C. difficile Toxin A / B ELISA Kit

Name: C. difficile Toxin A / B ELISA Kit
SKU: DEIA05711
Rating: 5 (1 reviews)

Regulatory status: For research use only, not for use in diagnostic procedures.

COA

SDS

Datasheet

Summary

Sample

Feces

Species Reactivity

Human

Detection Method

sELISA

Intended Use

This microplate-based ELISA (enzyme linked immunosorbent assay) kit is intended for the qualitative detection of both C. difficile Toxin A and Toxin B in feces. The assay is a useful tool as an aid of detection of C. difficile infection.

Contents of Kit

This test kit must be stored at 2 – 8°C upon receipt. For the expiration date of the kit refer to the label on the kit box. All components are stable until this expiration date.
Prior to use allow all reagents to come to room temperature. Regents from different kit lot numbers should not be combined or interchanged.
1. Toxin AB Antibodies Coated Microplate
One microplate with twelve by eight strips (96 wells total) coated with monoclonal anti-Toxin A and Toxin B. The plate is framed and sealed in a foil zipper bag with a desiccant. This reagent should be stored at 2 – 8 °C and is stable until the expiration date on the kit box.
2. Anti-Toxin AB Tracer Antibodies
One vial containing 12 mL ready-to-use horseradish peroxidase (HRP) conjugated monoclonal Toxin A and Toxin B antibodies in a stabilized protein matrix. This reagent should be stored at 2 – 8 °C and is stable until the expiration date on the kit box.
3. ELISA HRP Substrate
One bottle contains 12 mL of tetramethylbenzidine (TMB) with hydrogen peroxide. This reagent should be stored at 2 – 8 °C and is stable until the expiration date on the kit box.
4. ELISA Stop Solution
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.
5. Toxin AB Negative Control
One vial contains Toxin A and Toxin B negative control in a liquid bovine serum albumin based matrix with a non-azide preservative. This reagent should be stored at -20 °C or below for long- term storage.
6. Toxin AB Positive Control
One vial contains Toxin A and Toxin B positive control in a liquid bovine serum albumin based matrix with a non-azide preservative. This reagent should be stored at -20 °C or below for long- term storage.
7. Concentrated Fecal Sample Extraction Buffer
One bottle containing 10 mL of 10-fold concentrated fecal sample extraction buffer. This reagent should be diluted with 90 mL distilled water and mixed well. This yields as the fecal sample extraction buffer, calibrator diluent, calibrator 1, and negative control. The Fecal Sample Extraction Buffer may be stored at 2-8°C and is stable until the expiration date on the kit box.
8. ELISA Wash Concentrate
One bottle contains 30 mL of 30-fold concentrate. Before use the content must be diluted with 870 mL of distilled water and mixed well. Upon dilution this yields a working wash solution containing a surfactant in phosphate buffered saline with a non-azidepreservative. The diluted wash buffer should be stored at room temperature and is stable until the expiration date on the kit box.

Storage

Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in the sealed pouch to minimize exposure to air

Precision

The reproducibility of this assay is validated by measuring two samples both in a single assay of 16-replicate determinations (intra-assay) and in 10 different assays run on different dates (inter-assay).

Sensitivity

Forty stool samples were collected from normal subjects aging 23 to 64 years old. Ten fecal samples from patients with C. difficile infection were also collected. All fecal samples were prepared and assayed according to the procedures described in this manual. The results are summarized below.

The clinical sensitivity is 70% (7/10 × 100% = 70%)
The clinical diagnostic specificity is 100% (40/40 × 100% = 100%)
Sensitivity
A positive control of C. difficile toxin AB at a concentration of 200 ng/ml was serial diluted with negative control down to 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml and 6.25 ng/ml. All these diluted samples are measured with this kit. The results showed that 12.5 ng/mL had a negative interpretation, while 25 ng/mL showed a positive test result. Therefore, the analytical sensitivity of this kit is about 25 ng/mL.

General Description

Clostridium difficile is a common pathogen and a major cause of infectious diarrhea in hospitalized patients. While most strains produce both Toxin A and Toxin B, some only produce one or the other. These enterotoxins, which are both proinflamatory and cytotoxic, attack the mucosal lining of the intestines. If not identified and treated in a timely fashion, can result in permanent damage to the colon or colitis. This assay employs antibodies specific to both Toxin A and Toxin B.

Citations

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Background

Clostridium difficile is a Gram-positive, obligately anaerobic bacillus. It was first isolated from the feces of healthy newborns in 1935. It was originally named Bacillus difficile because of its obligate anaerobicity and difficulty in laboratory isolation and culture. Researchers confirmed the pathogenicity of this new species in experimental animals and the presence of exotoxins; however, it was not until 1978 that Clostridium difficile was first confirmed to be associated with antibiotic-associated diarrhea (AAD). Although Clostridium difficile was only identified as a human pathogen 40 years ago, Clostridium difficile infection (CDI)-related diseases have now become a serious public health problem. Currently, clinically, about 10%-25% of AAD and 90%-100% of pseudomembranous colitis (PMC) are caused by CDI, which is the leading cause of hospital-acquired bacterial infection and gastrointestinal inflammation-related death. The hospital infection rate has exceeded that of methicillin-resistant Staphylococcus aureus (MRSA). Since the beginning of the 21st century, with the emergence of highly virulent strains (BI/NAP1/027/toxin type m), regional disseminated outbreaks have occurred in North America, Australia, Europe and other countries, and the incidence and severity of CDI have increased dramatically worldwide. The severity of Clostridium difficile-associated disease (CDAD) can range from asymptomatic carriage, mild to moderate diarrhea, to explosive/fatal PMC, toxic megacolon, colon perforation, sepsis and septic shock. Severely infected patients require intestinal resection, and the mortality rate can reach 5%-40%. These symptoms are mainly caused by the exotoxins TcdA and TcdB secreted by pathogenic strains. The N-termini of the toxin proteins have Rho and Rac glucosyltransferase domains, which inactivate members of the Rho family guanosine triphosphatases (Rho GTPases) in the host cytoplasm through glycosylation, thereby causing the disintegration of the actin cytoskeleton, the destruction of tight junctions, the loss of intestinal epithelial integrity, and inducing acute colon inflammation. TcdA/B are members of the Large clostridial toxins (LCTs) family, which also includes the lethal (TcsL), hemorrhagic toxins (TcsH), alpha-toxin (TcnA) , and the large cytotoxin (tpeL) .

Figure 1. Clostridium difficile.(Sources: Wiep Klaas Smits, et al. 2016)

Among non-outbreak clinical isolates, about 20% of strains also produce a third toxin, called binary toxin or Clostridium difficile transferase (CDT), whose structure is similar to that of E-type gastropods Clostridium perfringens lota toxin, Clostridium Spiroforme toxin and Clostridium botulinum C2 cytotoxin are similar, including two independent protein subunits (CdtA and CdtB). CdtA has ADP-ribosyltransferase Active CdtA can irreversibly depolymerize the actin cytoskeleton; CdtB with binding ability can bind to host cells and promote the release of CdtA into the cytoplasm. The ribosylation of CDT can interfere with the polymerization of the actin network that constitutes the cell membrane, induce the formation of microtubule protrusions in intestinal epithelial cells, and allow more fibronectin to adhere to the cell surface, thus significantly enhancing the ability of Clostridium difficile to adhere to target cells. Therefore, carrying a binary toxin means that the strain is more virulent. CDI is a multifactorial, multistep, and multistage developmental process that includes disruption of normal colonic flora, germination and growth of spores (endogenous or exogenous infection), and subsequent colonization of the intestine. Its clinical manifestations are affected by many factors such as the patient's age, underlying diseases, immune status, and intestinal microorganisms. In addition, specific differences in virulence between C. difficile strains are also thought to be closely related to CDAD severity.

There is significant diversity in the tcdA/tcdB gene sequences of different C. difficile strains, which may lead to differences in virulence between strains. In addition, a breakthrough study on the regulatory mechanism of toxin genes came from the in-depth analysis of an approximately 19.6 kb segment on the C. difficile chromosome, which is called the pathogenicity locus (PaLoc) region. In addition to the genes encoding TcdA (tcdA) and TcdB (1cdB), PaLoc also contains an accessory gene including tcdR, tcdE, and tcdC. tcdR encodes a special RNA polymerase σ gene, which can initiate the transcription of tcdA/B genes and proteins, and upregulate the expression of toxin proteins; at the right end of PaLoc, a gene named tcdC encodes the negative direction of toxin gene expression regulatory factor, which is abundantly expressed in the exponential growth phase of bacteria and silenced at the beginning of the stationary phase. It has been confirmed that the tcdC sequence deletion is related to the high virulence of B1/NAP1/027/toxin type III strains; tcdE encodes a perforin-like protein ( holin-like), given that both holin proteins lack signal peptide sequences, it is currently believed that TedE may mediate the secretion of toxin proteins.

Alternative Names

C. difficile toxin
C. diff toxin
Clostridioides difficile toxin
Toxin A (TcdA) and Toxin B (TcdB)
Enterotoxin A (TcdA) and Cytotoxin B (TcdB)

References

1. Wiep Klaas Smits, et al. Clostridium difficile infection. Nature Reviews Disease Primers. 2016, volume 2, Article number: 16020 (2016)

Documents

pdf COA

pdf SDS

pdf Datasheet

Reagents for C. difficile Research

QA & Reviews

Q & A

Ask a Question

Q: We use mouse models of C difficile infection for our research. This kit mentions reactivity to humans. Has this kit been ever tried for mouse?

A: These kits do work with mouse and other mammalian species. So long as you have a big enough sample size for the C. Diff can be read.

Customer Reviews

References

Clostridium difficile infection: review

Eur J Clin Microbiol Infect Dis.

Authors: Czepiel J, Dróżdż M, Pituch H, Kuijper EJ, Perucki W, Mielimonka A, Goldman S, Wultańska D, Garlicki A, Biesiada G

Abstract

Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, anaerobic bacillus, which is widely distributed in the intestinal tract of humans and animals and in the environment. In the last decade, the frequency and severity of C. difficile infection has been increasing worldwide to become one of the most common hospital-acquired infections. Transmission of this pathogen occurs by the fecal-oral route and the most important risk factors include antibiotic therapy, old age, and hospital or nursing home stay. The clinical picture is diverse and ranges from asymptomatic carrier status, through various degrees of diarrhea, to the most severe, life threatening colitis resulting with death. Diagnosis is based on direct detection of C. difficile toxins in feces, most commonly with the use of EIA assay, but no single test is suitable as a stand-alone test confirming CDI. Antibiotics of choice are vancomycin, fidaxomicin, and metronidazole, though metronidazole is considered as inferior. The goal of this review is to update physicians on current scientific knowledge of C. difficile infection, focusing also on fecal microbiota transplantation which is a promising therapy.

Pathogenicity and virulence of Clostridioides difficile

Virulence

Authors: Buddle JE, Fagan RP

Abstract

Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhea, and is responsible for a spectrum of diseases characterized by high levels of recurrence, morbidity, and mortality. Treatment is complex, since antibiotics constitute both the main treatment and the major risk factor for infection. Worryingly, resistance to multiple antibiotics is becoming increasingly widespread, leading to the classification of this pathogen as an urgent threat to global health. As a consummate opportunist, C. difficile is well equipped for promoting disease, owing to its arsenal of virulence factors: transmission of this anaerobe is highly efficient due to the formation of robust endospores, and an array of adhesins promote gut colonization. C. difficile produces multiple toxins acting upon gut epithelia, resulting in manifestations typical of diarrheal disease, and severe inflammation in a subset of patients. This review focuses on such virulence factors, as well as the importance of antimicrobial resistance and genome plasticity in enabling pathogenesis and persistence of this important pathogen.

Clostridioides difficile Infection: Update on Management

Am Fam Physician

Authors: Mounsey A, Lacy Smith K, Reddy VC, Nickolich S

Abstract

Guidelines for the diagnosis and treatment of Clostridioides difficile infection have recently been updated. Risk factors include recent exposure to health care facilities or antibiotics, especially clindamycin. C. difficile infection is characterized by a wide range of symptoms, from mild or moderate diarrhea to severe disease with pseudomembranous colitis, colonic ileus, toxic megacolon, sepsis, or death. C. difficile infection should be considered in patients who are not taking laxatives and have three or more episodes of unexplained, unformed stools in 24 hours. Testing in these patients should start with enzyme immunoassays for glutamate dehydrogenase and toxins A and B or nucleic acid amplification testing. In children older than 12 months, testing is recommended only for those with prolonged diarrhea and risk factors. Treatment depends on whether the episode is an initial vs. recurrent infection and on the severity of the infection based on white blood cell count, serum creatinine level, and other clinical signs and symptoms. For an initial episode of nonsevere C. difficile infection, oral vancomycin or oral fidaxomicin is recommended. Metronidazole is no longer recommended as first-line therapy for adults. Fecal microbiota transplantation is a reasonable treatment option with high cure rates in patients who have had multiple recurrent episodes and have received appropriate antibiotic therapy for at least three of the episodes. Good antibiotic stewardship is a key strategy to decrease rates of C. difficile infection. In routine or endemic settings, hands should be cleaned with either soap and water or an alcohol-based product, but during outbreaks soap and water is superior. The Infectious Diseases Society of America does not recommend the use of probiotics for prevention of C. difficile infection.