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Bioanalytical Method: Detection of Total Anti-drug Antibodies

October 10, 2018
Introduction to Anti Drug-Antibody Assay
The total anti-drug antibody(TAb) assays detect the antibodies that are specific to the therapeutic molecules. These assays do not distinguish specific isotypes and detect all classes of antibodies present in the samples. The different platforms have been used for the detection of total antibodies such as bridging antibody assays, indirect ELISA assays, SPR assays, Immuno-PCR assay and most recently mass spectrometric assay. Although the detection system varies with different platforms, the detection still relies on the ADA and therapeutic molecules binding and interactions.
A tier-based approach is utilized for detection of ADA that includes
a) Screening Assay
b) Confirmation Assay
c) Titer Assay

a) Screening Assay: The screening assay is designed to detect and distinguish the presence or absence of anti-drug antibodies in the samples. The screening assays involve the incubation of samples with capture and detection reagents. The screening assays utilize the screening cut-point (Ref: Cut Point Page) to identify positive and negative samples. The screen-positive samples are subjected to confirmation assay to determine the specificity.

b) Confirmatory Assay: The confirmatory assays are designed to determine the "true positives" from screen-positive samples. The confirmatory assays are performed with the pre-incubation of samples with the therapeutic molecules. When the true positive samples are incubated with therapeutic molecules are expected to compete and inhibit the assay signals. In contrast, when the false positive sample are incubated with therapeutic molecules lacks signal inhibition. The confirmatory assay utilizes a confirmatory cut point which is discussed in further details elsewhere. (Ref: Cut Point Page)

c) Titer Assay: The titer assay is a semi-quantitative measure of the antibodies present in the samples and reported numerically as titer dilution. The accurate quantitative measurement of ADA is not possible due to the lack of the reference standards in the assay, therefore, the semi-quantitative measurement is performed by serially diluting the samples until it reaches the negative (below the titer cut point). The titer values are obtained by the interpolating titer dilution that crosses the cut point.

Bioanalytical Platform and Detection System:
Indirect ELISA/ECL method:
The indirect ELISA method utilizes the capture of antibodies using the drug and detection using the reagent that recognizes all classes of antibodies. The detection reagent may include an equimolar mixture of enzyme-conjugated anti-human IgG, anti-human IgM & anti-human IgE for the ELISA method. The anti-human IgG, anti-human IgM and anti-human IgE bind to the respective isotypes providing total antibody concentration. The characterization of ADA has shown the presence of a higher amount of IgG and IgM and a very low amount of IgE.
Alternatively, the enzyme-conjugated mixture of Protein A, Protein G, and Protein L have also been used for the detection of total antibodies in the sample. The Protein A and Protein G binds to the Fc region of the antibodies and has a high affinity for IgG. The protein L binds to the kappa short-chain region of the antibodies enabling the binding to the most class of antibodies.

Bridging Assay method:
The bridging ELISA/ECL methods are most widely used for the detection of total antibodies in the samples. The bridging assay format utilizes the specific binding of the bivalent/ multivalent antibodies to the drug to detect for capture and detection. In this type of assay, the equimolar concentration of biotinylated drugs and detection component conjugated drugs. This method has an advantage over the previous method where only bivalent binding proteins such as antibodies can bind to the drug. Any other monovalent endogenous interfering molecule may not result in positive antibody detection. But the caveat with bridging assay is that the multivalent binding proteins do interfere and result in false-positive results. In these scenarios, the further characterization utilizing drug-specific and antibody-specific (Protein A/G) immuno-inhibition provide the confirmation of the antibodies. The principle of ELISA and ECL detection methods are discussed on the page later.

Surface Plasmon Resonance Method:
The surface plasmon resonance method has also been used to detect total antibodies and isotyping. The technology utilizes the principle where the interaction of antigen/antibodies increases the mass on the adsorbed surface of the gold plate SPR chip resulting in a change in the inflection angle of the light which is recorded as a response unit. The method enables the detection of low-affinity antibodies which otherwise is not detected by ELISA or ECL methods. It also enables multiplexing including the isotype characterization and epitope mapping, automation and faster turn around.

Detection and Isotype Characterization of Anti-Drug Antibody using SRP (GE Healthcare)

Immuno-PCR Method:
The Immuno-PCR have previously used for the detection of anti-drug antibodies and showed high specificity and sensitivity. The immunoPCR utilizes a similar capture system such as biotinylated proteins and the detection reagent is cross-linked with the DNA probe which undergoes amplification. The presence of antibodies results in the amplification of complementary DNA segments with probes and the signal can be detected quantitative PCR.

Detection of ADA using Immuno-PCR

Bioanalytical Method: Detection of Total Anti-drug Antibodies Bioanalytical Method: Detection of Total Anti-drug Antibodies Reviewed by Biotechnology on October 10, 2018 Rating: 5

Medical Microbiology: Enteric Bacteria MCQ

October 09, 2018
Multiple Choice Question on Enteric Bacteria (E. coli, Salmonella spp and Shigella spp)

1) Intestines of human and other mammals are the natural habitat of enteric organisms, a large family of bacteria is present as a normal flora, which of the following is not the normal flora of intestine?
a) Escherichia spp
b) Salmonella spp
c) Staphylococcus spp
d) Proteus spp

2) Some enteric bacteria are part of normal inhabitants and incidentally cause disease but others such as.........are regularly pathogenic for humans.
a) Pseudomonas spp
b) Streptococcus spp
c) Salmonella spp
d) Proteus spp

3) All are the general characteristics of enteric bacteria, EXCEPT?
a) Catalase positive
b) Non-spore forming
c) Grow in media with bile salts
d) Nitrate negative

4) Enteric bacteria are mainly classified based on their ability to ferment various sugars including lactose. Which of the following bacteria is a non-lactose fermenter?
a) Klebsiella spp
b) Salmonella spp
c) Enterobacter spp
d) Citrobacter spp

5) Escherichia coli is one of the major enteric bacteria that causes diarrhea and is characterized according to its virulence properties. All are the types of E. coli, EXCEPT?
a) Enterohemorrhagic E. coli
b) Enteropathogenic E. coli
c) Enterotoxigenic E. coli
d) Enterolysogenic E. coli

6) After the bacteria is ingested, enteropathogenic E. coli (EPEC) attaches to the mucosal cells of the small intestine which results in malabsorption and diarrhea, mostly infecting infants in developing countries. Symptoms include watery diarrhea, vomiting, non-bloody stools which lasts for short duration mostly 1-3 days. What are the two important factors of pathogenesis?
a) The chromosomal locus of enterocyte effacement (LEE)
b) The bundle-forming pilus encoded by a plasmid adherence factor (EAF)
c) Both of the above
d) None of the above

7) Enteroinvasive E. coli (EIEC) are nonmotile, non-lactose or late lactose fermenters which are predominantly found in developing countries infecting children and travelers. Which of the following infection is similar to EIEC infection?
a) Bacillary dysentery
b) Shigellosis
c) Traveler’s diarrhea
d) Enteric fever

8) Enterobacteriaceae expresses a variety of virulent antigens, all of the following are the antigens, EXCEPT?
a) O antigen
b) K and Vi antigen
c) H antigen
d) D antigen

9) E. coli is one of the most common enteric bacteria in urinary tract infection (UTI) followed by Proteus mirabilis. All of the followings are the characteristic feature Proteus mirabilis, EXCEPT?
a) Facultative aerobes
b) Urease positive
c) Motile
d) Citrate positive

10) Shigellosis is caused by Shigella dysenteriae in humans causing fever, abdominal cramps, diarrhea sometimes with blood. The infection is attributed to the …........ activity of Shiga toxin which increases the severity by tissue invasion of the large intestine.
a) Exotoxic
b) Enterotoxic
c) Cytotoxic
d) Neurotoxic

11) Enterotoxigenic E. coli (ETEC) produces two major types of toxins, heat labile (LT) and heat stable (ST) toxins. LT or cholera-like toxin activates adenylate cyclase (cAMP) whereas ST activates.............causing Travelers’ diarrhea.
a) Ribosomal dysfunction
b) Decarboxylase reaction
c) Guanylate cyclase
d) Fermentation of sugars

12) Which of the following is a rapid lactose fermenter, motile enteric bacteria and is the major cause of a broad range of hospital-acquired infections such as pneumonia, urinary tract infections, and wound and device infections?
a) Streptococcus pyogenes
b) Pseudomonas aeruginosa
c) Mycobacterium tuberculosis
d) Enterobacter aerogenes

13) Salmonella typhi enter the human body through oral route penetrating into the intestine and reaching the lymphatics and the bloodstream ultimately causing the infection "Typhoid".Which of the following are most correct examples of the region of infection caused by the S. typhi?
a) Mononuclear phagocytic cells in the liver and Peyer’s patches of the small intestine
b) Liver, spleen, lymph nodes and large intestine
c) Isolated follicles and Peyer’s patches of a large intestine
d) None of the above

14) Which type of salmonellae is primarily infectious for humans?
a) Salmonella typhi A
b) Salmonella paratyphi A, B, and C
c) Salmonella paratyphi A and B
d) Salmonella paratyphi A
15) The symptoms of typhoid fever develop in one to three weeks after exposure to S. typhi, all of the given below are the major symptoms, EXCEPT?
a) Weight gain
b) Headache
c) High-grade fever
d) Rashes

16) Which of the following Shigella spp produces a heat labile exotoxin that affects both the gut and central nervous system resulting in diarrhea and meningismus?
a) Shigella sonnie
b) Shigella dysenteriae type 1
c) Shigella dysenteriae type 2
d) Shigella dysenteriae type 3

17) S. enteritidis and S. typhimurium causes enterocolitis and gastroenteritis in humans. What is the most common food for the transmission of this infection?
a) Raw meat
b) Eggs
c) Canned beans
d) Yogurt

18) The biochemical test used to identify and determine the ability of a bacteria to convert tryptophan into indole is?
a) IMViC test
b) MRVP test
c) TSI test
d) Citrate test

19) Which one is not the selective culture media for salmonellae and shigellae?
a) Deoxycholate citrate agar
b) Xylose-lysine decarboxylase agar
c) Salmonella –shigella agar
d) Blood agar

20) The Widal test is used for the detection of Salmonella typhi and other subspecies. This test is based on the principle where?
a) the antigens are detected using the neutralization assay
b) the antigen combines with its soluble antibody and form a lattice and develops a visible precipitate
c) the antigens bind to RBCs and form the agglutination
d) None of the above
Multiple Choice Answers

Medical Microbiology: Enteric Bacteria MCQ Medical Microbiology: Enteric Bacteria MCQ Reviewed by Biotechnology on October 09, 2018 Rating: 5
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