Mouse Anti-LPS IgG3 Antibody Assay Kit

Mouse Anti-LPS IgG3 Antibody Assay Kit - Cat Number: 6112 From Chondrex.

Research Field: Bacterial Research, Immunology

Clonality: N/A

Cross-Reactivity:

Host Origin: N/A

Applications: N/A

Isotype: N/A

Detection Range: 60 ng/ml-0.94 ng/ml

Sample Type: Serum, Plasma

Concentration: N/A

Immunogen:

DESCRIPTION: ELISA Kits to quantify mouse anti-E. coli LPS IgG/IgG1/IgG2a/IgG2b/IgG3 antibodies

6106: Mouse Anti-E. coli LPS IgG Antibody ELISA Kit
6107: Mouse Anti-E. coli LPS IgG1 Antibody ELISA Kit
6110: Mouse Anti-E. coli LPS IgG2a Antibody ELISA Kit
6111: Mouse Anti-E. coli LPS IgG2b Antibody ELISA Kit
6112: Mouse Anti-E. coli LPS IgG3 Antibody ELISA Kit
FORMAT: Pre-coated 96-well ELISA Plate with non-removeable strips
ASSAY TYPE: Indirect ELISA
ASSAY TIME: 4.5 hours
STANDARD RANGE: 6106/6107/6110/6111 : 100 ng/ml to 1.6 ng/ml
6112 : 500 ng/ml to 8 ng/ml
NUMBER OF SAMPLES: Up to 40 (duplicate) diluted samples/kit and up to 20 (duplicate) low dilution samples/kit
SAMPLE TYPES: Serum and Plasma
RECOMMENDED SAMPLE DILUTIONS: 1:100 (at least)
CHROMOGEN: TMB (read at 450 nm)
STORAGE: -20°C
VALIDATION DATA: 6106: Intra-assay (0.9-7%)/Inter-assay (2-11.7%)/Spiking Test (93-106%)
6107: Intra-assay (3.6-8.8%)/Inter-assay (9.3-13.1%)/Spiking Test (106-108%)
6110: Intra-assay (1-3.1%)/Inter-assay (2.6-6.1%)/Spiking Test (88-99%)
6111: Intra-assay (3.1-6.9%)/Inter-assay (1.9-9.8%)/Spiking Test (106-115%)
6112: Intra-assay (0.1-5.8%)/Inter-assay (3.9-9.1%)/Spiking Test (91-96%)

INTRODUCTION

Recent studies indicate environmental factors, especially intestinal microbiota and their toxins, may play pathogenic roles in autoimmune diseases such as rheumatoid arthritis (RA) (1-7), inflammatory bowel diseases (IBDs) (8, 9), systemic lupus erythematosus (SLE) (10), and other chronic disorders (11-13). It is possible that increased translocation of bacteria and bacterial toxins associated with high mucosal permeability and low immune function may be the primary and common pathogenesis of these autoimmune disorders (14).

Mice are ideal experimental animals to test this hypothesis due to the variety in genetic backgrounds, strains, and housing conditions, such as germ free, gnotobiotic, specific pathogen free, and conventional, all of which can affect susceptibility to potential pathogens. These differences in susceptibility may be attributed to the maturity of the immune system, depending on the environment. To facilitate and promote studies that determine immune responses to environmental agents, Chondrex.

Inc. provides mouse antibody ELISA kits against Escherichia coli O111:B4 lipopolysaccharide (E. coli LPS). In addition, Chondrex, Inc. also provides mouse antibody ELISA kits against E. coli O111:B4 (Cat # 6206, 6207, 6209 - 6212), staphylococcal enterotoxin A (Cat # 6218 - 6221), staphylococcal enterotoxin B
(Cat # 6214 - 6217), Porphyromonas gingivalis (Cat # 6225 - 6227), and Porphyromonas gingivalis LPS (Cat # 6222 - 6224). For further requests or consultation, please contact us at support@chondrex.com.
Mouse anti-E. coli LPS IgG Antibody ELISA Kit (Cat # 6106)
Mouse anti-E. coli LPS IgG1 Antibody ELISA Kit (Cat # 6107)
Mouse anti-E. coli LPS IgG2a Antibody ELISA Kit (Cat # 6110)
Mouse anti-E. coli LPS IgG2b Antibody ELISA Kit (Cat # 6111)
Mouse anti-E. coli LPS IgG3 Antibody ELISA Kit (Cat # 6112)

KIT COMPONENTS

PLATE MAPPING

Map the plate based on the number of samples and sample dilution. For example, if sample dilution is higher than 1:100, it is not necessary to run antigen un-coated wells, but if sample dilution is less than 1:100, it may be necessary to run antigen uncoated wells to determine the background noise reaction OD values of individual samples. An antigen uncoated plate (Catalog # 9026) for lower sample dilutions is not included. Please contact support@chondrex.com to place an order.

NOTES BEFORE USING ASSAY

NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placing the bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.
NOTE 6: For partial reagent use, please see the assay protocol’s corresponding step for the appropriate dilution ratio. For example, if the protocol dilutes 50 µl of a stock solution in 10 ml of buffer for 12 strips, then for 6 strips, dilute 25 µl of the stock solution in 5 ml of buffer. Partially used stock reagents may be kept in their original vials and stored at -20⁰C for use in a future assay.
NOTE 7: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and for disposal.

ASSAY OUTLINE

ASSAY PROCEDURE

Add Blocking Buffer: Add 100 µl of the Blocking Buffer (Solution A) to each well and incubate for 1 hour at room temperature
NOTE: If a sample with a dilution of 1:100 or less is assayed, antigen non-coated strips should be used. Solution A must be added to the non-coated wells without prior washing because any contaminants in the vessel containing the washing buffer will bind to the strips. For example, add 100 µl of Solution A to the antigen-coated strips (S1) and the corresponding uncoated strips (SB1). Incubate for 1 hour at room temperature.

2. Prepare Standard Dilutions: Dissolve one vial of standard with 1 ml of Sample/Standard Dilution Buffer (Solution B) to make the highest standard concentration - labeled “1”. Prepare serial dilutions by mixing 250 µl of the 1X standard with 250 µl of Solution B - labeled “2”. Then repeat this procedure to make five additional serial dilutions of standard. The 1X standard may be stored at -20°C for use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.

3. Prepare Sample Dilutions: Add 10 µl of mouse serum sample to 990 µl of Solution B (1:100) and keep it as a stock solution for future assays. Then, further dilute the sample with Solution B depending on the antibody levels. For example, take 250 µl of the sample stock solution and mix with 250 µl of Solution B to make a 1:200 dilution. If it is necessary to assay antibodies at less than 1:100 due to low antibody levels, antigen uncoated control strips will be necessary.

NOTE: Chondrex, Inc. recommends running a preliminary assay using various dilutions of sera (1:200, 1:1,000, 1:5,000) in order to determine the optimal dilution of your samples, especially before assaying a large number of samples.
4. Dilute Wash Buffer: Dilute 50 ml of 20X wash buffer in 950 ml of distilled water (1X wash buffer). Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.

5. Add Standards and Samples: Add 100 µl of Solution B (blank), standards, and samples to designated wells in duplicate. Incubate at room temperature for 2 hours.
NOTE: If a sample with a dilution of 1:100 or less is assayed, add 100 µl of the diluted samples to the antigen-coated strips (S1) and the corresponding uncoated strips (SB1).
6. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
7. Add Secondary Antibody: Dilute one vial of secondary antibody solution with 10 ml of Secondary Antibody Dilution Buffer (Solution

C). Add 100 µl of secondary antibody solution to each well and incubate at room temperature for 1 hour. 3. Prepare Sample Dilutions: Add 10 µl of mouse serum sample to 990 µl of Solution B (1:100) and keep it as a stock solution for future
assays. Then, further dilute the sample with Solution B depending on the antibody levels. For example, take 250 µl of the sample stock solution and mix with 250 µl of Solution B to make a 1:200 dilution. If it is necessary to assay antibodies at less than 1:100 due to low antibody levels, antigen uncoated control strips will be necessary.

NOTE: Chondrex, Inc. recommends running a preliminary assay using various dilutions of sera (1:200, 1:1,000, 1:5,000) in order to determine the optimal dilution of your samples, especially before assaying a large number of samples.
4. Dilute Wash Buffer: Dilute 50 ml of 20X wash buffer in 950 ml of distilled water (1X wash buffer). Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
5. Add Standards and Samples: Add 100 µl of Solution B (blank), standards, and samples to designated wells in duplicate. Incubate at room temperature for 2 hours.

NOTE: If a sample with a dilution of 1:100 or less is assayed, add 100 µl of the diluted samples to the antigen-coated strips (S1) and the corresponding uncoated strips (SB1).
6. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
7. Add Secondary Antibody: Dilute one vial of secondary antibody solution with 10 ml of Secondary Antibody Dilution Buffer (Solution
C). Add 100 µl of secondary antibody solution to each well and incubate at room temperature for 1 hour. 

8. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.