bio 1511 Elisa and Carbs

Carbohydrates

Sugars, starches, and other polysaccharides

Biomolecules

Four major groups: Lipids, Nucleic Acids, Carbohydrates, Amino Acids

Lipids

Form biological membranes and hormones; energy and insulation

Nucleic Acids

Building blocks of DNA and RNA

Amino Acids

Building blocks of proteins; structure, function, body regulation

Monosaccharide

1 structural unit

Disaccharide

2 structural units

Polysaccharide

3 or more structural units

Complex carbohydrates

Formed through dehydration synthesis

Reducing sugars

Aldehyde or ketone is not part of bond (is free) and can react with Benedict's reagent

Non-reducing sugars

Reactive aldehyde or ketone is part of bond formed in dehydration and cannot react with Benedict's reagent

Benedict's test

Non-reversible reaction test for reducing sugars resulting in color change. Basic citrate buffered solution.

Upon heating…

Positive reaction = green yellow orange red

Negative reaction = blue

Final color is dependent on number of reactive sites available

Green = few

Yellow = more

Orange = most

Red = greatest

Barfoed's test

Test containing copper ions but is acidic w/ pH of 4.5! Only free groups of ketone and aldehyde of monosaccharides can reduce blue cupric ions to red. Indicates if monosaccharide.

Positive reaction = forms red precipitate at the bottom of test tube

Negative reaction = lack of precipitate (negative w distilled water and other non carb)

SO DONT DIRECTLY IDENTIFY CARB AS DI OR POLY

Lugol's iodine test

Test based on certain polysaccharides to react w/ Lugol’s iodine such as amylase and glycogen. Solution has potassium iodide and iodine crystals initially amber colored. Iodine is mixed with starch/amylose helical structure.

Positive reaction (starch present) = blue-black

Short chain dextrin's + highly branched polysaccharides like glucose = rust, red violet color

Absence of coils/coil disruption = no color change (mono/di/water give negative results)

Aldehyde

C=O bonded to H

Ketone

C=O not bonded to H

Branched polysaccharides

Examples include glycogen and amylopectin, form sugar units and side chains attach

Unbranched polysaccharides

Examples include amylose (a component of starch) and cellulose, linear structure of polysaccharides

Basic formula of carbohydrates

(CH2O)n

What are the functions of carbs?

Energy storage, structural components

Benedict's test reagent

The reagent used in Benedict's test, which is blue and turns green, then orange, then red with more reactive sites in a solution.

What sugars react in Barfoed Test? And don’t?

Monosaccharides because they have free aldehyde and ketone groups to react to, but not disaccharides because their sugars that do not react in Barfoed's Test since they lack enough free groups.

Glycogen

A highly branched polysaccharide that is less reactive in Lugol's iodine Test.

Dehydration reactions

Chemical reactions that involve the removal of water to form new bonds.

Assays

Tests used to determine presence or absence of specific factor (no hypotheses, just predictions)

What does ELISA stand for and what is it?

Enzyme-Linked Immunosorbent Assay. It is an assay technique that relies on the binding of antibodies to antigens. Can indicate the presence or absence of target molecules, such as peptides, proteins, antibodies, and hormones.

positive reaction to ELISA?

The primary antibody is bonded to secondary antibody which is attached to an enzyme, and the activity produces a measurable response (such as a color change). = Presence of antigen 

Antigens

Foreign objects inducing immune response (virus/bacteria)

Antibodies

Specialized proteins produced by immune cells in response to the presence of antigens.

ELISA process and what results if positive plus indications of positive?

1) Antigen binds to test well

2) Primary antibody binds to antigen.

3) Secondary antibody with the enzyme binds to primary antibody

4) Attached enzyme interacts with enzyme substrate

Color change if You see a color change, fluorescence, or light signal.

• This indicates:

  • The target molecule is present.

  • The assay worked correctly.

• Means that the antigen is there or antibody target

Positive control

Sample with only antigen.

Negative control

Sample with only buffer.

Purpose of controls

To provide a standard positive and negative result to compare to your samples.

False positive

Test results indicate positive when sample is actually negative

Indicates incorrect procedure (forget to wash wells)/contamination or that the unrelated antibody reacts to antigen

False negative

Test results indicate negative when sample is actually positive.

Amt of antibody might be too low to produce positive result/mixed up procedure

Denaturation

The process that can permanently or reversibly affect enzyme activity.

Colorimetric test

A test where color changes indicate the presence of the secondary antibody.

Test sensitivity

Tests should be done multiple times with serial dilutions to assess test sensitivity to the amount of antibody.

Indicator enzyme

An enzyme that changes color to indicate the presence of the foreign object (disease, drug, etc.).

Primary antibody

Binds to the antigen in the ELISA process.

Secondary antibody

Binds to the primary antibody and is linked to an enzyme.

Real-World ELISA Applications

Pregnancy tests, illegal drug tests, detection of diseases, indoor air quality tests, food labeling quality control

Purpose of Writing Lab Reports

Lab reports communicate results of experiments and prepare you for future scientific writing.

Sections of a Lab Report

Include Introduction, Methods, Results, and Discussion with Conclusion.

Lab Report Topic

Effects of various factors on enzyme activity.

What’s a negative reaction for ELISA Test?

NO COLOR CHANGE MEANING NO PRESENCE OF ANTIGEN and primary has nothing to bind to so it just washes away

Lymphocytes

AKA B-Cells…

Responds to presence of antigen by producing antibodies that specifically respond to the antigens.

If it detects an antigen, it produces lots of antibodies that flood the bloodstream. Once binded at antigen active site it produces a “antigen-antibody complex” and any other processes to neutralize antigen

Macrophages

Cells that have receptors to attach to the antigen-antibody complex and engulf it to destroy the antigen

Agglutination

When large numbers of antibodies bind to multiple antigens cause clumping and a “lattice-like” molecule precipitates antigen changing it from insoluble to soluble and neutralizes it 

what do vaccines do via for antigens etc

They introduce part of the antigen, so that the B-cell will be able to recognize it if it encounters it in the future = faster memory response

supernatant

Clear liquid throughout the tube in a Benedicts Solution Test

What happens when you leave solution to sit undisturbed for several hours in Bendeicts Solution?

Red precipitate (or solid) of cuprous oxide forms at the bottom of the test tube

The amount of precipitate equals

Number of reactive sites and sugar originally present or oxidized