The ELISA (enzyme-linked immunosorbent assay) is a popular biochemistry assay that uses antibodies and color change to identify a substance. It involves using an enzyme-linked antibody to detect antigen-antibody binding, where the enzyme converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA has various applications such as screening blood donations, measuring hormone levels, and detecting infections and allergens.
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
Enzyme linked immunosorbent assay (elisa) and its clinical significancerohini sane
A comprehensive presentation on Enzyme Linked Immunosorbent Assay (ELISA) and its clinical significance for MBBS, BDS, B Pharm & Biotechnology students to facilitate self- study.
ELISA is the basic assay technique, known as enzyme-linked immunosorbent assay (also referred to as EIA: Enzyme Immunoassay) that is carried out to detect and measure antibodies, hormones, peptides and proteins in the blood.
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
Enzyme linked immunosorbent assay (elisa) and its clinical significancerohini sane
A comprehensive presentation on Enzyme Linked Immunosorbent Assay (ELISA) and its clinical significance for MBBS, BDS, B Pharm & Biotechnology students to facilitate self- study.
ELISA is the basic assay technique, known as enzyme-linked immunosorbent assay (also referred to as EIA: Enzyme Immunoassay) that is carried out to detect and measure antibodies, hormones, peptides and proteins in the blood.
Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
ELISA, principle and method by kk sahuKAUSHAL SAHU
What is ELISA.
Principle.
History.
Types of ELISA method.
1.Direct ELISA.
2.Indirect ELISA.
3.Sandwhich ELISA.
Conclusion.
References.
Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
Enzyme-Linked Immunosorbent Assay (ELISA) , Types of Elisa , Presentation on ...Rajesh Singh
ELISA stands for enzyme-linked immunosorbent assay.
It is a common laboratory technique which is used to measure the concentration of an analyte (antigens) in solution.
Where Ag-Ab interaction is monitored by enzyme measurement.
It is similar in principle to Radio Immuno Assay (RIA) but It depends on an enzyme rather than a radioactive label.
what is sandwich elisa, introduction to elisa, its type and main focus on sandwich elisa, , its process and advantages along with the disadvantages, its applications
Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
ELISA, principle and method by kk sahuKAUSHAL SAHU
What is ELISA.
Principle.
History.
Types of ELISA method.
1.Direct ELISA.
2.Indirect ELISA.
3.Sandwhich ELISA.
Conclusion.
References.
Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
Enzyme-Linked Immunosorbent Assay (ELISA) , Types of Elisa , Presentation on ...Rajesh Singh
ELISA stands for enzyme-linked immunosorbent assay.
It is a common laboratory technique which is used to measure the concentration of an analyte (antigens) in solution.
Where Ag-Ab interaction is monitored by enzyme measurement.
It is similar in principle to Radio Immuno Assay (RIA) but It depends on an enzyme rather than a radioactive label.
what is sandwich elisa, introduction to elisa, its type and main focus on sandwich elisa, , its process and advantages along with the disadvantages, its applications
The following presentation contains helpful information regarding Radioimmunoassay (RIA) and Enzyme-Linked Immunosorbent Assay (ELISA), including their history, introduction, advantages, procedures and applications.
gives a very brief info about western blotting procedures, attractive slides, with creative animation effects, i hope this ppt of mine works good for seminar and for educational purposes.
A path breaking technology which has made it possible for us to detect HIV. ELISA is an immunological assay nowadays even used to detect food proteins & is the science behind pregnancy color test. This presentation unlocks the working of this assay.
ELISA or Enzyme-linked Immunosorbent Assay is a qualitative and quantitative assay for detecting the presence of antigens (virus, hormones, enzymes, etc.) in a sample.
Blog praxilabs com_2021_09_20_elisa_principleAyaFarid2
The Enzyme Linked Immunosorbent Assay (ELISA) is 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. It is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
EATING DISORDERS (Psychiatry-7)by dr Shivam sharma.pptxShivam Sharma
For any queries ,contact shvmshrm@outlook.com
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## Introduction to Eating Disorders
Welcome to this comprehensive presentation on Eating Disorders, a critical and often misunderstood area of mental health. This presentation is designed to provide in-depth knowledge and insights into the various aspects of eating disorders, making it valuable for both postgraduate medical aspirants preparing for the INI-CET and the general public seeking to understand these complex conditions.
### Objectives:
1. **Understanding Eating Disorders**: Gain a clear understanding of what eating disorders are, their types, and their distinguishing characteristics.
2. **Etiology and Risk Factors**: Explore the underlying causes and risk factors that contribute to the development of eating disorders.
3. **Clinical Features and Diagnosis**: Learn about the clinical features, diagnostic criteria, and the importance of early detection.
4. **Management and Treatment**: Review the current approaches to managing and treating eating disorders, including medical, psychological, and nutritional interventions.
5. **Prevention and Awareness**: Discuss strategies for prevention, early intervention, and increasing awareness about eating disorders.
This presentation aims to bridge the gap between academic knowledge and practical understanding, providing you with the tools to recognize, diagnose, and effectively manage eating disorders. Whether you are preparing for a medical exam or seeking to educate yourself or others about these serious conditions, this presentation will equip you with essential information and practical insights.
Let's begin our journey into understanding eating disorders and the significant impact they have on individuals and society.
---
For any queries ,contact shvmshrm@outlook.com
Mastering Wealth: A Path to Financial FreedomFatimaMary4
### Understanding Wealth: A Comprehensive Guide
Wealth is a multifaceted concept that extends beyond mere financial assets. It encompasses a range of elements including money, investments, property, and other valuable resources. However, true wealth also includes non-material aspects such as health, relationships, and personal fulfillment. This guide delves into the various dimensions of wealth, exploring how it can be created, sustained, and enjoyed.
#### Defining Wealth
Traditionally, wealth is defined as the abundance of valuable resources or material possessions. It includes financial assets like cash, savings, stocks, bonds, and real estate. However, a broader understanding of wealth considers factors such as personal well-being, emotional health, social connections, and intellectual growth. This holistic view recognizes that true wealth is not solely about accumulating money but also about enhancing one's quality of life.
#### The Importance of Financial Wealth
Financial wealth remains a critical component of overall wealth. It provides security, freedom, and the ability to pursue opportunities. Key elements of financial wealth include:
1. **Savings**: Money set aside for future use. It is crucial for emergencies, large purchases, and financial goals.
2. **Investments**: Assets purchased with the expectation that they will generate income or appreciate over time. Common investments include stocks, bonds, mutual funds, real estate, and businesses.
3. **Income**: Regular earnings from work, investments, or other sources. Consistent income is essential for maintaining and growing wealth.
4. **Debt Management**: Effectively managing debt ensures that it does not erode financial wealth. This includes paying off high-interest debt and using credit wisely.
#### Creating Wealth
Creating wealth involves generating and accumulating financial and non-financial resources. The process can be broken down into several key strategies:
1. Education and Skill Development: Investing in education and skills enhances earning potential. Higher education, professional certifications, and continuous learning can lead to better job opportunities and higher salaries.
2. Entrepreneurship: Starting and running a successful business can be a significant source of wealth. Entrepreneurship requires innovation, risk-taking, and effective management.
3. Investing: Making smart investments is essential for wealth creation. This involves understanding different types of investments, assessing risks, and making informed decisions. Diversifying investments can reduce risk and increase potential returns.
4. Saving and Budgeting: Effective saving and budgeting help accumulate wealth over time. Setting financial goals, creating a budget, and sticking to it are foundational steps in wealth creation.
5. Real Estate: Investing in property can provide rental income and capital appreciation. Real estate is a tangible asset that can hedge against inflation
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
2. HISTORY
Prior to the development of the EIA/ELISA, the only option for conducting
an immunoassay was radioimmunoassay, a technique using radioactively-
labeled antigens or antibodies.
Avramais (1966, 1969) and Pierce (1967) developed methods to chemically
link antibodies to biological enzymes whose activities produce a measurable
signal with solutions containing appropriate substrates.
This signal has to be associated with the presence of antibody or antigen .
COMPONENTS OF ELISA
Antibody: IgG fraction of serum.
Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4 lysine
residues.
Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a catalyst to
oxidize substrate in the presence of Hydrogen peroxide to produce a blue color.
Reaction stopped with dilute acid to cause complex to turn yellow.
3. INTRODUCTION TO ELISA
A 96 - well microtiter plate
being used for ELISA.
A test that uses antibodies and color
change to identify a substance.
ELISA is a popular format of a
"wet-lab" type analytic biochemistry assay.
ELISA involves at least one antibody with
specificity for a particular antigen.
ELISA can perform other forms of ligand
binding assays instead of strictly
"immuno" assays.
4. “Wet lab" analytic biochemistry assay, ELISA involves detection of an "analyte"
in a liquid sample by a method that continues to use liquid reagents during the
"analysis“.
The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding
(antigen- antibody binding). The enzyme converts a colorless substrate
(chromogen) to a colored product, indicating the presence of Ag:Ab binding.
PRINCIPLE
5. Different antigen in sample
substrate
Colored
product
Primary antibody
Secondary antibody
6. There are variations of the ELISA test but the most basic type consist of an
antibody attached to the solid surface. This antibody has affinity for (will
latch onto) the substance of interest.
For example… Human Chorionic Gonadotropin (HCG), the commonly
measured protein which indicates pregnancy.
A mixture of purified HCG linked to an enzyme and sample (blood, urine,
etc.) under test are added to the test system. If no HCG is present in the test
sample the only HCG with linked enzyme will bind. The more HCG which
is present in the test sample the less enzyme linked HCG will bind.
HOW DOES ELISA WORK ?
7. TYPES OF ELISA
INDIRECT ELISA
DIRECT ELISA
SANDWICH ELISA
COMPETETIVE ELISA
NON -COMPETETIVE
ELISA
8. INDIRECT ELISA
Antigen is added to plate.
Added Blocking buffer.
Suitable primary antibody is added.
Secondary antibody- HRPO is then
added which recognizes and binds to
primary antibody.
TMB substrate is added, is converted
to detectable form.
Under standard condition ,the enzyme activity measured is proportional to
amount of specific antibody in the original serum.
9. ADVANTAGES OF INDIRECT DETECTION
Wide variety of labeled secondary antibodies are available commercially.
Versatile, since many primary antibodies can be made in one species and the
Same labeled secondary antibody can be used for detection.
Immunoreactivity of the primary antibody is not affected by labeling.
Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing for
signal amplification.
DISADVANTAGES OF INDIRECT DETECTION
Cross-reactivity may occur with the secondary antibody, resulting in
nonspecific signal.
An extra incubation step is required in the procedure.
10. DIRECT ELISA
1. Apply a sample of known antigen to
a surface.
2. Enzyme linked primary antibody is
applied to the plate.
3. Washed, After this wash, only the
antibody-antigen complexes remain
attached.
4. Apply a substrate which is
converted by the enzyme to elicit a
chromogenic signal.
Under standard condition ,the enzyme
activity measured is proportional to
amount of specific antibody in the
original serum.
11. ADVANTAGES OF DIRECT DETECTION
Quick methodology since only one antibody is used.
Cross-reactivity of secondary antibody is eliminated.
DISADVANTAGES OF DIRECT DETECTION
Immunoreactivity of the primary antibody may be reduced as a result of
labeling.
Labeling of every primary antibody is time-consuming and expensive.
No flexibility in choice of primary antibody label from one experiment to
another.
Little signal amplification.
12. SANDWICH ELISA
1. a. Plate is coated with suitable antibody.
b. Blocking buffer is added.
2. Sample is added to plate so antigen is bounded by capture antibody.
3. A suitable biotin labeled detection antibody is added to plate.
4. Enzyme HRPO is added and binds the biotin labeled detection antibody.
5. TMB substrate is added and converted by HRPO to colored product.
Under standard condition ,the enzyme activity measured is proportional to the Amount of
specific antigen in the original serum.
13. MATERIALS NEEDED FOR ELISA KIT
ELISA Plate
Positive control
Negative control
Dilution Buffer
Conjugate
TMB Substrate
Stop Solution
14. Wash
3X
Wash
4X
Wash
4X
Wash
4X
A EDCB
Alkaline phosphatase
substrate is added
and developed colour
is read at 405 nm
wavelength to
measure plasma
cencentration
Wells are
coated with
0.2 μg
primary
antibody
Diluted
plasma
is added to
coated wells,
which bind
to antibodies
0.1 μg of
biotinylated
(biotin = – )
antihuman
secondary
antibody
Incubated
overnight at 4˚C
Incubated at room temperature (24˚C)
2h 2h 1h
PROCEDURE OF ELISA
Add 1.2000
dilution of
streptavidin
conjugate to
alkaline
phosphatase
( E)
16. COMPETETIVE ELISACOMPETETIVE ELISA
COMPETETIVE ELISA
Solid phase coated with antibody
Add unknown amount of unlabeled
antigen and known amount of labeled
antigen
Free and labeled antigen are captured
Color formation by oxidation of substrate
into a colored compound
Under standard condition ,the enzyme activity measured is proportional to the
proportion of labeled antigen in the mixture of labeled and unlabled antigen.
17. ADVANTAGES:
Suitable for complex (crude or impure) samples, since
the antigen does not require purification prior to
measurement.
DISADVANTAGES
Each antigen may require a different method to couple it to
the enzyme.
18. COMPARISON BETWEEN VARIOUS TYPES OF ELISA
Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA
19. The ELISA assay yields three different types of data output:
Quantitative: ELISA data can be interpreted in comparison to a standard
curve in order to precisely calculate the concentrations of
antigen in various samples.
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.
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 RESULTS
20. ELISA data is typically graphed with Optical density Vs Log concentration to produce
a sigmoidal curve.
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.
21. 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.
22. PRECAUTIONS
Negative control with strong signal
The excessive background signal can be caused by inadequate rinsing of plates, reagents not
sufficiently diluted, inadequate blocking of plates or non-specific binding of enzyme
conjugate. The appearance of color in negative control wells may also indicate cross-reactivity
of secondary antibody with components in the antigen sample.
Positive control with no signal
Microwell plates not coated properly.
Reagents applied in wrong order or step omitted.
Secondary antibody not matched to the species of primary antibody.
Enzyme conjugate defective or inhibited by contaminant.
ELISA with weak signal
Wash buffer not adequately drained after every wash step.
Inadequate incubation times.
Enzyme conjugate defective or inhibited by contaminant.
Substrate defective or contaminated.
Microwell plates poorly coated.
Loss of capture antibody during blocking/washing.
23. PRACTICAL ASPECTS
Solid phases used in ELISA include cross-linked dextran or
polyacrylamide beeds, filter paper(cellulose) disc or polypropylene
tubes and disposable polystyrene microtitre plates.
Antigen or antibodies attached to solid phase by passive absorption.
Conjugate used must contain a highly reactive antibody coupled to
an enzyme with a highly turnover number(alkaline phosphatase and
horseradish peroxide are commonly used enzymes).
24. APPLICATIONS
Screening donated blood for evidence of viral contamination by
HIV-1 and HIV-2 (presence of anti-HIV antibodies)
Hepatitis C (presence of antibodies)
Hepatitis B (testing for both antibodies and a viral antigen)
Measuring hormone levels
HCG (as a test for pregnancy)
LH (determining the time of ovulation)
TSH, T3 and T4 (for thyroid function)
Detecting infections
Sexually-transmitted agents like HIV, syphilis and chlamydia
Hepatitis B and C
Toxoplasma gondii
Detecting illicit drugs.
Detecting allergens in food and house dust
25. A new technique uses a solid phase made up of an immunosorbent polystyrene
rod with 4-12 protruding ogives. The entire device is immersed in a test tube
containing the collected sample and the following steps (washing, incubation in
conjugate and incubation in chromigeneous) are carried out by dipping the ogives
in microwells of standard microplated prefilled with reagents.
Advantages of this technique:
The ogives can each be sensitized to a different reagent, allowing the
simultaneous detection of different antibodies and different antigens
for multi-target assays.
The sample volume can be increased to improve the test sensitivity in
clinical, food and environmental samples.
The use of laboratory supplies for dispensing sample
aliquots, washing solution and reagents in microwells is not
required, facilitating ready-to-use lab kits and on-site kits.
A NEW TECHNIQUE:- REVERSE ELISA