Biochemistry, Molecular Biology, Physiology, Microbiology, Immunology, Pharmacology & Drug Discovery
Preparation Lecture Notes, Multiple Choice Questions, Scientific Discoveries

Enzyme Replacement Therapy, Glycosaminoglycans, and MPS VII

December 23, 2018
Vestronidase alfa, an enzyme replacement therapy, is an approved drug for the treatment of MPS VII. The approval of this drug has set a precedent for future drug development for the regulators and drug developers. First, the pivotal clinical trial was conducted to evaluate the totality of the clinical data for determining the efficacy of the treatment, therefore, no primary clinical endpoint was established. Second, the drug obtains approval positive outcome on the secondary endpoint; changes in the urinary glycosaminoglycan, from placebo control. The glycosaminoglycan is also the pharmacodynamic biomarker that reflected the efficacy of the drug. Third, the novel multi-domain clinical responder index was designed to evaluate the treatment outcomes and included six minutes walk test (6MWT), forced vital capacity (FVC), shoulder range motion, visual acuity, BOT-2 fine, and gross motor activity. While MRDI evaluation per subject suggested the stabilization of clinical condition if not an improvement in all patients, improvement in individual parameters were not statistically significant.

Introduction
Vestronidase alfa is an approved enzyme replacement therapy for MPS VII (Sly disease). The MPS VII is a genetic disorder caused by a mutation of gene encoding beta-glucuronidase. The enzyme beta-glucuronidase is involved in the breakdown of large sugar molecules called glycosaminoglycans GAGs that include heparan sulfate, dermatan sulfate. The absence of the beta-glucuronidase enzyme leads to accumulation glycosaminoglycans in lysosomes resulting in increased lysosomal size and enlargement of tissues and organs. The common clinical manifestations include hepatomegaly, cardiac abnormalities, developmental defects, and neurological disorders. The primary goal of the treatment for MPS VII is to reduce the accumulation of these glycosaminoglycans in the tissues/organs.

Mechanism of action:
Vestronidase alfa is a recombinant human beta-glucuronidase enzyme that is produced from CHO cells which undergo post-translational sugar modifications. The post-translation sugar modification is required for recognition of target cells, entry into the target cells, and trafficking into the lysosome. The enzyme binds to a mannose-6-phosphate receptor present on the cell surface and localized into lysosome where it helps degrade the glycosaminoglycans, thereby alleviating the progression of the disease. The efficacy of the treatment is evident through clinical and non-clinical studies. The in-vitro studies and non-clinical studies have validated the receptor recognition, uptake and localization of the vestronidase alfa into the lysosome and clearance of glycosaminoglycans from target tissues.


Figure: Showing the disease state and treated state of the cell. In a disease state, the absence of the beta-glucuronidase results in accumulation of glycosaminoglycans in lysosomes. In the treated state of the cells, the recombinant enzymes enter the cells via a mannose-6-phosphate receptor and localized into lysosome where it recycles and clears these accumulated glycosaminoglycans.

Urinary GAGs as a pharmacodynamic biomarker
The mucopolysaccharidoses are a group of disorders caused by a deficiency of the enzyme of the metabolic pathway for degradation of glycosaminoglycans that leads to the accumulation these byproducts into the lysosome. Theoretically, measurement of glycosaminoglycans (heparan sulfate, chondroitin and keratan sulfate) in tissues and bodily fluids are a suitable biomarker for diagnosis, monitoring progression and treatment outcomes of the disease. But the measurement of glycosaminoglycans had limited value because of lack of appropriate methods, high background in healthy controls, variable sugar modifications, and chain lengths. It was known that deficiency of each enzyme in GAG metabolic pathway results in characteristics carbohydrate end products. But the lack of appropriate analytical methods precluded from showing clinical correlation and causal relationship of specific GAG deposition/excretion with diagnosis and progression of the disease. And the measurement of total urinary GAGs did not truly reflect the disease type, progression or improvement and limited it for a patient screening use only. As a breakthrough, the novel LC-LC MS assay offered a sensitivity, accurate and specific platform made measure and distinguish heparan sulfate, chondroitin sulfate and keratan sulfates feasible. Moreover, the platform allows measuring different non-reducing carbohydrate structure that are the characteristics for each MPS disorders. The LC-MS assay method enabled a sensitive and accurate measurement of uGAG in the clinical subject. Now, the uGAG is an attractive pharmacodynamic biomarker to evaluate the efficacy and show clear pharmacodynamic effect in MPS disorders.

Clinical Trial Design, Treatment Outcomes and Regulatory considerations
Vestronidase alfa enzyme was approved based on results from the three clinical studies, one phase 3 pivotal study and two phases 2 studies. The pivotal study was a multicenter, randomized, placebo-controlled, blind start single cross over phase 3 study that included 12 MPS VII subjects that meet the inclusion and exclusion criteria. The study design is given below (insert the study design). The subjects were dosed QOW (once every two weeks) through week 46. All groups received a minimum of 24 weeks of treatment with 4 mg/kg vestronidase alfa QOW.





Trail Design: Double Blind Placebo Controlled Single Switch Over Study

The primary objective of the phase 3 study was to determine the efficacy of the Vestronidase alfa in MPS VII subjects. The efficacy was determined the totality of the data on improvement from pre-dose baseline per subjects. The secondary endpoint was determined as a reduction of uGAG excretion following 24 weeks of therapy. The efficacy of the vestronidase alfa in MPS VII subjects was evaluated as a multi-domain clinical responder index (MDRI) following 24 weeks of vestronidase alfa exposure. The multi-domain clinical responder index encompasses 6-minute walk test, forced vital capacity (FVC), shoulder range motion, visual acuity, BOT-2 fine and gross motor activity. The minimum important difference for each of these parameters was established based on the available related data from other related MPS diseases.



Parameters of Multi-domain Clinical Responder Index
The variable age of enrolled subject and heterogeneity in the clinical manifestation of the patients limited the ability to effectively monitor the MID for multi-domain clinical responder indexes (MDRI) such as 6MWT, FVC, Gross and fine motor activity and visual activity because not all subject was able to successfully complete the administered test. These biological variables confounded with the outcome of these secondary endpoints and the MID was not statistically significant for these parameters. But the totality of the data provided showed the stabilization of the clinical condition of the patients even though a statistically significant improvement from the baseline were observed. Nonetheless, the consistent decrease in urinary GAGs and the totality of the clinical data per subjects suggested the stabilization of clinical condition if not the improvement in all patients.


uGAG: Percentage change from baseline

Regulators and experts highlighted the difficulties in ascertaining the clinical relevance of the observed effects with vestronidase alfa. The clinical history including onset, the progression of the disease is not well characterized/understood. Also, the timeline of the clinical trial may limit the possibility to observe the effect of the treatment on clinical outcomes. Therefore, observational natural history and registry studies were recommended to understand the disease symptoms and long term treatment outcome.

In conclusion, this drug approval underscores the significance of totality of the data to evaluate the efficacy and early identification of sensitive and measurable biomarkers in accelerated drug development and approvals.

Reference: EMA Assessment Report MEPSEVII
Enzyme Replacement Therapy, Glycosaminoglycans, and MPS VII Enzyme Replacement Therapy, Glycosaminoglycans, and MPS VII Reviewed by Biotechnology on December 23, 2018 Rating: 5

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
1)-c,
2)-c,
3)-d,
4)-b,
5)-d,
6)-c,
7)-b,
8)-d,
9)-a,
10)-b
11)-c,
12)-d,
13)-a,
14)-c,
15)-a,
16)-b,
17)-b,
18)-a,
19)-d,
20)-b

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

Medical Microbiology: Gram Positive Rods MCQs

September 26, 2018
Multiple Choice Questions on Gram Positive Rods

1) All are general characteristics of Bacilli, except
a) Anaerobes
b) Gram-positive
c) Spore-forming
d) Ubiquitous

2) Which one of the following Bacillus spp is NOT a medically important pathogenic bacteria?
a) Bacillus cereus
b) Bacillus thuringiensis
c) Bacillus thermophilus
d) Bacillus anthracis

3) Bacillus.............is commonly found in soil and is an insect pathogen which can infect humans also. This organism is often used as pest control in insecticidal products.
a) thuringiensis
b) subtilis
c) anthracis
d) cereus


4) Most of the species of Bacilli and Clostridia are.............., which means they obtain their nutrients from dead or organic matter.
a) Parasites
b) Saprophytes
c) Symbiotic
d) Mutualistic

5) Bacillus anthracis is a principle pathogen of a disease known as "anthrax" which primarily infects animals like goats and cattle and is transmitted to humans through ingestion or inhalation of spores. What are the two important virulence factors of this organism?
a) Capsule and enterotoxins
b) Exotoxins and enterotoxins
c) Endotoxins and capsule
d) Capsule and exotoxins

6) Bacillus cereus causes two types of foodborne intoxications, known as the emetic type and diarrheal type. What is the incubation period for the onset of symptoms ingestion in the emetic type of food intoxication?
a) 1-12 hours
b) 1-2 days
c) 1-5 hours
d) 1-24 hours

7) Bacillus….......is a common laboratory contaminant and is used as sterility testing. It is an endospore-forming bacteria and resistant to an extreme environmental condition.
a) subtilis
b) thermophilus
c) cereus
d) agalactiae

8) Which of the following species of Clostridia do not have flagella, but rapidly grows on nutrient media and mimics the growth of motile organisms?
a) Clostridium botulinum
b) Clostridium perfringens
c) Clostridium tetani
d) Clostridium difficile

9) All of the following is the type of infections caused by Clostridium perfringens, EXCEPT?
a) Gas gangrene
b) Food poisoning
c) Cellulitis
d) None of the above

10) The AVA BioThrax is the FDA approved vaccines for
a) Bacillus cereus
b) Clostridium tetani
c) Bacillus anthrax
d) Bacillus substilis


11) The shape and position of spores vary in different types of species and is useful in identification of Clostridia. Clostridium perfringens forms subterminal spores whereas Clostridium....... forms terminal spores.
a) tetani
b) botulinum
c) cereus
d) sordellii

12) The bacterial spore of..............can invade human body through a puncture wound and ultimately enter the central nervous system by releasing a potent toxin known as neurotoxins, thus blocking the release of neurotransmitters and resulting in cramping of muscles, causing loc jaw and other muscle related spasms.
a) Bacillus anthracis
b) Clostridium botulinum
c) Clostridium tetani
d) Bacillus subtilis

13) Which is the most common food associated with infant botulism?
a) Maple syrup
b) Honey
c) Canned baby food
d) Corn syrup

14) All of the given species of Clostridia produces botulinum toxins, except
a) Clostridium botulinum
b) Clostridium butyricum
c) Clostridium baratii
d) Clostridium difficile

15) The toxin responsible for food poisoning by Bacillus cereus is
a) Cytotoxins
b) Neurotoxins
c) Enterotoxins
d) None of the above



16) All of the statements given below for Bacillus anthracis are true, EXCEPT
a) Hemolytic colonies on Blood agar
b) Large gram-positive rods
c) Non-motile
d) Capsulated

17) Colony characteristics/characteristic of Clostridia
a) Grow well in Nutrient agar
b) Produce alpha hemolytic colonies
c) Grow under aerobic conditions
d) None of the above

18) Clostridium botulinum associated foodborne illness can occur within 18-24 hours of ingestion of a toxin produced by the bacteria in food. Which of the following is NOT the symptoms associated with C.botulinum toxin?
a) Poor vision
b) Fever
c) Difficulty swallowing
d) Bulbar paralysis

19) All of the following are the preventive measures for Tetanus, EXCEPT
a) Active immunization of toxoids
b) Prophylactic use of antitoxin
c) Administration of Bacitracin
d) None of the above

20) Clostridium perfringens toxins are released only after entering the host cell through wounds or from the intestinal tract, which of the following also known as alpha toxin can damage the cell membranes ultimately resulting in hemolysis and tissue destruction?
a) Lecithinase
b) Enterotoxin
c) Alpha
d) Beta


Multiple Choice Answers
1)- a,
2)-c,
3)-a,
4)-b,
5)-d,
6)-c,
7)-a,
8)-b,
9)-d,
10)-c
11)- a,
12)- c,
13)-b,
14)-d,
15)-c,
16)-a,
17)-d,
18)-b,
19)-c,
20)-a
Medical Microbiology: Gram Positive Rods MCQs Medical Microbiology: Gram Positive Rods MCQs Reviewed by Biotechnology on September 26, 2018 Rating: 5

Preparation of Surrogate Positive Control to assess Sensitivity of Antidrug Antibody Assay

September 19, 2018
Anti-drug antibody (ADA) assays are utilized for the detection and characterization of antibodies that are generated against the therapeutic molecules. These therapeutic candidates can be proteins, enzyme, viral packaged gene or RNA for interference or exon skipping. The detection and characterization of ADA against therapeutic candidates are crucial for immunogenicity profiling and investigation of immune response-related safety events. (Ref: Immunogenicity Assessment).

The assay sensitivity is the lowest concentration of antibodies that can be consistently detected by the method. The sensitive assay should detect ADAs in the clinical samples before they reach a level that can be associated with the altered PK/PD & safety profile (clinically relevant ADA). Evidence from clinical studies has shown that ADA levels as low as 100 ng/ml may be associated with altered PK/PD or safety profile. Therefore, the Food and Drug Agency (FDA) and the European Medical Agency (EMA) expect the sensitivity of any ADA assay less than 100 ng/ml and below 5-10 ng/ml for IgE assays.

In comparison to PK assays, the ADA assays do not have the reference standards that are representative of the endogenous counterparts and the assay sensitivity is determined using surrogate controls. And the surrogate positive controls are not the true representative of the ADA that is present in a clinical subject because of the diversity of antibodies binding affinities and recognition to epitopes. The assay sensitivity is assessed by testing the serial dilution of a surrogate controls antibody of known concentration, using a pooled sample matrix from drug-naive subjects. The assay sensitivity is calculated by interpolating linear portion of the dilution curve to the assay cut point and expressed as antibody detected/ml of the undiluted matrix.





Figure 1: Examples from ADA assays with different sensitivities based on the interpolated titer values at screening cut point


Strategies for preparation of surrogate positive control
The followings are the strategies used to obtain surrogate positive control for ADA assay. They include
-Obtaining antibodies from study subjects
-Use of hyperimmunized animals for antibody production
-Production of monoclonal and humanized antibodies
-Chemical cross-linking and conjugations

Obtaining antibodies from study subjects:
The ideal method is to purify the antibodies from clinical study subjects. These clinical subjects are dosed with the same therapeutic candidate molecules and the antibodies that are generated against these clinical subjects may be representative of clones and affinities. Unfortunately, each individual varies on extent, persistence, and type of antibodies produced against therapeutic candidate molecules driven partly by the genetic makeup of each individual. Another caveat is the limited availability of clinical samples, and the purification requires the large number and pools of clinical samples. Also obtaining clinical samples may not be feasible for the early stage of drug development with insufficient patient sample size or onset of antibodies. With these limitations, the obtaining and purifying antibodies from the clinical samples is always challenging.

Obtaining pre-existing antibodies for a normal individual:
Taking advantage of the biology, the screen and the use of pre-existing antibodies as positive control can be an alternative method. Literature has reported pre-existing antibodies against various antigens such as AAV capsids, PEGs, rheumatoid arthritis which may be used as positive control various ADA assays. The purified pre-existing antibodies are in limited quantities, have a low affinity toward target molecule and are biologically not relevant. Therefore, these antibodies may serve as positive control until the better positive controls are not obtained. These are especially useful for isotyping the antibodies when the detection system uses anti-human Igs and do not react with antibodies from other species. Using antibodies from other species underestimates the sensitivity of the assay.

Use of hyperimmunized animals for antibody production:
It is a widely adopted campaign for the production of specific polyclonal antibodies against therapeutic candidate molecule. The high dose of a therapeutic molecule is immunized to animals such as goats, mice, and rabbits. The bleed at different time points is collected for the screening of the antibodies. Depending on the type of antibody (IgM or IgM) purification, bleeding time and dosing are scheduled. For IgM purification, the bleed during the primary immune response is collected. In contrast to IgG, the bleed are collected after booster dose that allows the IgG class-switch and affinity maturation. It is always advised to collect multiple bleeds after dosing and screen for better quality antibodies. The species are selected based on the homology of the antigen and the number of antibodies required. In some instances, commonly used animals such as Cyno, goat, and sheep may not evoke sufficient immune response. In those cases, animals such as chicken are also used for the production of antibodies.

Production of Monoclonal antibodies and humanized antibodies:
The monoclonal antibodies are also widely used as a positive control in the ADA assays. The use of high-affinity monoclonal antibodies alone has shown to improve assay sensitivity but somewhat compromise drug tolerance of the assay. The monoclonal antibodies are produced using technology such as hybridoma, phage displays, etc. The hybridoma techniques utilize the fusion of prescreened target- specific B-cell from the immunized animals and HGPRT deficient myeloma cells. These cells are fused together and screened for a clone that successfully produces high-affinity antibodies. Since monoclonal antibodies target the single epitope of the drug, this is not representative of the clinical samples and may not be suitable control for all ADA assays. However, it may be appropriate for neutralizing antibody assay if the antibody generated specifically block the active /functional site.

Humanized monoclonal antibodies:
The monoclonal antibodies generated in the different model organisms may not be suitable when the assay is designed to specifically detect the Fc region of the antibody. This includes isotype characterization assay where ADA in the sample is captured using the drug and detected using reagent that recognizes the Fc region. In these scenarios, the utilization of monoclonal antibodies from mouse or goat origin may not work properly in the assay thereby not representing the sensitivity of the assay. Therefore, humanized antibodies become a viable alternative. The humanized antibodies can be produced by genetically engineering the variable heavy and light complementarity determining regions (CDR) and remaining constant coding sequence including Fc region. This method can be used to generate human IgG, IgM, and IgE that target the drug molecule and use for positive control.




Ref: Adapted from Kuby Immunobiology


Chemical cross-linking and conjugation:
The chemical cross-linking or conjugation is handfuls when the antibodies isotype from other species against the specific therapeutic molecule exist and there is a special need for the isotype positive controls. For example, when there is commercially available IgG but no IgE or IgM are available, these IgG can be fragmented (protease digestion) to obtain the Fab region and cross-linked with generic human IgE. This results in the cross-linked IgE molecule that also consists of Fab that binds to the drug. The fragmentation and cross-linking method may be used for short term solutions while other methods for the production of antibodies are being explored.



Figure: Example of a method for Protein cross-linking using SPDP from Thermo Scientific


Summary:
The positive controls are important for evaluating assay sensitivity and monitoring consistent performance of the assay. Obtaining suitable surrogate control is key to demonstrate the assay performance for analyzing samples from the clinical studies. The affinity purification, production of monoclonal and humanized antibodies and conjugation methods are commonly used for preparing positive controls and system suitability controls. The selection of appropriate depends on the type of assay, intended use, and feasibility.
Preparation of Surrogate Positive Control to assess Sensitivity of Antidrug Antibody Assay Preparation of Surrogate Positive Control to assess Sensitivity of Antidrug Antibody Assay Reviewed by Biotechnology on September 19, 2018 Rating: 5

Medical Microbiology- Microbial Culture and Identification: MCQ

September 18, 2018
Multiple Choice Questions on Microbial Culture and Identification

1) A bacteriological stain also known as Differential stain is used to identify acid-fast organisms, what is the name of the stain?
a)Negative stain
b) Gram stain
c) Ziehl-Neelsen stain
d) Schaeffer stain- Fulton stain

2) A Capsule is a.........layer lying outside the bacterial cell and can be found both gram positive and gram negative bacteria
a) Lipopolysaccharide layer
b) Polysaccharide
c) Phospholipid
d) None of the Above

3) Negative staining is the technique where the background of a specimen is stained whereas positive staining stains the whole specimen. Which dye is required for the negative staining technique?
a) Crystal violet
b) India ink
c) Methylene blue
d) Iodine

4) All of the following are common bacteriological stains, Except:
a) Bismarck brown
b) Methylene blue
c) Eosin
d) Crystal violet

5) Gram staining technique is used for the differentiation of gram-positive and gram-negative bacteria. What color do gram-negative bacteria show when observed under the microscope after the staining procedure?
a) Blue
b) Pink
c) Purple
d)Violet

6) MacConkey’s agar is both Selective and Differential media used primarily for the isolation of gram-negative bacteria. It consists of ...........which inhibits the growth of gram-positive bacteria.
a) Blood
b) Peptone
c) Bile salts
d) Tryptophan

7) All of the following are lactose fermenting bacteria, except
a) Klebsiella pneumoniae
b) Escherichia coli
c) Enterobacter aerogenes
d) Pseudomonas aeruginosa

8) Which of the given bacteria has flagella and shows positive motility test?
a) Staphylococcus aureus
b) Yersinia pestis
c) Klebsiella pneumoniae
d) Proteus vulgaris

9) The biochemical test helps in identification and classification of microorganisms, there are many different types of this test, which of the following tests show a negative result for Escherichia Coli?
a) Lactose
b) Indole
c) Citrate
d) Glucose

10)The cultural characteristics of Clostridium perfringens, when grown on Blood agar, are all of the given below, Except?
a) Microaerophilic
b) Spore-forming
c) Lecithinase positive
d) Stormy fermentation

11) …................is a rod-shaped gram-positive, a non-spore forming organism that can cause foodborne illness in human and animals. It can grow at very low temperature on a wide variety of food including raw meat, raw vegetables, unpasteurized milk, cheese, etc., and is considered one of the most virulent foodborne pathogenic bacteria.
a) Bacillus cereus
b) Listeria monocytogens
c) Clostridium botulinum
d) Lactobacillus acidophilus

12)Which of the given organism when grown in blood agar media is classified according to its hemolytic properties?
a) Bacillus anthracis
b) Proteus vulgaris
c) Streptococcus pyogens
d) Staphylococcus epidermidis

13) All of the statements for Bacillus anthracis are true except
a) Grows aerobically as well as anaerobically
b) Nonhemolytic colonies on Blood agar
c) Major foodborne pathogen
d) Endospore-forming bacteria

14) Which of the following bacteria is a part of human gut and mouth flora and is also used in making some fermented dairy products?
a) Candida albicans
b) Lactobacillus acidophilus
c) Staphylococcus aureus
d) Micrococcus luteus

15) …............is part of human normal gut flora and also a clinically important pathogen, it's infection can lead to symptoms such as itchy skin, itchy genital area, white coat on the tongue and mouth ulcers, constipation. When grown in the lab, the organism produces green pigmented colonies on CHROMagar.
a) Streptococcus mutans
b) Mycobacterium tuberculosis
c) Proteus mirabilis
d) Candida albicans

16) Which of the following organism of the Enterobacteriaceae family have the characteristics of swarming growth on culture media, produces urease enzyme, do not usually ferment lactose and is mainly the cause of urinary tract infections and nosocomial infections?
a) Proteus spp
b) Salmonella spp
c) Lactobacillus spp
d) Klebsiella spp

17) ................is a gram-negative bacillus, encapsulated, lactose fermenter and show mucoid colonies on media. It is also a part of the normal flora of the oral cavity, skin, and intestine.
a) Pseudomonas aeruginosa
b) Staphylococcus aureus
c) Streptococcus pneumoniae
d) Klebsiella pneumoniae

18) …......... is a blood agar media prepared by slowly heating the media at 80 degree Celsius until it becomes brown, it is used for the isolation of some important pathogenic organisms such as Haemophilus spp and Neisseria gonorrhoea.
a) Potato dextrose agar
b) Chocolate agar
c) Nutrient agar
d) Mac Conkey agar

19) All of the statements are true for Corynebacterium spp, except
a) Requires selective media containing tellurite
b) Gram-positive
c) Colonies appear black or gray
d) Motile

20. Mannitol Salt agar is both selective and differential media, it is used for the isolation of ................, the high salt concentration inhibits the growth of gram-negative bacteria.
a) Micrococcus spp
b) Streptococcus spp
c) Staphylococcus spp
d) Corynebacterium spp

Multiple Choice Answers
1(c), 2(b), 3(b), 4(a), 5(b), 6(c), 7(d), 8(d), 9(c), 10(a),
11(b), 12(c), 13(c), 14(b), 15(d), 16(a), 17(d), 18(b), 19(d), 20(c)
Medical Microbiology- Microbial Culture and Identification: MCQ Medical Microbiology- Microbial Culture and Identification: MCQ Reviewed by Biotechnology on September 18, 2018 Rating: 5

Immunology: Immunoglobulin Structure, Function: MCQ

September 15, 2018
Multiple Choice Question on Immunoglobulin Structure, Function

1) Immunoglobulin is the plasma protein that specifically binds to antigens. Identify the region of electrophoresis that consists of these major immunoglobulins.

a) Alpha region
b) Beta region
c) Gamma region
d) None of the above

2) The five classes of immunoglobulin include the following except
a) IgA
b) IgD
c) IgE
d) IgH

3) Which of the following class of immunoglobulin is pentameric structure?
a) IgA
b) IgD
c) IgH
d) IgM

4) Which of the following class of immunoglobulin is dimeric structure?
a) IgA
b) IgD
c) IgH
d) IgM

5) The IgA and IgMs consist of the following chain that allows its polymerization.
a) H chain
b) L chain
c) J chain
d) V chain

6) The monomeric immunoglobulin consists of heterodimers of heavy (H) and light (L) chain bound together by non-covalent interaction and disulfide bonds. Which of the following is the antigen binding site?
a) Fab
b) Fc
c) Hinge region
d) None of the above

7) The hinge region of the immunoglobulin consists of the disulfide bond that held the heterotetramer together. Also, it contributes to the flexibility of the antibody chain. Which of the following antibody class do not have a hinge region?
a) IgA
b) IgD
c) IgE
d) IgG

8) The hypervariable complementarity determining region is responsible for which of the following function
a) binding to antigen
b) binding to FcR
c) binding to complement
d) None of the above

9) Identify the protease that results in two different fragments of antibodies namely Fab and Fc fragments as shown in the figure below:

a) Pepsin
b) Trypsin
c) Papain
d) Fucin

10) Identify the protease that results in two different fragments of antibodies namely Fab and Fc fragments as shown in the figure below:

a) Pepsin
b) Trypsin
c) Papain
d) Fucin

11) The variable heavy and light chain make up the antigen recognition region which consists of six complementarity determining regions (CDRs) (three from each heavy and light chain). In addition, a stretch of amino acid sequence also known as framework region
a) assist in the recognition of antigen
b) act as a scaffold to support CDR
c) are highly variable
d) None of the above

12) The variable heavy and light chain make up the antigen recognition region. Which of the following is mostly involved in antigen binding?
a) Variable light chain
b) Variable heavy chain
c) Both of the above
d) None of the above

13) The contact area of the antigen binding area may consist of the protrusion or depression that complementarity matches the antigen. This contact area span approximately ................... based on well-studied Lysozyme/anti-Lysozyme interaction.
a) 5-12 amino acids
b) 15-22 amino acids
c) 25-60 amino acids
d) None of the above

14) The antigen-antibody interactions are considered inducible which means
a) The antigen binding site performed site that exactly fits the antigen.
d) The antigen binding site is rigid
c) The antigen binding site undergo confirmation changes after contact with the antigen
d) None of the above

15) Which of the following antibody have four constant regions (CH1, CH2, CH3, CH4)?
a) IgA
b) IgD
c) IgM
d) IgG

16) The effector function of the antibody requires its Fc region. The Fc region binds to cells or proteins to mediate its function. Which of the following is the effector function of the antibody?
a) Antigen binding to antibody promotes opsonization
b) Antigen binding to antibody activates complement
c) Antigen binding to antibody activates cell cytotoxicity
d) All of the above

17) Which of the following immunoglobulins are secretory and present in the milk?
a) IgG
b) IgM
c) IgE
d) IgA

18) The receptor that is responsible for transport of IgAs across the epithelial barriers:
a) Poly Fc receptor
b) Poly Ig receptor
c) Poly Fab receptor
d) All of the above

19) Which of the following antibody is produced as a primary immune response and have higher valency to remove clear antigens?
a) IgA
b) IgG
c) IgM
d) IgE

20) Which of the following is the passive immunity transferred from mother to its offspring?
a) Transplacental transfer of IgGs
b) Transfer of IgAs in the milk
c) Both a & b
d) None of the above

21) Which of the following antigen-bound antibodies bind to the Fc receptor present on the basophils and tissue mast cells, and releases various pharmacoactive mediators involved in anaphylaxis?
a) IgA
b) IgD
c) IgE
d) IgM

22) The Fc receptor is a plasma membrane glycoprotein that binds to different immunoglobulin and triggers effective functions. Which of the following Fc receptor is involved in the transfer of IgG from mother to fetus
a) Fc€R
b) FcRN
c) FcµR
d) FcγR

23) The immunoglobulin superfamily is the group of membrane proteins that possess one or more homologous immunoglobulin domain. Which of the following is NOT immunoglobulin superfamily?
a) T cell receptor
b) beta2 microglobulin
c) Insulin receptor
d) Platelet-derived growth factor

24) B-cell receptor consist of membrane-bound immunoglobulin and a small heterodimer protein required for signaling. Which of the following is the heterodimer protein?
a) Igα & Igβ
b) Igµ & Igγ
c) Igα & Igγ
d) None of Above

25) Multiple Myeloma is characterized by excessive production of immunoglobulin and presence of light chain in urine. Which of the following cells are responsible for the production of immunoglobulin
a) T cells
b) B-cells
c) Plasma cells
d) Dendritic cells

26) Which of the following complement is bound by IgG?
a) C2a
b) C2b
c) C3a
d) C3b

27) Which of the following subclass of IgG molecule is the most potent activator of complement pathway?
a) IgG1
b) IgG2
c) IgG3
d) IgG4

28) Which of the following subclass of IgG does not readily cross the placental barriers?
a) IgG1
b) IgG2
c) IgG3
d) IgG4

29) Which of the immunoglobulin isotype have the shortest half-life?
a) IgG
b) IgM
c) IgA
d) IgE

30) Which of the immunoglobulin isotype have the longest half-life?
a) IgG
b) IgM
c) IgA
d) IgE


Multiple Choice Answers

1)-c, 2)-d, 3)-d, 4)-a, 5)-c, 6)-a, 7)-c, 8)-a, 9)-c, 10)-a

11)-b, 12)-b, 13)-b, 14)-c, 15)-c, 16)-d, 17)-d, 18)- b, 19)-c, 20)-b

21)-c, 22)-b, 23)-c, 24)-a, 25)-c, 26)-d, 27)-c, 28)-b, 29)-d, 30)-a

Immunology: Immunoglobulin Structure, Function: MCQ Immunology: Immunoglobulin Structure, Function: MCQ Reviewed by Biotechnology on September 15, 2018 Rating: 5

Immunology: Host Defense Mechanism: MCQs

September 12, 2018
Multiple Choice Questions  on Host Defense Mechanism

1) According to ........................ theory, an individual lymphocyte expresses a membrane receptor that is unique to each antigen. The binding of the antigen to specific receptor provoke an immune response against that specific antigen.
a) Selective theory
b) Instructional theory
c) Clonal selection theory
d) None of Above

2) Immunity is the defense mechanism of the body that protect against foreign pathogens. The immunity can be classified into innate immunity and acquired immunity. Which the following is NOT true regarding innate immunity?
a) Broadly specific against the foreign antigen
b) Exist prior to the exposure o the antigens
c) Have memory cells
d) All of the above

3) Which of the following are the characteristics of acquired immunity?
a) Diversity
b) Memory
c) Specific
d) All of the above

4) Which of the following is not an example of innate immunity?
a) Phagocytosis
b) Antibodies
c) Interferon
d) Mucus membrane

5) Lysozyme is an enzyme present in the tears and mucous secretion that cleaves
a) Lipopolysaccharides
b) Cellulose
c) Peptidoglycan
d) None of the above

6) Lipopolysaccharides present in the cell membrane of gram-negative bacteria are recognized by .................... and elicit an inflammatory immune response
a) Phagocytosis
b) Antibodies
c) Mucus lining
d) Toll-Like receptors (TLR-2)

7) The cardinal signs of inflammation are rubor (redness), tumor (swelling), calor (heat) and dolor (pain). Which of the following is the characteristic feature of inflammation response?
a) Vasodilation
b) Increased capillary permeability
c) Recruitment of phagocytosis
d) All of the above

8) The adaptive immunity that involves the production of antibodies for the clearance of antigen is called
a) Cell-mediated cytotoxicity
b) Cell-mediated immunity
c) Humoral Immunity
d) None of the above


9) The antigen presenting cells (APCs) plays a crucial role in antibody-mediated and cell-mediated immune response. Which of the following is not the characteristic feature of APCs?
a) APCs internalize and degrade antigens
b) APCs present antigens to the T-cells via MHC-II molecules
c) APCs provide a co-stimulatory signal for T-cell activation
d) Excess co-stimulatory signals from APCs lead to hyperactivation of an immune response.


10) Which of the following is not the class of T cells?
a) T-helper cells
b) T-cytotoxic cells
c) T-suppressor cells
d) T-activator cells

11) Which of the following cells produces antibodies?
a) T-cells
b) Plasma cells
c) B cells
d) Memory cells

12) Which of the following is expressed in T cells that interact with antigen epitope?
a) T-cell receptor
b) T-cell antibodies
c) B-cell receptor
d) B-cell antibodies


13) Antibody functions as the effector of the humoral response by antigen binding and neutralizing it. The antigen can be eliminated by
a) Facilitating the antibodies update by phagocytes
b) Activating complements and inducing cell lysis
c) Preventing the binding and host cell attachment
d) All of the above

14) Which of the following immune cells are not derived from lymphoid progenitor cells?
a) B-cells
b) T-cells
c) Natural Killer cells
d) Neutrophils

15) The development of pluripotent hematopoietic stem cells into different cell types requires the expression of a different set of genes for lineage determination at appropriate time and order. The various transcription factor is required for the expression of these genes. Which of the following statement is TRUE
a) GATA-1 is required for myeloid lineage
b) Oct-2 is required for B cell differentiation to plasma cells
c) Ikaros is required for erythroid lineage
d) None of the above

16) The different lineage of the lymphocytes can be distinguished by characterizing the expression of their membrane molecules called the cluster of differentiation (CD). Which of the following CD is only found in B-cells?
a) CD-4
b) CD-8
c) CD-32
d) CD-45

17) Which of the following CD molecule is present in both cell types of T cells (Th and Tc) and acts as a receptor for the co-stimulatory signal from APCs?
a) CD2
b) CD4
c) CD28
d) CD45

18) Which of the following CD molecule is a signaling transduction molecule present in lymphoid lineage cells such as T-cell, B-cells, and NK
a) CD2
b) CD4
c) CD28
d) CD45

19) Which of the following cell plays a crucial role in antibody-dependent cell cytotoxicity
a) Macrophage
b) Natural Killer cells
c) B-cells
d) Dendritic cells

20) The tissue-specific macrophage-like tissues are essential for phagocytosis of the antigens. Which of the following cells are present in the kidney?
a) Alveolar macrophages
b) Kuffer cells
c) Histiocytes
d) Mesangial cells
21) Immunogens are the antigens that can evoke an immune response. Which of the following is not an immunogen?
a) Protein
b) Lipopolysaccharides
c) Polysaccharides
d) Hapten

22) The degree of immunogenicity generally depends on the degree of foreignness. Which of the following protein are highly conserved among species and have little immunogenicity when injected to cross-species?
a) Bovine Serum Albumin
b) Thyroglobulin
c) Insulin
d) Collagen

23) Although the degree of immunogenicity generally depends on the degree of foreignness, certain antigens/tissues are immunogenic against self-antigens. The example includes
a) Kidney
b) Cornea
c) Heart
d) Collagen

24) Which of the properties of an antigen makes it poorly immunogenic?
a) Distant species origin
b) High molecular weight proteins
c) Heteropolymers
d) Homopolymers

25) B-cells have cell surface antibodies that serve as the recognition molecule, and T cell recognizes when these antigens are presented via MHC molecules. Some glycolipids are also recognized by T-cell when presented by a non-MHC molecule known as
a) CD1
b) CD2
c) CD4
d) CD8

26) The discrete sites on the are recognized by antibody or T cells are called epitopes. Which of the following is TRUE regarding epitopes
a) B cell and T cells recognize the same epitopes
b) B cell and T cells recognize different epitopes
c) Epitope contains only sequential amino acids.
d) Epitopes bind to the antibody with covalent bonding

27) The epitopes that are recognized by B-cells are known as B-cell epitopes. The following is a true statement regarding B-cell epitope
a) they are generally hydrophilic amino acids present on the surface
b) they contain both sequential and conformational epitopes
c) they tend to present on the flexible region of the antigens
d) All of the above

28) The epitopes that are recognized by T-cells are known as T-cell epitopes. The following is a true statement regarding T-cell epitope:
a) the epitopes are recognized by T-cell as a trimeric complex of TCR, antigen and MHC molecule.
b) antigen processing is required for its presentation by APC
c) amino acid sequences of T-cell epitopes are generally internal of a protein molecule
d) All of the above

29) The following congenital defect of thymus development lead to T-cell deficiency:
a) Graves disease
b) DeGeorge's syndrome
c) Anaphylaxis
d) All of the above

30) Severe Combined immunodeficiency disorder (SCID) is a genetic disorder caused by a defective enzyme
a) Xanthine oxidase
b) Adenosine deaminase
c) Anaphyloticase
d) Lysozyme




Multiple Choice Questions Answer review
1)- c,
2)- c,
3)-d,
4)- b,
5)- c,
6)- d,
7)- d,
8)- c,
9)- d,
10)- d
11)-b,
12)-a,
13)-d,
14)-d,
15)-b,
16)-c,
17)-c,
18)-d,
19-b),
20)-d
21)-d,
22)-d,
23)-c,
24)-d,
25)-a,
26)-b,
27)-d,
28)-d,
29)-b,
30)-b
Immunology: Host Defense Mechanism: MCQs Immunology: Host Defense Mechanism: MCQs Reviewed by Biotechnology on September 12, 2018 Rating: 5

Medical Microbiology- Bacterial Pathogenesis: MCQ

September 09, 2018
Multiple Choice Question on Bacterial Pathogenesis

1) Which of the following microorganism is the major inhabitant of the skin?
a) Escherichia coli
b) Staphylococcus epidermidis
c) Staphylococcus aureus
d) Streptococcus pyogens

2) Which one of the following is not the normal flora of the skin?
a) Diptheriods
b) Staphylococci
c) Helicobacter
d) Micrococci

3) All of the following are gram-negative bacilli EXCEPT
a) Proteus spp
b) Pseudomonas spp
c) Neisseria spp
d) Klebsiella spp

4) Which of the following cocci shaped bacteria is usually seen in pairs?
a) Klebsiella spp
b) Neisseria spp
c) Pseudomonas spp
d) Clostridium spp

5) Which one of the following pathogenic organism is capable of living inside the living cell only(obligate intracellular pathogen)?
a) Salmonella
b) Mycobacterium
c) Rickettsia
d) Vibrio

6) ...................is the common normal flora of upper respiratory tract
a) Lactobacillus spp
b) Staphylococcus spp
c) Vibrio spp
d) None of the above

7) Which of the following organism releases endotoxin that causes muscular paralysis?
a) Clostridium botulism
b) Bacillus cereus
c) Streptococcus pyogens
d) Salmonella typhi

8) Which of the following enzyme is used to distinguish between different types of Staphylococcus isolates?
a) Proteases
b) Lipase
c) Hyaluronidase
d) Coagulase

9) Which of the following is the predominant urogenital flora present during newborn female infants?
a) Candida albicans
b) Lactobacillus acidophilus
c) Escherichia coli
d) Neisseria gonorrohea

10) All of the given organisms are the predominant normal flora of human feces EXCEPT
a) Pseudomonas sps
b) Bacteroides sps
c) Enterococcus sps
d) Bacillus sps

11) Vector-borne bacterial pathogenesis is caused only when bacteria are transmitted into a host cell through a vector. Among these ................ is a vector-borne pathogen.
a) Salmonella typhi
b) Yersinia pestis
c) Shigella dysenteriae
d) Escherichia coli

12) Some bacteria and fungi need an iron receptors molecule for their growth, what is it called?
a) Siderophores
b) Ionophores
c) Siderocytes
d) None of the above

13) Which toxin is produced by Streptococcus pyogens?
a) Shiga like toxin
b) Alpha toxin
c) Erythrogenic toxin
d) Cyanotoxin

14) Which bacteria causes toxic shock syndrome
a) Staphylococcus epidermidis
b) Staphylococcus aureus
c) Staphylococcus intermedius
d) None of the above

15) Catalase test is the biochemical test used for identification of various bacteria. Which of the following bacteria is negative to this test?
a) Enterobacter
b) Pseudomonas
c) Corynebacterium
d) Streptococci

16) Staphylococcus aureus releases a various toxin that is important for their pathogenesis. Following are toxin released by S aureus EXCEPT:
a) Botulinum
b) Alpha
c) Leucocidin
d) Enterotoxin

17) Which pathogen is a major cause of dental disease?
a) Staphylococcus epidermidis
b) Streptococcus mutans
c) Staphylococcus aureus
d) Streptococcus agalactiae

18) The microorganism that commonly causes an eye infection is
a) Chlamydia trachomatis
b) Staphylococcus aureus
c) Streptococcus pneumoniae
d) Streptococcus sanguinis

19) The most significant bacteria found in acne is ........ acnes.
a) Staphylococcus
b) Streptococcus
c) Propionibacterium
d) Bacillus

20) Which of the following bacteria is predominantly present in normal urine?
a) Escherichia coli
b) Staphylococcus epidermidis
c) Staphylococcus aureus
d) Streptococcus pyogens

Multiple Choice Answer Review
1- b) Staphylococcus epidermidis
2-c) Helicobacter
3- c) Neisseria spp
4-b) Neisseria spp
5-c) Rickettsia
6- b) Staphylococcus spp
7-a) Clostridium botulism
8-d) Coagulase
9- b) Lactobacillus acidophilus
10-a) Pseudomonas sps
11-b) Yersinia pestis
12-a) Siderophores
13-b) Alpha toxin
14-b) Staphylococcus aureus
15-d) Streptococci
16-a) Botulinum
17-b) Streptococcus mutans
18-a) Chlamydia trachomatis
19-c) Propionibacterium
20-a) Escherichia coli

Medical Microbiology- Bacterial Pathogenesis: MCQ Medical Microbiology- Bacterial Pathogenesis: MCQ Reviewed by Biotechnology on September 09, 2018 Rating: 5

Molecular Biology: MCQ on RNA Synthesis (Translation) & Maturation

September 05, 2018
Multiple Choice Question on
Transcription (RNA synthesis) and RNA processing 

1) Transcription of DNA results RNA that have multiple functions including
a) messenger RNA serves as a template for synthesis of proteins
b) tRNA serves as the adapter molecule for the addition of amino acids and elongation of the peptide chain
c) ribosomal RNA serves as machinery for protein synthesis
d) All of the above

2) Which of the following RNA serves the regulatory functions including splicing, gene silencing?
a) mRNA
b) tRNA
c) rRNA
d) small RNA

3) Which of the following statement is NOT true regarding transcription/RNA synthesis?
a) RNA synthesis occurs in the nucleus
b) Unlike DNA synthesis, the only selective sequence of DNA is transcribed to RNA
c) RNA synthesis requires a short stretch of RNA primers
d) DNA sequences, specific proteins, and small RNAs regulate RNA synthesis.

4) The pentose sugar moieties are the primary structural difference between DNA and RNA. In addition which of the following is primarily associated with RNA molecule?
a) RNA consist of thymine instead of uracil
b) RNA molecules are highly branched structure
c) RNA molecules have higher structural complexities
d) RNA molecules are anti-parallel and double-stranded


5) In prokaryotes, RNA polymerase catalyzes the synthesis of:
a) mRNA
b) rRNA
c) tRNA
d) All of the above

6) The RNA polymerase is a multi-subunit enzyme that recognizes a consensus nucleotide sequence (promoter region) upstream of the transcription start site. In prokaryotes, the consensus promoter sequence consists of 5-TATAAT-3' also known as
a) Enhancer box
b) Pribnow box
c) Transcription unit
d) None of the above

7) RNA polymerase catalyzes the synthesis of RNA by addition nucleotide monophosphate and release of pyrophosphate for nucleotide triphosphate. RNA polymerase
a) consists of 5'-3' exonuclease activity
b) lacks 3'-5' endonuclease activity
c) is a high fidelity enzyme
d) All of the above

8) In prokaryotes, a holoenzyme RNA polymerase consists of four core subunits namely 2α, 1β, 1β' and a promoter recognizing σ subunit. It may also require a termination factor for termination of the transcription factor. Which of the following is a transcription factor?
a) gamma factor
b) delta factor
c) epsilon factor
d) rho factor

9) In prokaryotes, TTGACA is an upstream consensus nucleotide sequence that is required for transcription ....... step
a) Initiation
b) Elongation
c) Termination
d) Capping

10) The termination of transcription occurs in both rho-dependent and rho-independent manner. Which of the following is NOT true regarding the termination of transcription
a) rho proteins recognize C-rich region near 3'end of the newly synthesized RNA
b) rho-independent termination occurs when the transcription reaches the palindromic structure leading to the formation of hairpins
c) rho protein competes with RNA polymerase for binding to nucleotides
d) None of the above

11) Rifamycin is an antibiotic used for the treatment of tuberculosis. It binds to .... subunit of RNA polymerase and inhibits the initiation of transcription.
a) α,
b) β
c) σ
d) ζ

12) In eukaryotes, the RNA synthesis process is more complex than prokaryotes. The RNA synthesis process is regulated by chromatin structure, upstream and downstream sequences, binding partners, etc. Which of the following is TRUE regarding the transcription process in eukaryotes:
a) Most actively transcribed genes are found in a loosely relaxed form of chromatin called euchromatin
b) The most inactive segment of DNA is found in compact chromatin structure called heterochromatin
c) Histone modification such as methylation, acetylation regulate the RNA transcription by modulating chromatin structure
d) All of the above

13) In eukaryotes, three different RNA polymerases are involved in the synthesis of a different class of RNAs namely: rRNA, tRNA, and mRNA. The RNA polymerase that is required for the synthesis of mRNA is
a) RNA polymerase I
b) RNA polymerase II
c) RNA polymerase III
d) None of the above

14) In eukaryotes, the consensus promoter sequences (TATA box) that are required for initiation of transcription is generally present
a) 10 nucleotide upstream of transcription start site (TSS)
b) 25 nucleotide upstream of TSS
c) 10 nucleotide downstream of TSS
d) 25 nucleotide downstream of TSS

15) Enhancers are special cis-acting DNA sequences that increase the rate of transcription by RNA polymerase. Which of the following is true regarding enhancers?
a) 10 nucleotide upstream elements
b) 25 nucleotide downstream elements
c) present closer or 1000s nucleotide upstream or downstream of TSS
d) All of the above

16) The capping of nucleotide prevents the rapid cleavage of mRNA and catalyzed by guanylyltransferase. Identify the nucleotide cap that is attached at the 5'end of mRNA.
a) 5-methyl guanosine
b) 7- methyl guanosine
c) 5- acetyl guanosine
d) 7- acetyl guanosine

17) The polyadenylation is a post transcription modification that stabilizes the mRNA and prevent from the cleavage. The consensus PolyA sequence is
a) (AAGAAA)n
b) (AACAAA)n
c) (AATAAA)n
d) (AAUAAA)n

18) In eukaryotes, the primary transcripts are processed to remove intervening sequence resulting in mRNA and the process is known as splicing. The complex of RNA, nucleoproteins that execute splicing process is called:
a) Primosome
b) Splicing fork
c) Spliceosome
d) None of the above

19) The role of small nuclear ribonucleoprotein particles (snRNPs) is
a) to bind intronic sites and exon segments
b) facilitate the looping of the two exons into the correct alignment for splicing
c) All of the above
d) None of the above

20) The auto-antibodies against the small nucleoproteins are present in
a) beta thalassemia
b) systemic lupus erythematosus
c) Phenylketonuria
d) None of the above

21) The antibody binding diversity is a result of a type splicing that produces mRNA variants and protein variants by processing different segment of exons. The process is known as
a) Diversity splicing
b) Alternative splicing
c) Conservative splicing
d) None of the above

22) CAAT box is present in many
a) Prokaryotic promoters upstream of TATA box
b) Prokaryotic promoters are downstream of TATA box
c) Eukaryotic promoters are upstream of TATA box
d) Eukaryotic promoters are downstream of TATA box


Multiple Choice Answers
1-d) All of the above
2-d) small RNA
3- c) RNA synthesis requires a short stretch of RNA primers
4- a) RNA consist of thymine instead of uracil
5- d) All of the above
6-b) Pribnow box
7-b) lacks 3'-5' endonuclease activity
8- d) rho factor
9-a) Initiation
10)-c) rho protein competes with RNA polymerase for binding to nucleotides
11- b) β
12- d) All of the above
13-b) RNA polymerase II
14-b) 25 nucleotide upstream of TSS,
15-c) present closer or 1000s nucleotide upstream or downstream of TSS,
16-b) 7- methyl guanosine
17-d) (AAUAAA)n
18-c) Spliceosome
19-c) All of the above
20-b) systemic lupus erythematosus,
21- b) Alternative splicing
22-a)Prokaryotic promoters upstream of TATA box



Molecular Biology: MCQ on RNA Synthesis (Translation) & Maturation Molecular Biology: MCQ on RNA Synthesis (Translation) & Maturation Reviewed by Biotechnology on September 05, 2018 Rating: 5
Powered by Blogger.