ASCI 440 Lab Final

negative

Blue bottle for mastitis

bead with antibodies (all of them go on the bottom row)

White bottle for mastitis

beads without antibodies (all of them go on the top row)

Green bottle for mastitis

positive

Red bottle for mastitis

blue

Elisa positive wells are what color?

colorless

Eliza negative well are what color?

connects to antibody and reacts with the enzyme to change color

SA (secondary antibody) purpose

400 mL

What amount of mL should you start with for a 1:5 dilution to end with 500mL total

single radial immunodiffusion

what does RID stand for?

determines concentration of antibodies, antigens, compliment proteins

what does RID do?

True

T/F: standard antigen solution have known concentrations

Agar containing antibody molecules (molten Agar)

what type of Agar is used for RID

precipitation reaction

what kind of reaction occurs when optimal amount of antibody and antigen are bound in RID

agar with no antibodies

What kind of agar does Double Immunodiffusion use?

line of precipitation is formed

what occurs at the zone of equivalence in double immunodiffusion?

towards

antibody and antigen diffuse ___ each other

precipitation lines will fuse and result in arc shaped precipitation band

Identical antigens in double immunodiffusion

two precipitation lines will be formed and they will cross each other

Non-identical antigens in double immunodiffusion

share one or more epitopes. incomplete cross or an arc with a spur

partially identical antigens in double immunodiffusion

10^4 cells/mL

Magic number

# cells x dilution x 10^4 cells/mL

equation to find cells/mL

cells will burst because of osmosis

why don't we dilute with water?

equivalence

Zone: the number of the antigen determinants was equal to the number of active antibodies centres, irrespective of the heterogeneity of the latter

antigen-excess

Zone: more antigens than antibodies

antibody-excess

Zone: more antibodies than antigens

dilution factor = x / (x+Vfinal)

formula for serial dilutions

enzyme-linked immunosorbent assay

what dies ELISA stand for?

they change color

why are enzymes used in ELISA testing?

pregnancy

what antibody-based tests can you buy at your local pharmacy?

55

what percent of the blood is plasma?

45

what percent of blood do the erythrocytes make up?

what is the buffy coat

the appearance of the layer which is made up of white blood cells

lymphocytes

first peak of flow cytometry graph

monocytes

second and smallest peak of the flow cytometry graph

granulocytes: eosinophils, neutrophils, and basophils

last and widest peak of the flow cytometry graph

CD4

Co-receptor for the T-Cell receptor, specifically T-helper cells

CD8

marker for cytotoxic T cells

Ig

marker for plasma

CD16

marker for monocytes, neutrophils, and NK cells

marks cytotoxic and helper T cells

CD3

monocyte

lymphocyte

neutrophil

eosinophil

basophil

revolving nose piece

objective lens

stage clip

stage

iris diaphragm

condenser lens

light source

base

ocular eye piece

observation tube

arm

coarse focus

adjust with coarse focus on 10x, then only use fine focus to adjust when you switch lenses

fine focus

stage control knob

par focal

The capacity of an instrument to maintain focus regardless of the magnification used - if it’s focused on 10x it should still be focused in 4x

phagocytosis and stimulate lymphocytes

monocyte function

antibody producing cells and helper cells

lymphocyte function

phagocytosis

neutrophil function

allergic response, C3b receptor activation

Eosinophil function

IgE Fc receptor, kills indirectly via blood flow regulation

Basophil function

1-3

range of mastitis grading

lymphocytes, neutrophils, monocytes

3 most common immune cells in the blood

how to harvest a buffy coat

1. Spin collected blood (EDTA added) at 1000g for 10 minutes with the brake off

2. Using a transfer pipette, remove most of the plasma and decant

3. Carefully remove buffy coat and decant into test tube

explain how flow cytometry works

• Sample Prep: Cells are suspended in fluid, often "tagged" with fluorescent dyes/antibodies.

• Single-File Flow: Cells pass one-by-one through a narrow stream.

• Laser Interrogation:

  • Light Scatter: Laser hits cell; forward scatter = size, side scatter = internal complexity.

  • Fluorescence: Dyes on cells glow, indicating specific molecules.

• Detection: Detectors convert scattered/fluorescent light into electrical signals.

• Data Analysis: Computer processes signals into plots to identify, count, and measure cell characteristics.

proper pipetting tips

ALWAYS use the best pipette for the volume you need to transfer

ALWAYS use the correct tips

NEVER turn a pipette upside down – tips down always

NEVER try to set the pipette beyond its range

what does the CMT test measure