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A pregnancy test stick with 2 red lines; one is labeled test line and the other is labeled control line. A key on the stick states that 2 lines means pregnant and 1 line means not pregnant
A lateral flow test detecting pregnancy-related hormones in urine. The control stripe verifies the validity of the test and the test line determines the presence of pregnancy-related hormones in the urine. (credit: modification of work by Klaus Hoffmeier)
A diagram showing lateral flow test of a urine sample. The top panel shows a positive test and the bottom shows a negative test. In both a urine sample is added; in the positive sample there are antigens present. The label reads hCG urine sample is applied to absorbent sample pad. In both cases the urine sample flows across the test area. The first region is a mix area that contains hCG-second antibody-AuNPs (hCG-GC). These then flow across the hCG strip in both samples. Next, we reach the test line. Here there are antibodies that bind the antigens in the positive sample but bind nothing in the negative sample. In the positive sample the hCG-GC also bind to the antigen, forming an antibody sandwich around the antigen. The presence of the hCG-GC causes a color change here in the positive sample but not in the negative sample.  Finally, the control line contains antibodies that bind the hCG-GC directly; so these bind in both the positive and negative samples. The control line turns a color in both.
Immunochromatographic assays, or lateral flow tests, allow the testing of antigen in a dilute solution. As the fluid flows through the test strip, it rehydrates the reagents. Antibodies conjugated to small particles bind the antigen in the first stripe and then flow onto the second stripe where they are bound by a second, fixed antibody. This produces a line of color, depending on the color of the beads. The third, control stripe binds beads as well to indicate that the test is working properly. (credit: modification of work by Yeh CH, Zhao ZQ, Shen PL, Lin YC)
  • What physical process does the lateral flow method require to function?
  • Explain the purpose of the third strip in a lateral flow assay.

[link] compares some of the key mechanisms and examples of some of the EIAs discussed in this section as well as immunoblots, which were discussed in Detecting Antigen-Antibody Complexes .

Immunoblots&Enzyme Immunoassays
Type of Assay Mechanism Specific Procedures Examples
Immunoblots Uses enzyme-antibody conjugates to identify specific proteins that have been transferred to an absorbent membrane Western blot: Detects the presence of a particular protein Detecting the presence of HIV peptides (or peptides from other infectious agents) in patient sera
Immunostaining Uses enzyme-antibody conjugates to stain specific molecules on or in cells Immunohistochemistry: Used to stain specific cells in a tissue Stain for presence of CD8 cells in host tissue
Enzyme-linked immunosorbent assay (ELISA) Uses enzyme-antibody conjugates to quantify target molecules Direct ELISA: Uses a single antibody to detect the presence of an antigen Detection of HIV antigen p24 up to one month after being infected
Indirect ELISA: Measures the amount of antibody produced against an antigen Detection of HIV antibodies in serum
Immunochromatographic (lateral flow) assays Techniques use the capture of flowing, color-labeled antigen-antibody complexes by fixed antibody for disease diagnosis Sandwich ELISA: Measures the amount of antigen bound by the antibody Detection of antibodies for various pathogens in patient sera (e.g., rapid strep, malaria dipstick)
Pregnancy test detecting human chorionic gonadotrophin in urine

Part 3

Although the indirect ELISA for HIV is a sensitive assay, there are several complicating considerations. First, if an infected person is tested too soon after becoming infected, the test can yield false-negative results. The seroconversion window is generally about three weeks, but in some cases, it can be more than two months.

In addition to false negatives, false positives can also occur, usually due to previous infections with other viruses that induce cross-reacting antibodies. The false-positive rate depends on the particular brand of test used, but 0.5% is not unusual. Thomas, Justin G., Victor Jaffe, Judith Shaffer, and Jose Abreu, “HIV Testing: US Recommendations 2014,” Osteopathic Family Physician 6, no. 6 (2014). Because of the possibility of a false positive, all positive tests are followed up with a confirmatory test. This confirmatory test is often an immunoblot (western blot) in which HIV peptides from the patient’s blood are identified using an HIV-specific mAb-enzyme conjugate. A positive western blot would confirm an HIV infection and a negative blot would confirm the absence of HIV despite the positive ELISA.

Unfortunately, western blots for HIV antigens often yield indeterminant results, in which case, they neither confirm nor invalidate the results of the indirect ELISA. In fact, the rate of indeterminants can be 10–49% (which is why, combined with their cost, western blots are not used for screening). Similar to the indirect ELISA, an indeterminant western blot can occur because of cross-reactivity or previous viral infections, vaccinations, or autoimmune diseases.

  • Of the 1300 patients being tested, how many false-positive ELISA tests would be expected?
  • Of the false positives, how many indeterminant western blots could be expected?
  • How would the hospital address any cases in which a patient’s western blot was indeterminant?

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Key concepts and summary

  • Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen or a fluorogen.
  • Immunostaining is an EIA technique for visualizing cells in a tissue ( immunohistochemistry ) or examining intracellular structures ( immunocytochemistry ).
  • Direct ELISA is used to quantify an antigen in solution. The primary antibody captures the antigen, and the secondary antibody delivers an enzyme. Production of end product from the chromogenic substrate is directly proportional to the amount of captured antigen.
  • Indirect ELISA is used to detect antibodies in patient serum by attaching antigen to the well of a microtiter plate, allowing the patient (primary) antibody to bind the antigen and an enzyme-conjugated secondary antibody to detect the primary antibody.
  • Immunofiltration and immunochromatographic assays are used in lateral flow tests , which can be used to diagnose pregnancy and various diseases by detecting color-labeled antigen-antibody complexes in urine or other fluid samples

Fill in the blank

To detect antibodies against bacteria in the bloodstream using an EIA, we would run a(n) ________, which we would start by attaching antigen from the bacteria to the wells of a microtiter plate.

indirect ELISA

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Short answer

Why is it important in a sandwich ELISA that the antigen has multiple epitopes? And why might it be advantageous to use polyclonal antisera rather than mAb in this assay?

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The pregnancy test strip detects the presence of human chorionic gonadotrophin in urine. This hormone is initially produced by the fetus and later by the placenta. Why is the test strip preferred for this test rather than using either a direct or indirect ELISA with their more quantifiable results?

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Questions & Answers

with the aid of a well labeled diagram describe the conducting system
Maridad Reply
what is cellular immunity
namugenyi Reply
Cellular Immunity. -Lymphocytes act against target cell. -Acts directly by killing infected cells.
abdinor
What are NK cells
Peter
Natural killer cells
Rahaba
what are Antigen determinant
mary
cellular immunity is the state where the lymphocytes destroy the infected or targeted cell
cynthia
any examples of oedema
cynthia
introduction of microbial diversity-1
Bhavanimangali Reply
List the type of micro organism arround us and how they can be seen and with what kind of instrument
clinton Reply
how is the arrangements of bacteria in bacilli
Vaidah Reply
Provide some examples of bacterial structures that might be used as antibiotic targets and explain why.
Vaidah
Coccobacilli, Club-Shaped bacilli, Bacilli with rounded ends, Fuilform bacilli, Bacilli with ends square.
Enoch
three main antibiotic targets in bacteria: The cell wall or membranes that surrounds the bacterial cell. The machineries that make the nucleic acids DNA and RNA. The machinery that produce proteins
Rushikesh
The bacterial cell wall. Protein production. and DNA synthesis. Why, this is because most drugs (antibiotics) affects the cell wall of the bacteria, which makes the bacteria weak or susceptible in human body.
Enoch
UV rays affecting the..
Mali Reply
what is microbiology
Baba Reply
Microbiology is the study of microorganisms, which are prokaryotic and eukaryotic cell which includes bacteria, fungi, viruses and pathogenic protozoa.
Enoch
Microbiology is the branch of Life science which deals with scientific study of many Microorganisms.
Rushikesh
what is types of microbiology
Alsheikh
Immunology, Bacteriology, Virology, Mycology, Algology etc
Enoch
Virology, Immunology, Bacteriology, Algology, Mycology, Protoozology etc
Enoch
and what is mycology
Alsheikh
Immunology, Serology, Virology, Microbial Genetics, Parasitology, Bacteriology, Mycology, Molecular, Cell Biology, Agricultural, Water,Soil, Food Industrial ,Pharmaceutical, Applied, Environmental, Clinical, Medical,Marine Microbiology, Microbial Systematics, Etc, are & many types of Microbiology.
Rushikesh
study of fungi is called mycology
Munna
Mycology is the branch of Microbiology which deals with scientific study of Fungi.
Rushikesh
Study of microorganisms,which we can't see with our naked eye is called microbiology
Munna
Mycology is the scientific study of Fungi.
Enoch
virology is the study of viruses
Oppah
what is microbiology? microbiology is the study of small microorganisms that we can not with our naked eyes.
Leticia
what is taxonomical classification of microbiology
Bami
The algae, protozoa, slime moulds, fungi, bacteria, archaea and viruses ,are taxonomic classification of Microorganisms
Rushikesh
We have Bacteria, Archaea, Protozoa, Algae, Fungi, Viruses.
Enoch
microbiology is the study of microbes too small to be seen by naked eyes
Maridad
microbiology is a branch of biology which deals with study of smallest living microrganisms such as bacteria protozoa fungi and viruses
Chaitra
microbiology is the study of microorganisms which can't be seen by our naked eyes
Nakaweesi
Micro - Minute Bio - Life Logus - Study
Roshan
what is the meaning of antimicrobial susceptibility testing
Devshree Reply
seven gram positive bacteria
Okocha Reply
seven examples of gram negative bacteria
Okocha
seven examples of gram negative bacteria
Okocha
Physical conditions that would enable selective Isolation of staphylococcus epidermis
shongile Reply
Nutritional requirements that would enable selective Isolation of staphylococcus epidermis
shongile
Nutritional requirements that would enable selective Isolation of staphylococcus epidermis
shongile
what is constant flux but
Jane Reply
Digestion of food is completed in __
Amina Reply
Small Intestine
Enoch
large inteatine
Abdiwali
small intestine
Betelhem
Small intestine
Abdirisaq
small intestine
Marah
small intestine specific in illum
Mariam
difference btwn hausteria and appears
Raviha Reply
numerical and molecular taxanomy
Dhanshri Reply
difference btwn hausteria and appesorium
Raviha
full life cycle of plasmodium parasite
Emmah Reply

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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