MAHA ELISA Kit
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- Target
- MAHA (Mouse Anti-Human Antibody (MAHA))
- Reactivity
- Human
- Detection Method
- Colorimetric
- Method Type
- Sandwich ELISA
- Detection Range
- 0.5-27.0 μg/mL
- Minimum Detection Limit
- 0.5 μg/mL
- Application
- ELISA
- Purpose
- This ELISA (enzyme-linked immunosorbent assay) kit isproduced for the quantitative determination of mouse antihumanantibody (MAHA) levels in mouse serum or plasmasamples. It detects both MAHA-IgG and MAHA-IgMsubtypes. The test is used for detection of mice with positiveMAHA that may affect prescribed studies involvinghumanized monoclonal antibody, which is essential to avoidmisleading immunoassay test results and conclusion.This kit can also be used for specimens collected from otheranimals such as rat, dog, cat, rabbit, primate, etc.
- Brand
- ED™
- Sample Type
- Serum, Plasma
- Components
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1. Human IgG Coated Microplate. One bottle contains 12 mL of 0.5 M sulfuric acid. This reagent should be stored at 2-8 °C or room temperature and is stable until the expiration date on the kit box
MAHA Standards.Five vials containing different levels of MAHA in a liquid protein matrix with a non-azide based preservative. Refer to vial for exact concentration for each standard. These reagents should be stored at 2-8 °C and are stable until the expiration date on the kit box
MAHA Controls.Two vials containing different levels of MAHA in a liquid protein matrix with a non-azide based preservative. Refer to vials for exact concentration range for each control. Both controls should be stored at 2-8 °C and are stable until the expiration date on the kit box. - Material not included
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1. Precision single channel pipettes capable of delivering 25 μL, 50 μL, 100 μL, and 1000 μL etc
2. Repeating dispenser suitable for delivering 100 μL
3. Disposable pipette tips suitable for above volume dispensing
4. Disposable 12 x 75 mm or 13 x 100 glass tubes
5. Disposable plastic 100 mL and 1000 mL bottle with caps
6. Aluminum foil
7. Deionized or distilled water
8. Plastic microtiter well cover or polyethylene film
9. ELISA multichannel wash bottle or automatic (semi-automatic) washing system
10. Spectrophotometric microplate reader capable of reading absorbance at 450 nm.
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- Sample Volume
- 50 μL
- Assay Time
- 4 h
- Plate
- Pre-coated
- Protocol
- This ELISA is designed, developed and produced for thequantitative measurement of MAHA in serum and plasmasamples. The assay utilizes the two-site sandwichtechnique with two selected antibodies that bind to MAHA.Assay standards, controls and patient samples are directlyadded to wells of a microplate that is coated with highlypurified human IgG. After the first incubation period, theMAHA binds to the human IgG on the wall of microtiter welland unbound proteins in each microtiter well are washedaway. Then a horseradish peroxidase (HRP) labeled humanantibody is added to each microtiter well and a sandwich ofwell coated human IgG MAHA HRP-Conugated humanantibody is formed. The unbound HRP conjugated humanantibody is removed in the subsequent washing step. For thedetection of this immunocomplex, the well is then incubatedwith a substrate solution in a timed reaction and thenmeasured in a spectrophotometric microplate reader. Theenzymatic activity of the immunocomplex bound to MAHA onthe wall of the microtiter well is directly proportional to theamount of MAHA in the sample. A standard curve isgenerated by plotting the absorbance versus the respectiveMAHA concentration for each standard on point-to-point or 4parameter curve fit. The concentration of MAHA in testsamples is determined directly from this standard curve.
- Reagent Preparation
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(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details. - Sample Collection
- Only 50 μL of mouse serum or plasma is required for MAHA measurement in duplicate. No special preparation of subject is necessary prior to specimen collection. In the case of serum, whole blood should be collected and must be allowed to clot for a minimum of 30 minutes at room temperature before the serum is separated by centrifugation (850 1500xg for 10 minutes). The serum should be separated from the clot within three hours of blood collection and transferred to a clean test tube. Serum or plasma samples should be stored at 2 - 8 °C if the assay is to be performed within 72 hours. Otherwise, the samples should be stored at - 20 °C or below until measurement. Avoid repeated (more than three times) freezing and thawing of specimen.
- Assay Procedure
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(1) Place a sufficient number of human IgG coated microwell strips/wells in a holder to run MAHA standards, controls and unknown samples in duplicate.
(2) Test Configuration
(3) Add 25 μL of standards (Cat. 30491-30495), controls (Cat. 30496-30497) and patient samples into the designated microwell.
(4) Add 100 μL of assay buffer to each well
(5) Cover the plate with one plate sealer and incubate plate at room temperature, shaking for 45 minutes.
(6) Prepare MAHA tracer antibody working solution by 1:21 fold dilution of the HRP-conjugated human antibody with the tracer Antibody Diluent. For each strip, it is required to mix 1 mL of the tracer antibody diluent with 50 μL of the tracer antibody in a clean test tube.
(7) Remove plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 μL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(8) Add 100 μL of above diluted MAHA tracer antibody to each of the wells.
(9) Cover the plate with a plate sealer and an aluminum foil to and incubate plate at room temperature, shaking for 45 minutes.
(10) Remove plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 μL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(11) Add 100 μL of ELISA HRP Substrate into each of the wells.
(12) Cover the plate with a plate sealer and also with an aluminum foil to avoid exposure to light.
(13) Incubate plate at room temperature for 20 minutes.
(14) Remove the aluminum foil and plate sealer. Add 100 μL of ELISA Stop Solution into each of the wells. Mix gently.
(15) Read the absorbance at 450 nm within 10 minutes in a microplate reader.NOTE: to reduce the background, one can set the instrument to dual wavelength measurement at 450 nm with background wavelength correction set at 595 nm or 620 nm or 630 nm. - Calculation of Results
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- Calculate the average absorbance for each pair of duplicate test results
2. Subtract the average absorbance of the STD 1 (0 ?g/mL ) from the average absorbance of all other readings to obtain corrected absorbance
3. The standard curve is generated by the corrected absorbances of all standard levels on the ordinate against the standard concentration on the abscissa using point-to-point or log-log paper. Appropriate computer assisted data reduction programs may also be used for the calculation of results. We recommend using Point-to-Point curve fit.The MAHA concentrations for the controls and test samples are read directly from the standard curve using their respective corrected absorbance.
- Calculate the average absorbance for each pair of duplicate test results
- Assay Precision
- The intra-assay precision was validated by measuring one control sample in a single assay with eight replicate determinations. The inter-assay precision is validated by measuring one control sample in duplicate in 6 individual assays.
- Restrictions
- For Research Use only
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- Precaution of Use
- The reagents must be used in a professional laboratory environment and are for research use only. Source material (e.g. highly purified bovine serum albumin) of bovine serum was derived in the contiguous 48 United States. It was obtained only from healthy donor animals maintained under veterinary supervision and found free of contagious diseases. Wear gloves while performing this assay and handle these reagents as if they are potential infectious. Avoid contact with reagents containing TMB, hydrogen peroxide, or sulfuric acid. TMB may cause irritation to skin and mucous membranes and cause an allergic skin reaction. TMB is a suspected carcinogen. Sulfuric acid may cause severe irritation on contact with skin. Do not get in eyes, on skin, or on clothing. Do not ingest or inhale fumes. On contact, flush with copious amounts of water for at least 15 minutes. Use Good Laboratory Practices
- Storage
- 4 °C
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- Target
- MAHA (Mouse Anti-Human Antibody (MAHA))
- Target Type
- Antibody
- Background
- Development of humanized monoclonal antibodies (IgG) andtheir fragments as in vivo diagnosis procedure(radionuclides) and treatment for patients with variousdiseases has made great progress. In animal study, even asingle dose injection of a humanized monoclonal antibody orits fragment may induce immune response directed againstthis foreign protein (immunogen). In the circulation, thepresence of mouse antibody against human antibody wouldbind to the injected humanized antibody therapeutics ordiagnosis and diminish the efficacy of either in vivo diagnosisor treatment. The present of MAHA in mouse serum orplasma specimens may cause both false positive and falsenegative immunoassay test results depending on assayprinciples and monoclonal/polyclonal antibodies used in theassay system.Therefore, the presence of MAHA would eventually lead tounreliable pre-clinical data collection and study conclusion.This MAHA ELISA is a ready-to-use test kit with wellbreakablemicrotiter plate and simple test procedures. It alsoprovides a wide measurement range without high dosehook effect.
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