biotechnology

1.   Highest capacity vector is

a) Cosmid

b) YAC

c) Yeast integrative vector

d) Bacteriophage vector

Ans: b

• Cosmid: A plasmid with a cos site of lambda phage.Insert size: 30-45 kb
• YAC: yeast artificial chromosome. Insert size: 1 Mb
• Bacteriophage vectors: refers to lambda and M13 phage vectors
• Lambda phage vectors: Insert size: 8-23 kb
• M13 vectors used for obtaining single stranded copies of cloned DNA that are suited for DNA sequencing
• BAC: Bacterial Artificial Chromosome. Insert size: 300 kb

2.   The C value denotes the total number of DNA in a

        a) Diploid

        b) Haploid

        c) Aneuploid

        d) Polyploid

      Ans:b

• C value: Haploid DNA content of an organism or the amount of DNA in haploid nuclei like a gamete.
• C value paradox: refers to the phenomenon that huge genomic content has nothing to do with the complexity of an organism.For eg: Protists has genome size much larger than humans.

3. Cdc mutants are useful for the study of 

a) Chromosome breakpoint

b) Apoptopsis

c) Various stages of Cell cycle

d) Homeodomain 

 Ans: c  cdc refers to cell division cycle

4.  RNA is very much susceptible to hydrolysis in alkali because

a) It contains Uracil residues in its structure

b) Its 2’ OH groove participate in intramolecular cleavage of phosphodiester backbone

c) Cleavage occurs in the glycosilic bonds of purine bases

d)Cleavage occurs in the glycosilic bonds of pyrimidine bases

Ans:b

• Because of the presence of 2’ OH group in the ribose sugar
• DNA is more stable than RNA because of the presence of H at 2’ position in DNA rather than OH in RNA.

5. Which one of the following is not a requirement of a PCR reaction?

a) DNA template

b) NTPs

c) MgCl2

d) Taq polymerase

Ans:d

Here the best option is MgCl2 even though it is required for DNA polymerase activity. (It can be avoided)

6. The heterozygosity of any locus can be ascertained by

 a) SNPs

b) RFLPs

c) FISH analysis

d) Either RFLP analysis or SNP

Ans: d

• SNP: single nucleotide polymorphism refers to the variation in the lengths of some DNA btw individuals due to single base changes

          Application: used in DNA finger printing

• RFLP: restriction fragment length polymorphism: refers to the variation in the restriction fragment length between individuals of a species.
•  FISH: florescence insitu hybridization: hybridizing fluorescently labelled DNA probes on to human chromosome preparations allows genes to be mapped directly to their chromosomal locations.

7.  Hormone pairs requires for a callus to differentiate are

 a) Auxin and Cytokinin

b) Auxin and Gibberellin

c) Cytokinin and Gibberellin

d) Ethylene and Gibberllin 

Ans: a

Auxin induces rooting whereas cytokinins promote shooting if supplied in optimum ratio.

8. Embryo rescue is a useful technique to 

 a) Grow /generate hybrids between different plant species

 b) Complete the growth of embryos susceptible to defects in seed development

 c) Break the dormancy of seeds

 d) All of the above

Ans:d 

9. Antibody diversity is generated by           

a) protein splicing

b) somatic mutations

c) allelic exclusion

d) interchromosomal recombination          

   Ans: b

• Somatic mutation: a mechanism by which point mutations are introduced into rearranged immunoglobulin variable region genes during activation and proliferation of B cells. It contributes significantly to antibody diversity.
• Allelic exclusion: a process that permits the expression of only one of the allelic forms of a gene. It contributes to specificity of IgGs

10. The precursor for penicillin-G biosynthesis during fermentation process is            

a)Phenylacetic acid

b)Acetic acid

c)Phenoxy acetic acid

d)None of the above

Ans:c 

11.Plastome is                

a)Plasmalemma protein

b)A type of plasmid

c)An organellar genome

d)None of the above

Ans: plastome: Genetic material of plastid like Chloroplast

12. Which of the following process require energy

a) ligation

b) restriction digestion

c) hybridization

d) transformation

Ans:a

Ligation: sealing of single stranded nicks or breaks by ligase enzyme. The process requires ATP and NADP⁺.

13) Enhanced axillary branching for multiple shoot production is promoted by               

a)2,4-D

b)Abscisic acid

c)BA (Benzyl adenine)

d)Gibberellic acid

Ans:a

2,4-D is an auxin and auxin is responsible for sustaining apical dominance there by promoting axillary branching

14.  Viral replication within cells is inhibited by             

a)IL-4

b)IL-1

c)IFN alpha

d)TNF alpha

Ans:c 

Interferons: are antiviral agents (proteins) secreted by virus infected cells and induces a virus resistant state to the surrounding cells by inhibiting its replication.

15.  In the course of cell cycle, the level of the protein cyclin abruptly falls during             

a) G1phase

b) G2 phase

c) S phase

d) M phase

 Ans:d                                    

16. Protein binding regions of DNA are identified by one of the following techniques

a) Finger printing

b) Foot printing

c) Southern blotting

d) Western blotting

  Ans: (b) Foot Printing

• Foot Printing: The identification of a protein-binding site on a DNA molecule by determining which phosphodiester bonds are protected from cleavage by DNase I.
• Southern Blotting: The technique used for identifying specific DNA fragment using labelled probe.

17.Si RNA (s) interfere at

a) Transcriptional level

b) Post -transcriptional level

c) DNA replication level

d)Translational level

Ans: (b) Post -transcriptional level

18. Plant secondary metabolites

a) Help to increase the growth rate of plant

b) Help in plant reproduction processes

c) Provide defence mechanisms against microbial attack

d) Make the plant susceptible to unfavourable conditions

Ans: (c) Provide defence mechanisms against microbial attack

19. Mobile genetic elements present in human genome are

P) LINES

Q) SINES

R) P elements

S) IS elements

(a) Q, R (b) P, Q  (c) P, R (d) Q, S

Ans: (b) P, Q

20. Meristems escape virus invasion because

a)Vascular system is absent in the meristem

b) of low metabolic activity in the meristem

c) of low endogenous auxin level

d) the virus inactivating system has low activity in the meristem

Ans: b) of low metabolic activity in the meristem

21. The enzyme that can be used in 5’end labeling of DNA are

P) Alkaline phosphatase

Q) DNA ligase

R) Terminal transferase

S) Polynucleotide kinase

(a) P, S (b) R, Q (c) P, R (d) R, S

Ans: (a) P, S

22. Identify the natural plant growth regulators from the following list

 P) Zeatin

Q) Benzylamino purine (BAP)

R) Indole Acetic Acid (IAA)

S)  2,4-Dichlorophenoxyacetic acid (2,4-D)

(a)P, Q (b) Q, S (c) P, R(d) R,S

Ans: c) P, R

23. Cells of meristemoid are best described as

a) differentiated and non dividing

b) Dedifferentiated and dividing

c) differentiated and dividing

d) dedifferentiated and non dividing

Ans: c) differentiated and dividing

24. Ultra filtration process cannot be used for

a)Fractionation of Proteins

b)Desalting

c) Harvesting of cells

d)Selective removal of solvents

Ans: c) Harvesting of cells

25. The number of replicons in a typical mammalian cell is

a) 40-200

b) 400

c) 1000-2000

d) 50000-100000

Ans: a) 40-200

26. What product will result from complete hydrolysis of soluble dextran

a) Sucrose only

b) Fructose only

c) Glucose &Fructose only

d) Glucose only

Ans: d) Glucose only

27. The mobility of DNA in agarose gel electrophoresis is solely based on its

a) Charge

b) Conformation

c) Size

d) None of these

Ans: c) Size

28. Which of the following fluorescent probes is used to monitor the progress of amplification of Real Time PCR?

a) SYBR Green

b) FITC

c) Cyan Blue

d) Rhodamine

Ans: b) FITC

29. Expression of which of the following reporter genes does not require addition of specific substrate for detection

a) Luciferase

b) β- Glucuronidase

c) β-Glucosidase

d) Green Fluorescent Protein (GFP)

        Ans: a) Luciferase

                                                                                 

30. Zinc fingers are characteristic of

(a) Blood clotting Proteins

(b) RNA Chaperones

(c) DNA binding proteins

(d) Lysosomal hydrolyses

Ans: DNA binding proteins

• Chaperons: proteins that assist in proper folding of proteins.

31. Multiplication of genetically identical copies of a cultivar by asexual reproduction is known as

(a) aclonal propagation

(b) clonal propagaation

(c) polyclonal propagation

(d) vegetative propagation

Ans: clonal propagaation

32. Parthenogenetic embryos in plant are those are formed by

(a) unfertilized eggs

(b) fertilized eggs

(c) male gametophyte

(d) sporophytic cells

Ans: unfertilized eggs

• Parthenogenesis: The development of an individual from an egg without fertilization.

33. Which one of the following is the growth factor used for growth of tissues and organs in plant tissue culture?

(a) Cytokinin

(b) Cysteine

(c) Cytidylate

(d) Cyclic AMP

Ans: Cytokinin

• Cysteine :Sulphur containing amino acid
• Cytidylate: an enzyme with kinase activity
• Cyclic AMP: second messenger in intracellular signal transduction

34. Which of the following techniques is best suited for immobilizing an affinity ligand?

(a) Physical adsorption

(b) Gel entrapment

(c) Cross-linking with a polymer

(d) Covalent linkage to a spacer arm

Ans: Covalent linkage to a spacer arm                                                   

 35.  To be a cloning vector, a plasmid does NOT require

a) an origin of replication

b) a restriction site

c) an antibiotic resistance marker

d) to have a high copy number

Ans: d) to have a high copy number

Properties of a Cloning Vector

• Should be able to replicate autonomously
• Easy to transform
• Should have suitable marker
• Unique restriction sites
• For expression vector, control elements like promoter, operator etc should be there.

36. Enzyme papain is used with success to

a) increase in meat production

b) leaven bread

c) ripen papaya fruit

d) tenderize meat

Ans: d) tenderize meat

• Source: papaya fruit

37. For protoplast fusion to be successful in plant cells

a) fusion agents other than polyethylene glycol should be used

b) cellwall of the two strains of cells should be compatible

c) DNA between the two cells should be compatible

d) osmolarity of the medium is not important

Ans: DNA between the two cells should be compatible

38. In animal cell culture, the addition of serum to media is essential for providing

a) amino acids for protein synthesis

b) nucleotides for DNA synthesis

c) growth factors

d) All of the above

Ans: All of the above

39. Which one of the following reactions is used for the purpose of recycling enzymes in bioprocesses?

a) isomerisation

b) immobilisation

c) phosphorylation

d) polymerisation

Ans: Immobilisation

• Technique used for the fixation of enzymes or cells on to solid supports
• Advantage: reuse of enzyme for many reactions
• Methods of enzyme immobilization:-adsorption, covalent bonding, entrapment, membrane confinement

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GATE Questions: Biochemistry

 1. Which of the following inhibitor uncouples electron transport and oxidative phosphorylation?

(a) Azide

(b) Dinitrophenol (DNP)

(c)Oligomycin

(d) Rotenone

Ans: Dinitrophenol (DNP)

• Azide  (N₃^-), cyanide (CN^-) and CO all inhibit cytochrome oxidase
• DNP is an un coupling agent as they stop ATP synthesis without disrupting electron transport.  Here the energy derived from electron transport is released as heat.
• Rotenone and amytal inhibits electron transport at NADH dehydrogenase 
• Uncoupling protein thermogenin in brown adipose tissue for maintaining body temperature in cold thriving animals
• Non shivering thermogenesis: The production of heat by uncoupling

2. Which of the following activate Protein kinace C

(a) Inositol 1,4,5-triphosphate

(b) Diacylglycerol

(c)Inositol

(d)Cyclic AMP

Ans: Inositol 1,4,5-triphosphate (IP₃)

• All options are intracellular signalling molecules called second messengers

3. Transcription initiation sites can be determined by

(a) Foot Printing

(b) Northern Blotting

(c) Primer extension

(d) Nick translation

Ans: Foot Printing

• DNA foot printing: The identification of protein binding site on a DNA molecule Here RNA polymerase enzyme (a protein) binds to the transcription initiation site at the beginning of transcription.
• Northern Blotting: identifying specific RNA sequence in a sample using a probe
• Primer extension: by DNA polymerase
• Nick translation: The repair of nick (ss breaks) using DNA pol I, generally to introduce labelled nucleotides into a DNA molecule

4. One common feature between B and T cells is that

(a) both cells produce antibodies

(b) both cells possess MHC class II

(c) both B cell receptor and T cell receptor undergo rearrangement

(d) both cells can produce cytokines

Ans:  both B cell receptor and T cell receptor undergo rearrangement

• Only B cell can produce antibodies (specifically plasma cells)

•  Only B cells possess MHC class II as it can also function as antigen presenting cells
• Only T cell can produce cytokines that eventually activates B cells

5. In Hybridoma technology, the myeloma cells used

(a) lack HGPRTase

(b) lack the ability to produce Ig

(c) lack both HGPRTase and ability to produce Ig

(d) lack thymidine kinase

Ans: lack HGPRTase

• Myeloma cells will die out in HAT selection medium as it lacks HGPRTase (enzyme in nucleotide synthesis) and B cells will undergo normal cell death and only hybrid cells survive in HAT medium (refer monoclonal antibody production)                                   

6. Which amino acid residue is most likely to be found in the interior of a water soluble globular protein?

a) Ser

b) Arg

c) Asp

d) Val

Ans: Valine: A hydrophobic amino acid preferring interior of the globular protein

• Arginine and asparagine are hydrophilic and tend to be in the exterior, serine is also reactive due to the presence of OH group (polar).

More about refer  Amino acids

7.  Of the peptide sequences given below, which one is the digestive enzyme trypsin most likely to cleave?

a) ----Val-Lys-Pro-Met----

b) ----Arg-Val-Phe-Tyr----

c) ----Glu-Gly-Trp-Gly----

d) ----Trp-Asp-Gln-Pro---- 

Ans: b) ----Arg-Val-Phe-Tyr----

• Digestive enzyme Trypsin cleave on the C terminal side of basic amino acids (Arg, Lys) residues
• Chymotrypsin cleave on the C terminal side of aromatic amino acids (Phe, Trp, Tyr) residues
• Cyanogen bromide cleave C terminal side of Met residues

8. Which pair of amino acids will have the highest absorbance at 280 nm? (Assume equimolar concentarions)

a) Thr & His

b) Phe & Pro

c) Trp &Tyr

d) Phe & His

Ans: c) Trp &Tyr

• Absorbance at 280nm is by aromatic amino acids. Here Tryptophan and tyrosine are the aromatic amino acids (Phe is the other aromatic amino acid)
• Order of absorbance: Trp>Tyr>Phe
• The aromatic rings of Trp and Tyr contain delocalised π electrons that strongly absorb UV light (280nm).

9. Vitamin D is derived from which of the following precursors by the action of UV-light?

a) 7-Dehyrocholestrol

b) Lanosterol

c) Glycocholate

d) Squalene epoxide

Ans: a) 7-Dehyrocholestrol

• Vit-D (Cholecalciferol)

For more, refer our post: Vitamins

10. The molecular defect in familial hypercholesterolemia is due to the lack of functional

a) VLDL receptor

b) IDL receptor

c) LDL receptor

d) HDL receptor

Ans: c) LDL receptor

• Familial hypercholesterolemia an inherited disorder 
• Condition: elevated level of cholesterol in the blood as LDL cholesterol cannot be taken up by the tissues.

------------------------------------------ Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------

Immunology Glossary Terms

Immunology: (“immunis” Latin=’exempt’): Brach of biomedical science that deals with the study of all aspects of immune system in all organisms.

Immune system: refers to the collection of mechanisms involving cells, tissues and organs that protects organisms against disease by identifying and killing pathogens and tumour cells.

Immunity:  to the state or quality of being resistant (immune) from infectious disease, either by virtue of previous exposure (adaptive immunity) or as an inherent trait (innate immunity).

Immunization: the process of producing a state of immunity in a subject..

Immunogen: A substance capable of eliciting an immune response. All immunogens are antigens, but some antigens are not immunogens (e.g., haptens).

Immunogenicity: The capacity of a substance to induce an immune response under a given set of conditions.

Innateimmunity/inborn/genetic/heritable: Nonspecific host defences that exist prior to exposure to an antigen and involve anatomic, physiologic, endocytic and phagocytic and inflammatory mechanisms.

Adaptive immunity/acquired/specific: Host defences that are mediated by B and T cells following exposure to antigen and that exhibit specificity, diversity, memory and self/non-self-recognition.------------------------------------------ Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------

جامعة دمياط - كلية التربية النوعية - أ.م.د. إيهاب فتحى جبر أبو العدب

جامعة دمياط - كلية التربية النوعية

الإسم:	أ.م.د. إيهاب فتحى جبر أبو العدب
الجهة:	جامعة دمياط - كلية التربية النوعية - الاقتصاد المنزلى
المؤهل:	الدكتوراه - التخصص كيمياء (حيوية ) - عام 2006 - من كلية العلوم ماجستير - التخصص كيمياء( كيمياء حيوية) - عام 1994 - من كلية العلوم بكالوريوس - التخصص كيمياء - عام 1990 - بتقدير جيد - من كلية العلوم
الوظيفة:	استاذ مساعد لقب علمي
البريد الإلكتروني:	ehab10f@mans.edu.eg
الوظائف الإشرافية:	عضو كنترول الفرقة الثالثة جميع الشعب الفصل الدراسى الثانى للعام الجامعى 2011/2012 من 10/5/2012 حتى الأن
	عضو بكنترول الفرقة الأولى جميع الشعب وتخلفات الفرق الأخرى للفصل الدراسى الثانى للعام الجامعى 2010/2011 من 21/5/2011 حتى الأن
	عضو مجلس قسم الاقتصاد المنزلى للعام الجامعى 2011/2012 من 18/3/2011 حتى 29/8/2011
	عضو بكنترول الفرقة الأولى جميع الشعب الفصل الدراسى الأول للعام الجامعى 2010/2011 من 22/12/2010 حتى الأن
	عضو لجنة المختبرات العلمية بالكلية للعام الجامعى 2010/2011 من 29/8/2010 حتى 31/7/2011
	عضو لجنة المختبرات العلمية للعام الجامعى 2009/2010 من 23/8/2009 حتى الأن
	عضو كنترول الفرقة الثالثة(قديم حديث) جميع الشعب2008/2009 من 6/12/2008 حتى 31/7/2009
	عضو لجنة المختبرات العلمية للعام الجامعى 2008/2009 من 21/8/2008 حتى الأن
	عضو كنترول التخلفات جميع الفرق2008/2009 من 6/8/2008 حتى 31/7/2009
	عضو مجلس ادارة وحدة ضمان الجودةوالأعتماد بالكلية من 25/6/2008 حتى 22/6/2009
	عضو بلجنة الطباعة والإعلام فى المؤتمر السنوى التاسع بكلية التربية النوعية بدمياط(تطوير كليات التربية النوعية فى ضوء معايير الجودة والاعتماد) من 29/4/2008 حتى 30/4/2008
	عضو لجنة الخطة البحثية للكلية بلجنة الجودة والاعتماد للعام الجامعى 2007/2008 من 31/12/2007 حتى الأن
	منسق برنامج الاقتصاد المنزلى بالكلية بالتعاون مع وحدة ضمان الجودة من 26/12/2007 حتى الأن
	عضو بكنترول الفرقة الأولى جميع الشعب للعام الجامعى 2007/2008 من 5/12/2007 حتى الأن
	عضو مجلس قسم الأقتصاد المنزلى من 24/10/2007 حتى 23/10/2008
السيرة الذاتية

------------------------------------------ Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------

Amino acids structures

------------------------------------------ Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------

الحرم المكي

------------------------------------------Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------

تصميم مترو جديد في اليابان‎

------------------------------------------ Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------

اليابان تطلق لاب توب يشحن بالماء

اليابان تطلق لاب توب يشحن بالماء

وكالات طوكيو / فكر مصممو ذلك الكمبيوتر في توفير الطاقة وكذلك استخدام الطاقة النظيفة لشحن الأجهزة في المستقبل، كتوفير للكهرباء فمع زيادة استخدام الأجهزة قد يعاني العالم من نقص في الكهرباء ! فما هو الحل ! الحل هو استخدام كوب واحد من المياه لشحن البطارية !! قد تندهش من الفكرة بالطبع ولكن ربما تقتنع بها وهو ما سوف يعمل عليه لإنتاج أول صيحة منه ليصبح حقيقة قريباً .

شرح مصممو ذلك الجهاز وهما اليابانيان هايرم كيم وسين جي بيك كيفية عمله إن النظام يستخدم خزان المياه الخارجية وهو يعتبر بطارية ذلك الجهاز، والجميع يعلم أن المياه عنصراها هما الهيدروجين والأكسجين ، وهما بالتحديد مولد الطاقة لذلك الجهاز، فما إن يتم وضع البطارية التي هي خزان الجهاز في كوب المياه يقوم ذلك الخزان بامتصاص المياه، وبعد وضعه وتركيبه في الجهاز يقوم بتوليد الطاقة من خلال صمام خاص بالجهاز عن طريق الأكسجين والهيدروجين و تفاعلهما داخل ذلك الصمام.

وفي الصور توضح لكم البطارية أو خزان المياه الذي يخزن المياه بداخله ويمكنك من خلال الشريط الأخضر المضئ فوقه التعرف ما إذا كان الخزان قد امتلئ أم لا .

قد يكون ذلك الجهاز مدهش لكم فقط لإنه يمكنه توليد طاقته من المياه فقط ، ولكن هذه ليست فقط مزاياه فنحن في البداية فقط، ما يميز ذلك الجهاز أيضاً هو سهولة حملة من مكان لآخر وذلك لانه يتم طيه بسهولة.

قد تسأل لماذا ذلك الجاهز في تم تصميمه باللون الأخضر فقط !! بالطبع بعد سرد الموضوع والشرح يمكنك بسهوله الإجابة على ذلك السؤال فكما تعتمد الطبيعة والبيئة علي المياه بعنصريه وأيضاً الأكسجين الذي هو أحد عناصر المياه، فتري ذلك الجهاز يعتمد أيضا عليهما ولذلك تم ربطه بالطبيعة والأهم إنه يساعد في الحفاظ على البيئة وأستخدام الطاقة النظيفة فقط.

------------------------------------------ Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------

An Introduction to ELISA

• Overview
• Basic ELISA Procedure
• Antibodies and Antigens in ELISA
• Four typical ELISA formats
• ELISA Detection Options
• ELISA Results
• Sensitivity

Overview

The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique which is used to measure the concentration of an analyte (usually antibodies or antigens) in solution. The basic ELISA, or enzyme immunoassay (EIA), is distinguished from other antibody-based assays because separation of specific and non-specific interactions occurs via serial binding to a solid surface, usually a polystyrene multiwell plate, and because quantitative results can be achieved. The steps of the ELISA result in a colored end product which correlates to the amount of analyte present in the original sample.

ELISAs are quick and simple to carry out, and since they are designed to rapidly handle a large numbers of samples in parallel, they are a very popular choice for the evaluation of various research and diagnostic targets. Figure 1 shows a typical ELISA result.

ELISAs were first developed in the early 1970s as a replacement for radioimmunoassays. They remain in wide use in their original format and in expanded formats with modifications that allow for multiple analytes per well, highly sensitive readouts, and direct cell-based output.

Basic ELISA Procedure

ELISAs begin with a coating step, where the first layer - either an antigen or an antibody - is adsorbed to a polystyrene 96 well plate. (Adsorption is the passive attachment of a liquid to a solid surface creating a thin film.) Coating is followed by blocking and detection steps as shown in the simple schematic diagram below.

Since the assay uses surface binding for separation, several washes are repeated between each ELISA step to remove unbound materials. During this process it is essential that excess liquid is removed in order to prevent the dilution of the solutions added in the next stage. For greatest consistency specialized plate washers are used.

ELISAs can be quite complex, including various intervening steps and the ability to measure protein concentrations in heterogeneous samples such as blood. The most complex and varying step in the overall process is detection, where multiple layers of antibodies can be used to amplify signal.

Figure 2. ELISA overview flowchart and schematic.

Antibodies and Antigens in ELISA

All ELISAs rely on the specific interaction between an epitope, a small linear or three dimensional sequence of amino acids found on an antigen, and a matching antibody binding site. The antibodies used in an ELISA can be either monoclonal (derived from unique antibody producing cells called hybridomas and capable of specific binding to a single unique epitope) or polyclonal (a pool of antibodies purified from animal sera that are capable of binding to multiple epitopes).

There are four basic ELISA formats, allowing for a certain amount of flexibility which can be adjusted based on the antibodies available, the results required, or the complexity of the samples.

It is possible to use both monoclonals and polyclonals in an ELISA; however, polyclonals are more typically used for the secondary detection layer in indirect ELISAs, while monoclonal antibodies are more typically used for capture or primary detection of the antigen.

Four Typical ELISA Formats

The ELISA provides a wealth of information in its simplest formats, but it can also be used in more complex versions to provide enhanced signal, more precise results, or if certain reagents are not available. The four typical ELISA formats are described briefly below. The end result for all the ELISAs is shown in figure 3, a single well, or a series of wells in a multiwall dish, with color intensity varying in proportion to the amount of antigen/analyte in the original sample.

Direct ELISA
An antigen coated to a multiwell plate is detected by an antibody that has been directly conjugated to an enzyme. This can also be reversed, with an antibody coated to the plate and a labeled antigen used for detection, but the second option is less common.

This type of ELISA has two main advantages:

• It is faster, since fewer steps are required
• It is less prone to error, since there are fewer steps and reagents

Indirect ELISA
Antigen coated to a polystyrene multiwell plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal.

This method has several advantages:

• Increased sensitivity, since more than one labeled antibody is bound per primary antibody
• Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody
• Cost savings, since fewer labeled antibodies are required

Sandwich ELISA
Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule. The first antibody, termed the capture antibody, is coated to the polystyrene plate. Next, the analyte or sample solution is added to the well. A second antibody layer, the detection antibody, follows this step in order to measure the concentration of the analyte. Polyclonals can also be used for capture and/or detection in a sandwich ELISA provided that variability is present in the polyclonal to alow for both capture and detection of the analyte through different epitopes.

If the detection antibody is conjugated to an enzyme, then the assay is called a direct sandwich ELISA. If the detection antibody is unlabeled, then a second detection antibody will be needed resulting in an indirectsandwich ELISA.

This type of assay has several advantages:

• High specificity, since two antibodies are used the antigen/analyte is specifically captured and detected
• Suitable for complex samples, since the antigen does not require purification prior to measurement
• Flexibility and sensitivity, since both direct and indirect detection methods can be used

Competition or Inhibition ELISA
This is the most complex ELISA, and is used to measure the concentration of an antigen (or antibody) in a sample by observing interference in an expected signal output. Hence, it is also referred to as an inhibition ELISA. It can be based upon any of the above ELISA formats, direct, indirect, or sandwich, and as a result it offers maximum flexibility in set up.

It is most often used when only one antibody is available to the antigen of interest or when the analyte is small, i.e. a hapten, and cannot be bound by two different antibodies.

A simple example of a competitive ELISA is shown in figure 7. In this case samples are added to an ELISA plate containing a known bound antigen. After coating, blocking, and washing steps, unknown samples are added the plate. Detection then follows pretty much as with other ELISA formats. If the antigen in the sample is identical to the plate-adsorbed antigen, then there will be competition for the detection antibody between the bound and free antigen. If there is a high concentration of antigen in the sample, then there will be a significant reduction in signal output of the assay. Conversely, if there is little antigen in the sample, there will be minimal reduction in signal.

Therefore, with a competition ELISA, one is actually measuring antigen concentration by noting the extent of the signal reduction. If the detection antibody is labeled, then this would be a direct competition ELISA and if unlabeled, then this would be an indirect competition ELISA.

For further examples of competition ELISAs, and a thorough explanation of this technique, please refer to The ELISA Guidebook (Crowther 2001).

Figure 7. Competition ELISA. Bound and free antigen compete for binding to a labeled detection antibody.

ELISA Detection Options

ELISAs, by definition, take advantage of an enzymatic label to produce a detectible signal that is directly correlated to the binding of antibody to an antigen. There are a few different types of enzymes and enzyme substrates that are typically used for ELISAs and a few slightly different methods for incorporating the enzyme step into the process. The final assay signal is measured with a spectrophotometric or fluorescent plate reader (depending upon the substrate chosen).

One aspect of ELISA terminology that often leads to confusion is the variability in the way the terms direct and indirect are applied. We will adhere to the use of these terms as they apply to the detection portion of the assay as indicated below:

Direct detection
Antibodies are directly labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP); this is the most common ELISA detection strategy. HRP and AP substrates typically produce a colorimetric output that is read by a spectophotometer. Detection can also occur by fluorescently-labeled antibodies [here the assay is usually termed a fluorescence-linked immunosorbent assay (FLISA)]

Indirect detection
Antibodies are coupled to biotin, followed by a streptavidin-conjugated enzyme step; this is most common.

Additionally, it is possible to use unlabeled primary antibodies followed by enzyme-coupled or biotinylated secondary antibodies. If the secondary antibody is biotinylated, then a tertiary step is required for detection. In this case treatment with the streptavidin-enzyme conjugate, followed by an appropriate substrate.

ELISA Results

The ELISA assay yields three different types of data output:

1) Quantitative:
ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.

2) Qualitative:
ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen.

3) Semi-quantitative:
ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.

ELISA data is typically graphed with optical density vs log concentration to produce a sigmoidal curve as shown in Figure 8. Known concentrations of antigen are used to produce a standard curve and then this data is used to measure the concentration of unknown samples by comparison to the linear portion of the standard curve. This can be done directly on the graph or with curve fitting software which is typically found on ELISA plate readers.

Figure 8. A typical ELISA standard curve.

Sensitivity

ELISAs are one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody –antigen interaction. In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results. As mentioned earlier, indirect detection will produce higher levels of signal and should therefore be more sensitive. However, it can also cause higher background signal thus reducing net specific signal levels.

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