Beta-2-Microglobulin

The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of mouse b2M in serum, plasma, urine and other biological fluids. For research use only


Controlling of reaction time:
Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading. 6. TMB Substrate is easily contaminated. Please protect it from light. 7. The environment humidity which is less than 60% might have some effects on the final performance, therefore, a humidifier is recommended to be used at that condition.

CALCULATION OF RESULTS
This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between b2M concentration in the sample and the assay signal intensity.
Average the duplicate readings for each calibrator, control, and samples. Create a calibration curve with the log of b2M concentration on the y-axis and absorbance on the x-axis. Draw a best fit curve through the points and it can be determined by regression analysis. If samples have been diluted, the concentration read from the calibration curve must be multiplied by the dilution factor.

Sensitivity
The minimum detectable dose of b2M is typically less than 48.9 pg/mL.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by subtracting two standard deviations to the mean optical density value of twenty zero calibrator replicates and calculating the corresponding concentration.

Specificity
This assay has high sensitivity and excellent specificity for detection of b2M.
No significant cross-reactivity or interference between b2M and analogues was observed.

Note:
Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between b2M and all the analogues, therefore, cross reaction may still exist.
2. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is only for reference. 3. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer. 4. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism. TMB Substrate should remain colorless till it is reacted with the enzyme which binds to the microplate. 5. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microplate from the storage bag until needed. 6. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 O.D. at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment. 7. Variation in sample preparation and each step of experimental operation may cause different results. In order to get better reproducible results, the operation of each step in the assay should be controlled. 8. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intraassay variance among kits from different batches might arise from above factors, too. 9. Kits from different manufacturers with the same item might produce different results, since we haven't compared our products with other manufacturers. 10. The calibrator of the kit and immunogen used for antibody preparation are commonly recombinant proteins, as different fragments, expression systems, purification methods might be used in recombinant protein preparation, we can not guarantee the kit could detect recombinant protein from other companies. So, it is not recommended to use the kit for the detection of recombinant protein. 11. Please predict the concentration of target molecules in samples, or arrange a preliminary experiment, it is a good way to solve specific problem, e.g. the concentration of samples are beyond the detection range of the kit. 12. The kit might not be suitable for detection of samples from some special experiment, for instance, knock-out experiments, due to their uncertainty of effectiveness. 13. The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

FOR RESEARCH USE ONLY.
KAMIYA BIOMEDICAL COMPANY

INTENDED USE
The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of mouse b2M in serum, plasma, urine and other biological fluids. For research use only.

Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1

STORAGE
All the reagents should be kept according to the labels on vials. The Calibrator, Detection Reagent A, Detection Reagent B, Reagent Diluent and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4°C. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. Opened test kits will remain stable for 1 month, provided it is stored as described above.

TEST PRINCIPLE
This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to b2M has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled b2M and unlabeled b2M (Calibrators or samples) with the pre-coated antibody specific to b2M. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of b2M in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of b2M in the sample.

SAMPLE COLLECTION AND STORAGE Serum
Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1,000 x g. Assay freshly prepared serum immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1,000 x g at 4°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Urine
Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤-20°C. Avoid repeated freezethaw cycles.

Other biological fluids
Centrifuge samples for 20 minutes at 1,000 x g. Collect the supernates and assay immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Note:
1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination. 2. When performing the assay, bring samples to room temperature. 3. Sample hemolysis will influence the result, so hemolytic specimen should not be used. 4. It is highly recommended to use serum instead of plasma for the detection based on quantity of our in-house data.

REAGENT PREPARATION
Bring all kit components and samples to room temperature (18-25°C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.

Calibrator
Reconstitute the Calibrator with 1.0 mL of Calibrator Diluent, kept for 10 minutes at room temperature, shake gently (not to foam). The concentration of the calibrator in the stock solution is 10,000 pg/mL. Please prepare 5 tubes containing 0.6 mL Calibrator Diluent and produce a triple dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted calibrator such as 10,000 pg/mL, 3

Detection Reagent B
Briefly spin or centrifuge the stock Detection Reagent B before use. Dilute to the working concentration with Assay Diluent B (1:100).

Wash Solution
Dilute 20 mL of Wash Solution Concentrate (30X) with 580 mL of de-ionized or distilled water to prepare 600 mL of Wash Solution (1X).

TMB substrate
Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:
1. Prepare calibrator within 15 minutes before assay. Pease do not dissolve the reagents at 37°C directly. 2. Making serial dilution in the wells directly is not permitted. 3. Detection Reagent A and B are sticky solutions, therefore, slowly pipette them to reduce the volume errors. 4. Please carefully reconstitute Calibrators or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10 μL for one pipetting. 5. The reconstituted Calibrators, Detection Reagent A and Detection Reagent B can be used only once. 6. If crystals have formed in the Wash Solution concentrate (30X), warm to room temperature and mix gently until the crystals are completely dissolved. 7. Contaminated water or container for reagent preparation will influence the detection result.

SAMPLE PREPARATION
1. We are only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the calibration curve, users must determine the optimal sample dilutions for their particular experiments. 4. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
5. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
6. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products. Incubation time and temperature must be controlled. 4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and falsely elevated absorbance reading.

Controlling of reaction time:
Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading. 6. TMB Substrate is easily contaminated. Please protect it from light. 7. The environment humidity which is less than 60% might have some effects on the final performance, therefore, a humidifier is recommended to be used at that condition.

CALCULATION OF RESULTS
This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between b2M concentration in the sample and the assay signal intensity.
Average the duplicate readings for each calibrator, control, and samples. Create a calibration curve with the log of b2M concentration on the y-axis and absorbance on the x-axis. Draw a best fit curve through the points and it can be determined by regression analysis. If samples have been diluted, the concentration read from the calibration curve must be multiplied by the dilution factor.

Sensitivity
The minimum detectable dose of b2M is typically less than 48.9 pg/mL.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by subtracting two standard deviations to the mean optical density value of twenty zero calibrator replicates and calculating the corresponding concentration.

Specificity
This assay has high sensitivity and excellent specificity for detection of b2M.
No significant cross-reactivity or interference between b2M and analogues was observed.

Note:
Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between b2M and all the analogues, therefore, cross reaction may still exist.

IMPORTANT NOTE
1. The final experimental results will be closely related to validity of the products, so the kit should be used prior to the expiration date. And please store the kits exactly according to the instruction.
2. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is only for reference. 3. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer. 4. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism. TMB Substrate should remain colorless till it is reacted with the enzyme which binds to the microplate. 5. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microplate from the storage bag until needed. 6. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 O.D. at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment. 7. Variation in sample preparation and each step of experimental operation may cause different results. In order to get better reproducible results, the operation of each step in the assay should be controlled. 8. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intraassay variance among kits from different batches might arise from above factors, too. 9. Kits from different manufacturers with the same item might produce different results, since we haven't compared our products with other manufacturers. 10. The calibrator of the kit and immunogen used for antibody preparation are commonly recombinant proteins, as different fragments, expression systems, purification methods might be used in recombinant protein preparation, we can not guarantee the kit could detect recombinant protein from other companies. So, it is not recommended to use the kit for the detection of recombinant protein. 11. Please predict the concentration of target molecules in samples, or arrange a preliminary experiment, it is a good way to solve specific problem, e.g. the concentration of samples are beyond the detection range of the kit. 12. The kit might not be suitable for detection of samples from some special experiment, for instance, knock-out experiments, due to their uncertainty of effectiveness. 13. The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

INTENDED USE
The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of mouse b2M in serum, plasma, urine and other biological fluids. For research use only.

COMPONENTS Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1

STORAGE
All the reagents should be kept according to the labels on vials. The Calibrator, Detection Reagent A, Detection Reagent B, Reagent Diluent and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4°C. The unused strips should be kept in a sealed bag with the desiccant provided to minimize exposure to damp air. Opened test kits will remain stable for 1 month, provided it is stored as described above.

TEST PRINCIPLE
This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to b2M has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled b2M and unlabeled b2M (Calibrators or samples) with the pre-coated antibody specific to b2M. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of b2M in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of b2M in the sample.

SAMPLE COLLECTION AND STORAGE Serum
Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1,000 x g. Assay freshly prepared serum immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1,000 x g at 4°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Urine
Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at ≤-20°C. Avoid repeated freezethaw cycles.

Other biological fluids
Centrifuge samples for 20 minutes at 1,000 x g. Collect the supernates and assay immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Note:
1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination. 2. When performing the assay, bring samples to room temperature. 3. Sample hemolysis will influence the result, so hemolytic specimen should not be used. 4. It is highly recommended to use serum instead of plasma for the detection based on quantity of our in-house data.

REAGENT PREPARATION
Bring all kit components and samples to room temperature (18-25°C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.

Calibrator
Reconstitute the Calibrator with 1.0 mL of Calibrator Diluent, kept for 10 minutes at room temperature, shake gently (not to foam). The concentration of the calibrator in the stock solution is 10,000 pg/mL. Please prepare 5 tubes containing 0.6 mL Calibrator Diluent and produce a triple dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted calibrator such as 10,000 pg/mL, 3,333.3 pg/mL, 1,111.1 pg/mL, 370.4 pg/mL, 123.5 pg/mL, and the last EP tubes with Calibrator Diluent is the blank as 0 pg/mL.  10,000 3,333.3 1,111.1 370.4 123.5 0

Detection Reagent A
Reconstitute the Detection Reagent A with 150 µL of Reagent Diluent, kept for 10 minutes at room temperature, shake gently (not to foam). Dilute to the working concentration with Assay Diluent A (1:100).

Detection Reagent B
Briefly spin or centrifuge the stock Detection Reagent B before use. Dilute to the working concentration with Assay Diluent B (1:100).

Wash Solution
Dilute 20 mL of Wash Solution Concentrate (30X) with 580 mL of de-ionized or distilled water to prepare 600 mL of Wash Solution (1X).

TMB substrate
Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:
1. Prepare calibrator within 15 minutes before assay. Pease do not dissolve the reagents at 37°C directly. 2. Making serial dilution in the wells directly is not permitted. 3. Detection Reagent A and B are sticky solutions, therefore, slowly pipette them to reduce the volume errors. 4. Please carefully reconstitute Calibrators or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10 μL for one pipetting. 5. The reconstituted Calibrators, Detection Reagent A and Detection Reagent B can be used only once. 6. If crystals have formed in the Wash Solution concentrate (30X), warm to room temperature and mix gently until the crystals are completely dissolved. 7. Contaminated water or container for reagent preparation will influence the detection result.

SAMPLE PREPARATION
1. We are only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the calibration curve, users must determine the optimal sample dilutions for their particular experiments. 4. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
5. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
6. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products. 2. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is only for reference. 3. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer. 4. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism. TMB Substrate should remain colorless till it is reacted with the enzyme which binds to the microplate. 5. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microplate from the storage bag until needed. 6. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 O.D. at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment. 7. Variation in sample preparation and each step of experimental operation may cause different results. In order to get better reproducible results, the operation of each step in the assay should be controlled. 8. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intraassay variance among kits from different batches might arise from above factors, too. 9. Kits from different manufacturers with the same item might produce different results, since we haven't compared our products with other manufacturers. 10. The calibrator of the kit and immunogen used for antibody preparation are commonly recombinant proteins, as different fragments, expression systems, purification methods might be used in recombinant protein preparation, we can not guarantee the kit could detect recombinant protein from other companies. So, it is not recommended to use the kit for the detection of recombinant protein. 11. Please predict the concentration of target molecules in samples, or arrange a preliminary experiment, it is a good way to solve specific problem, e.g. the concentration of samples are beyond the detection range of the kit. 12. The kit might not be suitable for detection of samples from some special experiment, for instance, knock-out experiments, due to their uncertainty of effectiveness. 13. The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

FOR RESEARCH USE ONLY.
KAMIYA BIOMEDICAL COMPANY