Mouse Anti-PG-LPS IgM Antibody Assay Kit

Mouse Anti-PG-LPS IgM Antibody Assay Kit - Cat Number: 6223 From Chondrex.

Research Field: Bacterial Research, Immunology

Clonality: N/A

Cross-Reactivity:

Host Origin: N/A

Applications: N/A

Isotype: N/A

Detection Range: 100 ng/ml-1.6 ng/ml

Sample Type: Serum, Plasma

Concentration: N/A

Immunogen:

DESCRIPTION: ELISA kit to quantify mouse anti-PG LPS IgG/IgM/IgG3 antibodies

6222: Mouse Anti-PG LPS IgG Antibody ELISA Kit
6223: Mouse Anti-PG LPS IgM Antibody ELISA Kit
6224: Mouse Anti-PG LPS IgG3 Antibody ELISA Kit
FORMAT: Pre-coated 96-well ELISA Plate with removeable strips
ASSAY TYPE: Indirect ELISA
ASSAY TIME: 4.5 hours
STANDARD RANGE: 6222/6224: 500 ng/ml to 8 ng/ml
6223: 100 ng/ml 1.6 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: 1:200 (at least)
CHROMOGEN: TMB (read at 450 nm)
STORAGE: -20°C
VALIDATION DATA: 6222: Intra-Assay (5.6-9.5%)/Inter-Assay (1.8-5.2%)/Spiking Test (98-105%)
6223: Intra-Assay (2.7-4.1%)/Inter-Assay (2.9-9.5%)/Spiking Test (90-97%)
6224: Intra-Assay (3.5-7%)/Inter-Assay (5.7-9.4%)/Spiking Test (98-108%)
NOTES: N/A

INTRODUCTION

Recent studies indicate that environmental factors, especially intestinal microbiota and their toxins, may play roles in the development of 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). In fact, germ-free mice fail to develop ankylosing enthesopathy, a naturally occurring joint disease in mice with many parallels to human ankylosing spondylitis (15). Moreover, several studies indicate that increased intestinal mucosal permeability due to stress, surgery, and minor gastrointestinal disorders such as irregularity, may contribute to excess translocation of gut bacteria and their toxins into the body (16-18).

These events suggest the pathological importance of intestinal bacteria with regard to autoimmune disorders.
Among the many environmental factors, gram-negative bacteria and their toxins, such as Porphyromonas gingivalis (P. gingivalis), Escherichia coli (E. coli), and lipopolysaccharide (LPS), induce inflammation by increasing levels of inflammatory cytokines, and may be involved in various types of chronic autoimmune disorders. In fact, chronic P. gingivalis oral infection prior to arthritis induction increases the likelihood of Th17 cell responses, ultimately accelerating collagen-induced arthritis (CIA) development in mice (19). Interestingly, orally administered P. gingivalis also perturbs gut microbiomes in CIA mice, affecting the gut immune system and the gut microbiota composition (20).

Beyond autoimmune disorders, P. gingivalis components have also been identified in the brains of Alzheimer’s Disease patients. The chronic P. gingivalis infection may change the permeability of the brain-blood barrier and reduce the efflux of β-amyloids from the centralnervous system into systemic circulation (21). In order to advance these microbiome-related research fields, Chondrex, Inc. provides mouse anti- P. gingivalis (33277) and P. gingivalis - LPS (33277) antibody ELISA kits to elucidate the host immune response to potential environmental pathogens. For a complete list of available mouse anti-bacteria and bacterial toxins antibody ELISA kits, such as E. coli (O111:B4), E. coli -LPS (O111:B4), and staphylococcal enterotoxins, please visit www.chondrex.com or contact us at support@chondrex.com.

KIT COMPONENTS

IDENTIFICATION OF ANTIGEN-COATED STRIPS

ASSAY OUTLINE

PLATE MAPPING

Map the plate based on the number of samples and sample dilution. For example, if sample dilution is higher than 1:200, it is not necessary to run antigen un-coated wells, but if sample dilution is less than 1:200, 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 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 1:200 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” below. 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

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 1:200 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 blotting 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 blotting 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.