between 1.0 ng/mL and >200 ng/mL (e.g., women < 40 years of age, women with PCOS, children) must be diluted before testing.

Recently, the picoAMH has been cleared by the U.S. FDA for the clinical assessment of menopausal status. Within the U.S., the item will be referenced as MenoCheck picoAMH ELISA AL-124 (note that there is no -i at the end). For the international markets, especially Europe with the CE mark, the catalog number is and will continue to be AL-124-i. We are adding the MenoCheck data to the existing IFU to complement the existing IFU data. The IFU can be accessed by clicking here.

We know that several countries will require an RUO version because the product may not be registered with the Ministry of Health. Also, because the picoAMH ELISA is so versatile for clinical assessment of menopausal status as well as clinical research into areas such as oncofertility, primary ovarian insufficiency, and related fields, we have maintained a ‘research use only’ version of the picoAMH ELISA to ensure that laboratories and researchers avoid any potential conflicts with off-label uses. The IFU for the RUO version can be accessed by clicking here.

Summary:

If you are outside the U.S. and you want to measure…. Then you should order…. Regulatory Status

Women with normal range AMH up to PCOS samples U.S. AMH ELISA, AL-105-i CE Mark

Women for the clinical measurement of AMH for any condition where the concentration is very low, to include Menopause. The labeling references multiple applications. picoAMH (MenoCheck) ELISA, AL-124-i CE Mark

This is the Research Use Only version of the original picoAMH ELISA assay. The intended use is a generic “for the measurement of AMH in serum”. Some countries need an RUO label in case the assay is not registered. This also allows labs to use it for off-label uses. picoAMH ELISA, AL-124-r Research Use Only

For picoAMH ELISA AL-124 Customers in the United States

Ansh Labs recently received FDA clearance for the picoAMH ELISA kit to be used as an aid in the determination of menopausal status in women thus the kit has now been rebranded as MenoCheck ELISA, AL-124. To learn more about this test, please visit www.MenoCheck.com or download the product flyer by clicking here. The IFU can be downloaded by clicking here.

Because the original picoAMH ELISA is so versatile for clinical assessment of menopausal status as well as clinical research into areas such as oncofertility, primary ovarian insufficiency, and related fields, we have maintained a ‘Research Use Only’ version of the picoAMH ELISA to ensure that laboratories and researchers avoid any potential conflicts with off-label uses.

Summary:

If you are in the United States and you want to measure…. Then you should order…. Regulatory Status within U.S.

Women with normal range AMH up to PCOS samples US AMH ELISA, AL-105 Research Use Only

Women for menopausal status MenoCheck picoAMH ELISA, AL-124 FDA Cleared

Women undergoing chemotherapy, have secondary amenorrhea (e.g., premature ovarian failure, hypothalamic amenorrhea, etc), or with pre-testing dilution women with normal to high levels of AMH (e.g., healthy young women, women with PCOS, children) picoAMH ELISA, AL-124-r Research Use Only

For Ultra-Sensitive AMH ELISA AL-105 Customers in the United States

Ansh Labs has two main assays for human AMH, the original Ultra-Sensitive (US) AMH kit and an assay designed with even more sensitivity for measuring the very low AMH levels in peri-menopausal women, the picoAMH kit. The US AMH assay can be used to measure AMH levels between 0.06 and 23 ng/mL without dilution. The picoAMH assay is designed to measure AMH levels between 0.006 and 1.0 ng/mL without dilution, but specimens with AMH levels between 1.0 ng/mL and >200 ng/mL (e.g., women < 40 years of age, women with PCOS, children) must be diluted before testing.

Recently, the picoAMH has been FDA cleared as an aid in the determination of menopausal status in women. This assay is now offered as the MenoCheck picoAMH ELISA AL-124. To learn more about this test, please visit www.MenoCheck.com or download the product flyer by clicking here. The IFU can be downloaded by clicking here.

Because the original picoAMH ELISAs is so versatile for assessment of menopausal status as well as research into areas such as oncofertility, primary ovarian insufficiency, and related fields, we have maintained a ‘Research Use Only’ version of the picoAMH ELISA to ensure that laboratories and researchers avoid any potential conflicts with off-label uses.

Summary:

If you are in the United States and you want to measure…. Then you should order…. Regulatory Status within U.S.

Women with normal range AMH up to PCOS samples US AMH ELISA, AL-105 Research Use Only

Women for menopausal status MenoCheck picoAMH ELISA, AL-124 FDA Cleared

Women undergoing chemotherapy, have secondary amenorrhea (e.g., premature ovarian failure, hypothalamic amenorrhea, etc), or with pre-testing dilution women with normal to high levels of AMH (e.g., healthy young women, women with PCOS, children) picoAMH ELISA, AL-124-r Research Use Only

Bovine AMH Table 1

Reference / Method / Pub Year / Title
(19) / Necklaws / 1986 / Detection of Mullerian inhibiting substance in biological samples by a solid phase sandwich radioimmunoassay
(18) DSL/Beckman 2008 Intrafollicular Steroids and Anti-Mullerian Hormone During Normal and Cystic Ovarian Follicular Development in the Cow
(25) DSL/Beckman 2008 Antral Follicle Count Reliably Predicts Number of Morphologically Healthy Oocytes and Follicles in Ovaries of Young Adult Cattle

Collaboration

Building relationships with customers and key opinion leaders to discover, develop and validate new assays

Developing intellectual property

Training in biomarker immunoassay and laboratory procedures

Immunoassay and Dx Device Development

Custom configuration and assay development

Validate assays for specific diagnostic purpose(s)

Validation of assays in preclinical research animal models

Testing to support regulatory submissions: pre-clinical and/or clinical

Biospecimen Management

Specimen design and collection support, including blood spot testing

Specimen aliquoting and/or distribution services

Biobanking services

Option for return of residual specimen

Chain of custody documentation

Testing

Internal immunoassay of human and animal blood and blood derived biospecimens, focused on:

Ansh assays for biomarkers in Women’s Health: Fertility, Oncofertility, Menopause and Gonadotoxicity, PCOS, Endometriosis, etc.

Ansh biomarkers in Pregnancy: pre-eclampsia, gestational diabetes.

Ansh IGF family and related Biomarkers: metabolic diseases

Ansh TGF-β superfamily biomarkers: in vitro fertilization/embryo development, folliculogenesis, oncology

CLIA-certified, SOP-guided testing of client specimens

Customized testing quality control and quality assurance

Customized validation, as needed, for biobanked specimens

Specimen volume-sparing protocols

Interpretive support

Color developing slowly

Plates are not at the correct temperature

Ensure plates are at room temperature and that the reagents are at room temperature before use. Do not incubate on a lab bench near cool air vents that could lower laboratory temperatures and affect the enzyme-substrate reaction.

Conjugate too weak

Prepare the substrate solutions as instructed before use. Ensure the stock solutions are not expired and that they have been stored correctly. Make sure the prepared reagents are the correct concentration.

Contamination of solutions

Presence of contaminants, such as sodium azide and peroxidase can affect the substrate reaction. Avoid using reagents containing these preservatives

Weak Color Development

Was substrate added at the correct point in the assay?

See the assay procedure provided in the instruction manual.

Were the antibody and conjugate added at the correct time?

See the assay procedure provided in the instruction manual. The antibody and conjugate provided are often color-coded for convenience and to help reduce laboratory errors.

Did all of the components belong to the specific kit being used?

When customers order more than one type of kit, they can sometimes confuse the reagents between kits.

How long was the substrate incubation?

It is possible that Stop Solution was added to the plate without allowing the full substrate incubation.

What were the conditions of the substrate incubation?

If a plate is left to incubate on a cold lab bench or under a drafty area during ambient incubations, signal values (e.g. optical density) may be lower than expected.

Were reagents brought to room temperature prior to use?

It is important to ensure that all reagents are brought to room temperature prior to use, or as mentioned in the product specific instruction manual. Usually leaving the kit out on the bench top at ambient temperature for about half an hour prior to setting up the assay will be sufficient, when the reagents can be stored at 4°C. Frozen volumes take a little more time to come to room temperature. Do not thaw frozen reagents in a water bath. If a different standard/sample diluent is used (such as culture media) this must also be warmed.

Was the plate read at the correct wavelength?

See the assay procedure provided in the instruction manual to ensure you’re reading the plate at the correct wavelength. It may be necessary to check the filters in your plate reader and the program using during reading. If others are using the instrument, they may make changes to the settings for their experiment.

Were the proper volumes of reagents added?

See the assay procedure provided in the instruction manual.

What were the conditions of the incubations?

If the incubation times and temperatures are not observed, this can lead to lower than expected signal values (e.g. optical density). Pay attention that in air-conditioned rooms the temperature does not drop below 21°C.

How was the plate shaken during incubations (if required)?

If customers do not have a plate shaker, they will often use an orbital flask shaker or some other piece of equipment. This is not a problem as long as the liquid is vigorously displaced about 3/4 of the way up the sides of the wells without coming out. It is very important that the plate is secured into place. If the plate is not shaken and it is required in the procedure, a longer incubation may be necessary to bring the reagents to equilibrium.

How long after the addition of Stop Solution was the plate read?

The plate needs to be read at the correct wavelength as soon as possible after the addition of the Stop Solution. We generally recommend that the plate be read within 10 minutes.

High absorbance values for samples and/or positive control (absorbance does not go down as the sample is diluted down the plate)

The concentration of samples or positive control is too high and out of range for the sensitivity of the assay. Re-assess the assay you are using OR reduce the concentration of samples and control by dilution before adding to the plate. Take the dilution into account when calculating the resulting concentrations.

Low absorbance values/low RLUs

Target protein not expressed or not immunoreactive in the sample or species used

Check the expression profile of the target protein to ensure it will be expressed in your samples. If there is low level of target protein expression, increase the amount of sample used, or you may need to change to a more sensitive assay.

Insufficient antibody

Check the recommended amount of antibody is used. The concentration of antibody may have to be increased to optimize performance.

Substrate solutions not fresh or combined incorrectly

Prepare the substrate solutions as instructed before use. Ensure the stock solutions are not expired and that they have been stored correctly. Make sure the prepared reagents are the correct concentration.

Reagents not fresh or not at the correct pH

Ensure reagents have been prepared correctly and not expired.

Short incubation time

Ensure you are incubating the antibody for the recommended amount of time, if an incubation time is suggested. The incubation time may require increasing for optimal results.

Incubation temperature too low

These assays are optimized for room temperature. Ensure the incubations are carried out at the correct temperature. Do not expose to air vents or cross currents that may affect the incubating temperatures. Incubation temperature may require some optimization. Ensure all reagents are at room temperature before proceeding.

Stop solution not added

Addition of stop solution increases the intensity of color reaction and stabilizes the final color development.

High background across entire plate

Conjugate too strong or incubated for too long

Check dilution of conjugate. Use at the recommended dilution. In colorimetric assays, color will develop rapidly.

Substrate solution or stop solution is not fresh

Use fresh substrate and stop solutions. If either solution is not clear, (i.e., yellowish) it an indication the reagent is contaminated.

Reaction not stopped in time, or at all

Color will keep developing if the substrate reaction is not stopped.

Plate left too long before reading on the plate reader

Always read the plate within the optimal reading timeframe suggested. In chemiluminescence assays, RLUs will decline rapidly after a certain amount of time. In colorimetric assays, the color will keep developing at a slow rate even after adding the stop solution.

Contaminants from laboratory glassware

Ensure reagents are fresh and prepared in clean glassware free of any contaminants or residual detergents.

Substrate incubation carried out in direct sunlight

Substrate incubation should be carried out away from bright light or direct sunlight.

Incubation temperature too high

These assays are optimized for room temperature. Ensure the incubations are carried out at the correct temperature. Do not expose to air vents or cross currents that may affect the incubating temperatures. If incubators are used set at the correct temperature. Incubation temperature may require some optimization.

Edge Effects

Where was the plate incubated?

Often times the conditions for ambient incubations can be less than ideal. If there is a draft in the area or the plate is incubated on a cold lab bench, this can lead to uneven color development.

If multiple plates were run, were they stacked on top of each other during incubation?

Multiple plates should only be incubated in a single layer. This will assure that no area of the plate is at a different temperature than any other.

If a non-ambient incubation was required, was the plate properly sealed?

Making sure that the plate sealer is tightly covering all of the wells will help to discourage uneven evaporation of the well contents, or condensation for colder incubation conditions.

Poor Standard Curve

What was used as the standard diluent?

Diluents other than the supplied assay buffer may contain interfering substances that can affect the standard curve.

How was the precision of the standard curve?

If the %CV values for the standard curve signal values (e.g. optical density) are consistently above 5%, it may be a good idea to pay particular attention to pipetting technique. If the standard curve signal values were acceptable but the sample precision was not, the problem relates to the sample. Also, see recommendations under “Poor Precision”.

Were the Blank and NSB values subtracted out?

If the net signal values (e.g. optical density) are not used, the signal values will appear higher than those presented in the sample data in the instruction manual.

How were the standard dilutions prepared?

It is important that test tubes of an appropriate size and material are used. Standard dilutions must be properly mixed (e.g. vortexed) while preparing the serial dilutions. It is also crucial that the standard dilutions be prepared and used within the time specified in the product specific instruction manual. Never store unused standard dilutions for a later use.

Poor Precision

Were the wells washed properly?

All wells receive the same treatment during the wash step. If some are washed less than others, this can translate to poor precision. It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels.

Were the wells aspirated sufficiently after the wash steps?

It is very important that as little Wash Buffer as possible remains in the wells after aspiration. Residual buffer can cause dilution of subsequent reagents. After the last wash step, it is a good idea to hit the plate several times over a piece of paper toweling to remove excess buffer.

How were reagents pipetted into wells?

In order to eliminate precision error, customers need to remember to pre-rinse all pipet tips used in the assay. We usually recommend that the customer draw up the liquid into the tip and aspirate it three times prior to addition into the well. Regular pipet calibration and maintenance is also essential to ensure that the tips fit properly and that the correct volumes are dispensed. Be sure reagents are not splashed between wells or outside of the wells during pipeting (especially if using repeater pipets).

Drift

Were reagents brought to room temperature prior to use?

If the reagents are not at a constant temperature prior to their addition into the wells, the results from one side of the plate to the other can differ depending on the temperatures at addition.

Was the set-up of the assay interrupted?

If the assay is interrupted at any point during the addition of reagents, it is possible that differing results will be seen before the interruption versus after. The wells that had reagents added before the interruption will have been incubating for longer than those after.

High background in Zero (NSB, blank)

Contamination of reagents/samples

May be contamination of reagents or samples, or cross contamination from splashing between wells. Use fresh reagents and pipette carefully.

Insufficient washing of plates

Ensure well areas are washed adequately by filling the wells with wash buffer. Ensure all residual antibody solutions are removed before washing.

Too much antibody used leading to non-specific binding

Check the recommended amount of antibody suggested. Try using less antibody, or diluting antibody in appropriate buffer.

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