IgG1 ELISA Kit

Produktnummer ABIN625006

Human IgG1 ELISA Kit, Colorimetric Assay für die Quantifizierung von Human IgG1 und es wurde in 6+ Publikationen zitiert.

Kurzübersicht für IgG1 ELISA Kit (ABIN625006)

Target: IgG1

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 0.05 ng/mL - 40 ng/mL

Applikation: ELISA

Proben: Plasma, Cell Culture Supernatant, Serum

Produktdetails IgG1 ELISA Kit

Untere Nachweisgrenze: 0.05 ng/mL

Verwendungszweck: Human IgG1 ELISA Kit for cell culture supernatants, plasma, and serum samples.

Analytische Methode: Quantitative

Spezifität: Detect human IgG1. No cross reactivity was found againt IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE. Will not detect Fc tagged proteins/peptides.

Sensitivität: 50 pg/mL

Produktmerkmale:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Bestandteile:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

,

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Benötigtes Material:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Anwendungsinformationen

Applikationshinweise: Suggested Plasma Dilution: ~10,000,000 fold

Plattentyp: Pre-coated

Protokoll:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Aufbereitung der Reagenzien:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: 1x Assay Diluent (Item E) should be used for dilution of serum/plasma samples. Suggested dilution for normal serum/plasma: 10,000,000 fold. For example, add 1 µL of serum/plasma into a tube with 99 µL 1x Assay Diluent to prepare a 100-fold diluted sample. Mix through and then pipette 1 µL of prepared 100-fold diluted sample into a tube with 99 µL 1x Assay Diluent to prepare a 10,000 fold diluted sample. Mix through and then pipette 1 µL of prepared 10,000-fold diluted sample into a tube with 999 µL 1x Assay Diluent to prepare a final 10,000,000 fold diluted sample. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C and then add 600 µL 1x Assay Diluent into Item C vial to prepare a 300 ng/mL stock standard solution. Dissolve the powder thoroughly by a gentle mix. Add 80 µL IgG1 standard (300 ng/mL) from the vial of Item C, into a tube with 520 µL 1x Assay Diluent to prepare a 40 ng/mL standard. Pipette 400 µL 1x Assay Diluent into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. 1x Assay Diluent serves as the zero standard (0 ng/mL). 80 µL standard + 520 µL 200 µL 200 µL 200 µL 200 µL 200 µL 200myl 40 13.3 4.44 1.48 0.49 0.16 0.05 0 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) ) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 2,000-fold with 1x Assay Diluent. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 6 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent to prepare a final 2,000 fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Testdurchführung:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Ergebnisberechnung:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent Human IgG1 concentration (ng/mL) O D =4 50 n m 0.1 1 10 0 0.1 1 10 100
Sensitivity: The minimum detectable dose of IgG1 is typically less than 0.05 ng/mL. C. LINEARITY Sample Type Serum Plasma 1:2 Average % of Expected 88 82 Range ( %) 77-97 72-94 1:4 Average % of Expected 86 81 Range ( %) 75-96 71-92 D. REPRODUCIBILITY Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Testpräzision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Avoid repeated freeze-thaw cycles.

Lagerung: -20 °C

Informationen zur Lagerung: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Haltbarkeit: 6 months

Referenzen für IgG1 ELISA Kit (ABIN625006)

Olivares, Castillejo, Varea, Sanz: "Double-blind, randomised, placebo-controlled intervention trial to evaluate the effects of Bifidobacterium longum CECT 7347 in children with newly diagnosed coeliac disease." in: The British journal of nutrition, Vol. 112, Issue 1, pp. 30-40, (2014) (PubMed).

Zhang, Tang, Goryacheva, Niessner, Knopp: "Anodic-stripping voltammetric immunoassay for ultrasensitive detection of low-abundance proteins using quantum dot aggregated hollow microspheres." in: Chemistry (Weinheim an der Bergstrasse, Germany), Vol. 19, Issue 7, pp. 2496-503, (2013) (PubMed).

Prakash, Andrabi, Kumar, Kabra, Lodha, Vajpayee, Luthra: "Binding antibody responses to the immunogenic regions of viral envelope in HIV-1-infected Indian children." in: Viral immunology, Vol. 24, Issue 6, pp. 463-9, (2012) (PubMed).

Kim, Lo, Wear, Stojadinovic, Weina, Izadjoo: "Improvement of anti-Burkholderia mouse monoclonal antibody from various phage-displayed single-chain antibody libraries." in: Journal of immunological methods, Vol. 372, Issue 1-2, pp. 146-61, (2011) (PubMed).

Liao, Tsai: "Clinical effectiveness of Tyrophagus putrescentiae allergy by local nasal immunotherapy using strips of Dermatophagoides pteronyssinus." in: The Journal of asthma : official journal of the Association for the Care of Asthma, Vol. 48, Issue 9, pp. 957-64, (2011) (PubMed).

Barry, Soloviev: "Quantitative protein profiling using antibody arrays." in: Proteomics, Vol. 4, Issue 12, pp. 3717-26, (2004) (PubMed).

Details zu IgG1

Target: IgG1

Substanzklasse: Antibody

Hintergrund:

Gene Symbols: IGHG1

Protein Name (Synonyms): Ig gamma-1 chain C region

UniProt: P01857