Immunoassay Reagent Test Kits – PRODUCT CATALOG

Thank you for your interest in Ansh Labs immunodiagnostic products. We are experts in developing protein hormone immunoassays for research and clinical diagnostics. We are mainly focused on research areas involving hormones of the TGF-Beta superfamily, PAPP-A family of proteases, and the IGF system. Our assay methods are used for many biomedical research purposes ranging from pre-clinical biomarker studies to translational research in the following fields:

Reproductive endocrinology
Pregnancy complications
Atherosclerotic cardiovascular disease
Neurodegenerative diseases

Cat#	Brand	Description	Intended Use	Size/ Unit	Pkg	Price
AL-205	AnshLite™	AMH, Ultra-Sensitive CLIA	RUO	96-well	kit	Inquire
AL-105		AMH, Ultra-Sensitive ELISA	RUO	96-well	kit	Inquire
AL-205	AnshLite™	AMH, Bovine CLIA	RUO	96-well	kit	Inquire
AL-105		AMH, Bovine ELISA	RUO	96-well	kit	Inquire
AL-205	AnshLite™	AMH, Equine CLIA	RUO	96-well	kit	Inquire
AL-105		AMH, Equine ELISA	RUO	96-well	kit	Inquire
AL-205	AnshLite™	AMH, Rat/Mouse CLIA	RUO	96-well	kit	Inquire
AL-105		AMH, Rat/Mouse ELISA	RUO	96-well	kit	Inquire
AL-210	AnshLite™	Activin A CLIA	RUO	96-well	kit	Inquire
AL-110		Activin A ELISA	RUO	96-well	kit	Inquire
AL-217	AnshLite™	Follistatin CLIA	RUO	96-well	kit	Inquire
AL-117		Follistatin ELISA	RUO	96-well	kit	Inquire
AL-223	AnshLite™	Inhibin A CLIA	RUO	96-well	kit	Inquire
AL-123		Inhibin A ELISA	RUO	96-well	kit	Inquire
AL-107	AnshLite™	Inhibin B CLIA	RUO	96-well	kit	Inquire
AL-217		Inhibin B ELISA	RUO	96-well	kit	Inquire
AL-208	AnshLite™	MBP CLIA	RUO	96-well	kit	Inquire
AL-108		MBP ELISA	RUO	96-well	kit	Inquire
AL-206	AnshLite™	PAPP-A CLIA	RUO	96-well	kit	Inquire
AL-106		PAPP-A ELISA	RUO	96-well	kit	Inquire
AL-209	AnshLite™	PAPP-A2 CLIA	RUO	96-well	kit	Inquire
AL-109		PAPP-A2 ELISA	RUO	96-well	kit	Inquire
AL-201	AnshLite™	picoPAPP-A CLIA	RUO	96-well	kit	Inquire
AL-101		picoPAPP-A ELISA	RUO	96-well	kit	Inquire

RUO: Unless otherwise stated in our catalog or other company documentation accompanying Ansh Labs product(s), the products are intended for Research Use Only, and not for in vitro diagnostic purposes, or therapeutic uses of any type.

Immuno Assay Development

ELISAs are used to determine the concentration of a target antigen in a sample. This is a highly sensitive and simple assay type to perform.

We utilize the ‘sandwich’ type ELISA to determine the antigen concentration in unknown samples. This ELISA method is fast and accurate, and if a purified antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample.

The sandwich ELISA requires two antibodies that bind to unique epitopes on the antigen. After producing dozens of antibodies, we select the optimal pair of monoclonal antibodies for capture and detection of the exact epitope(s) that give the assay the specificity and sensitivity required to measure the analyte in biological specimens. We purify the capture antibody and bind it to the bottom of a microtiter plate well. Antigen is added and allowed to complex with the bound antibody. Unbound products are then washed away, and a second antibody used for detection is added and allowed to bind to the antigen to form a “sandwich” complex. The assay is quantitated by measuring the amount of labeled second antibody bound to the matrix.

We accomplish this using a horseradish peroxidase (HRP)-based system for enzymatic catalysis of a colorimetric substrate such as TMB (tetramethylbenzidine), or our proprietary AnshLite™ luminogenic substrate.

Sandwich-type Assay Validation Process

Spike and recovery is performed using the zero standard, low, mid, and high standard concentrations spiked into at least 8 different samples in duplicate. This test should be repeated at least two times to determine if results are reproducible and acceptable.
Linearity tests are performed on at least 8 different samples in duplicate. A minimum of three dilutions of the neat samples should be performed for this test. This same test should be repeated at least two times to verify the results are reproducible and acceptable.
Intra-assay variation is tested by running 8 different samples (of varying concentration) in replicates of ten across the microtiter plate and determining the % CVs of the samples. Acceptable CVs are <10%.
Inter-assay variation is determined by evaluating at least 8 samples (of varying concentration) in duplicate on at least three different microtiter plates on different days using the same reagent lots. Acceptable CVs are <10%.
The limit of detection of an analyte in a sample that can be detected with a 95% probability (n=20) is determined by processing nine serum samples in the low calibrator range following CLSI EP17 guidelines. Ten assay runs were performed over six days with all samples run in duplicate per run.
We determine specificity of the assay by running standards or stock materials of several similar analytes of available species in the assay to determine if any similar analytes can be detected by the assay. Some cases, when we have samples available for this purpose, we determine species immunoreactivity.
If appropriate, we will compare sample results using our kits to sample values obtained using a commonly available assay to assess the correlation of sample values.
When appropriate, we will compare serum vs. plasma values to determine the correlation between different sample types, biological fluids, or cell/tissue culture media.

Sandwich assay troubleshooting guide:

Usual Problem	Common Cause(s)	Resolution(s)
1. 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.
2. 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 is 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.
3. 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.
4. 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.
5. 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.
6. 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.

Thank you for your interest in Ansh Labs immunodiagnostic products. We have expertise in developing protein hormone immunoassays for research and clinical diagnostics. We are focused in research areas involving hormones of the TGF-Beta Superfamily, PAPP-A Family, and the IGF System. Our assay methods are used for many biomedical research purposes ranging from pre-clinical biomarker studies to translational research in the following fields:

Reproductive endocrinology
Pregnancy complications
Atherosclerotic cardiovascular disease
Neurodegenerative diseases

Cat#	Brand	Description	Intended Use	Size/ Unit	Pkg	Price
AL-205-i	AnshLite™	AMH, Ultra-Sensitive CLIA	CE	96-well	kit	Inquire
AL-105-i		AMH, Ultra Sensitive ELISA	CE	96-well	kit	Inquire
AL-214	AnshLite™	AMH, Bovine CLIA	CE	96-well	kit	Inquire
AL-114		AMH, Bovine ELISA	CE	96-well	kit	Inquire
AL-215	AnshLite™	AMH, Equine CLIA	CE	96-well	kit	Inquire
AL-115		AMH, Equine ELISA	CE	96-well	kit	Inquire
AL-213	AnshLite™	AMH, Rat/Mouse CLIA	CE	96-well	kit	Inquire
AL-113		AMH, Rat/Mouse ELISA	CE	96-well	kit	Inquire
AL-210-i	AnshLite™	Activin A CLIA	CE	96-well	kit	Inquire
AL-110-i		Activin A ELISA	CE	96-well	kit	Inquire
AL-217-i	AnshLite™	Follistatin CLIA	CE	96-well	kit	Inquire
AL-117-i		Follistatin ELISA	CE	96-well	kit	Inquire
AL-223-i	AnshLite™	Inhibin A CLIA	CE	96-well	kit	Inquire
AL-123-i		Inhibin A ELISA	CE	96-well	kit	Inquire
AL-207-i	AnshLite™	Inhibin B CLIA	CE	96-well	kit	Inquire
AL-107-i		Inhibin B ELISA	CE	96-well	kit	Inquire
AL-208	AnshLite™	MBP CLIA	RUO	96-well	kit	Inquire
AL-108		MBP ELISA	RUO	96-well	kit	Inquire
AL-206-i	AnshLite™	PAPP-A CLIA	CE	96-well	kit	Inquire
AL-106-i		PAPP-A ELISA	CE	96-well	kit	Inquire
AL-209-i	AnshLite™	PAPP-A2 CLIA	CE	96-well	kit	Inquire
AL-109-i		PAPP-A2 ELISA	CE	96-well	kit	Inquire
AL-201	AnshLite™	picoPAPP-A CLIA	RUO	96-well	kit	Inquire
AL-101		picoPAPP-A ELISA	RUO	96-well	kit	Inquire

Immuno Assay Development

ELISAs are used to determine the concentration of a target antigen in a sample. This is a highly sensitive and simple assay type to perform.

Sandwich-type Assay Validation Process

Spike and recovery is performed using the zero standard, low, mid, and high standard concentrations spiked into at least 8 different samples in duplicate. This test should be repeated at least two times to determine if results are reproducible and acceptable.
Linearity tests are performed on at least 8 different samples in duplicate. A minimum of three dilutions of the neat samples should be performed for this test. This same test should be repeated at least two times to verify the results are reproducible and acceptable.
Intra-assay variation is tested by running 8 different samples (of varying concentration) in replicates of ten across the microtiter plate and determining the % CVs of the samples. Acceptable CVs are <10%.
Inter-assay variation is determined by evaluating at least 8 samples (of varying concentration) in duplicate on at least three different microtiter plates on different days using the same reagent lots. Acceptable CVs are <10%.
The limit of detection of an analyte in a sample that can be detected with a 95% probability (n=20) is determined by processing nine serum samples in the low calibrator range following CLSI EP17 guidelines. Ten assay runs were performed over six days with all samples run in duplicate per run.
We determine specificity of the assay by running standards or stock materials of several similar analytes of available species in the assay to determine if any similar analytes can be detected by the assay. Some cases, when we have samples available for this purpose, we determine species immunoreactivity.
If appropriate, we will compare sample results using our kits to sample values obtained using a commonly available assay to assess the correlation of sample values.
When appropriate, we will compare serum vs. plasma values to determine the correlation between different sample types, biological fluids, or cell/tissue culture media.

Sandwich assay troubleshooting guide:

Usual Problem	Common Cause(s)	Resolution(s)
1. 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.
2. 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 is 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.
3. 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.
4. 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.
5. 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.
6. 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.

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Immunoassay Reagent Test Kits – PRODUCT CATALOG

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