membrane transport.ppt A B C D E F G H I

Membrane transport

1. TRANSPORT ACROSS
MEMBRANE
Prepared by
Dr.Jayshree Nellore
Professor
Department of Biotechnology
Sathyabama Institute for science and
Technology
Chennai-600119

2. Permeability of the Cell Membrane-
Differentially PermeableDifferentially Permeable

3. Permeability of the Cell Membrane

4. •Diffusion
– the passive movement of molecules from
a higher to a lower concentration until
equilibrium is reached.
–How can we explain diffusion?
–Gases move through plasma membranes
by diffusion.
•Osmosis– A special case of diffusion
DIFFUSION

5. •Diffusion is the tendency for molecules to spread
out evenly into the available space
•Although each molecule moves randomly, diffusion
of a population of molecules may be directional
•At dynamic equilibrium, as many molecules cross
the membrane in one direction as in the other

6. •Substances diffuse down their concentration
gradient, the region along which the density of
a chemical substance increases or decreases
•No work must be done to move substances
down the concentration gradient
•The diffusion of a substance across a biological
membrane is passive transport because no
energy is expended by the cell to make it
happen

7. Process of diffusion

10. Molecules of dye
Membrane (cross section)
WATER
(a) Diffusion of one solute
(b) Diffusion of two solutes
Net diffusion Net diffusion
Net diffusion Net diffusion
Net diffusion Net diffusion
Equilibrium
Equilibrium
Equilibrium

11. Gas exchange in lungs by diffusion

12. Osmosis
•Osmosis is a special term used for the diffusion of water through cell
membranes.
•Although water is a polar molecule, it is able to pass through the
lipid bilayer of the plasma membrane. Aquaporins
— transmembrane proteins that form hydrophilic channels — greatly
accelerate the process, but even without these, water is still able to
get through.
•Water passes by diffusion from a region of higher to a region of
lower concentration. Note that this refers to the concentration of
water, NOT the concentration of any solutes present in the water.
•Water is never transported actively; that is, it never moves against
its concentration gradient. However, the concentration of water can
be altered by the active transport of solutes and in this way the
movement of water in and out of the cell can be controlled.
Example: the reabsorption of water from the kidney tubules back into
the blood depends on the water following behind the active transport
of Na
+
.

13. Lower
concentration
of solute (sugar)
Higher
concentration
of solute
Sugar
molecule
H
2
O
Same concentration
of solute
Selectively
permeable
membrane
Osmosis

14. Question:
What’s in a Solution?
Answer:
•solute+solventsolution
•NaCl +H
2
0 saltwater

15. Water Balance of Cells Without Walls
•Tonicity is the ability of a surrounding solution to
cause a cell to gain or lose water (is a RELATIVE
term, comparing two different solutions)
Isotonic solution: Solute concentration is the same
as that inside the cell; no net water movement
across the plasma membrane
Hypertonic solution: Solute concentration is
greater than that inside the cell; cell loses water
Hypotonic solution: Solute concentration is less
than that inside the cell; cell gains water

16. Water Balance of Cells with Walls
•Cell walls help maintain water balance
•A plant cell in a hypotonic solution swells until
the wall opposes uptake; the cell is now turgid
(firm)
•If a plant cell and its surroundings are isotonic,
there is no net movement of water into the cell;
the cell becomes flaccid (limp), and the plant
may wilt
•In a hypertonic environment, plant cells lose
water; eventually, the membrane pulls away
from the wall, a usually lethal effect called
plasmolysis

17. Hypotonic
solution
Osmosis
Isotonic
solution
Hypertonic
solution
(a) Animal cell
(b) Plant cell
H
2
O H
2
O H
2
O H
2
O
H
2
O H
2
O H
2
O H
2
O
Cell wall
Lysed Normal Shriveled
Turgid (normal) Flaccid Plasmolyzed

18. •Hypertonic or hypotonic environments create
osmotic problems for organisms
•Osmoregulation, the control of solute
concentrations and water balance, is a necessary
adaptation for life in such environments
•The protist Paramecium, which is hypertonic to its
pond water environment, has a contractile
vacuole that acts as a pump

19. Hypertonic
•A solution with a greater solute
concentration compared to another solution.
3% NaCl
97% H
2O
Red Blood Cell
5% NaCl
95% H
2
O
solutionsolution
Which
way
will
the
water
move
?

20. Hypotonic
•A solution with a lower solute concentration
compared to another solution.
3% Na
97% H
2O
Red Blood Cell
1% Na
99% H
2
O
solutionsolution
Which
way
will
the
water
move
?

21. Isotonic
•A solution with an equal solute concentration
compared to another solution.
3% Na
97% H
2
O
Red Blood Cell
3% Na
97% H
2
O
solutionsolution
Which
way
will
the
water
move
?

22. •Function—Transport. Are specific,
combine with only a certain type of
molecule.
•Types
–Facilitated transport--passive
–Active transport—requires energy
Carrier Proteins

23. Facilitated Diffusion of Ions
Facilitated diffusion of ions takes place
through proteins, or assemblies of proteins,
embedded in the plasma membrane. These
transmembrane proteins form a water-
filled channel through which the ion can
pass down its concentration gradient.

24. •Carrier proteins undergo a subtle change in shape that
translocates the solute-binding site across the
membrane
Eg.
 Maltoporin. This homotrimer in the outer membrane of
E. coli forms pores that allow the disaccharide maltose and a
few related molecules to diffuse into the cell. `
The plasma membrane of human red blood cells contain
transmembrane proteins that permit the diffusion of glucose
from the blood into the cell.
•Some diseases are caused by malfunctions in specific
transport systems, for example the kidney disease
cystinuria

25. EXTRACELLULAR
FLUID
CYTOPLASM
Channel protein
Solute
Solute
Carrier protein
(a) A channel
protein
(b) A carrier protein

26. The transmembrane channels that permit
facilitated diffusion can be opened or closed.
They are said to be "gated".
Some types of gated ion channels:
•ligand-gated
•mechanically-gated
•voltage-gated
•light-gated

27. Ligand-gated ion channels:
Many ion channels open or close in response to binding a small
signaling molecule or "ligand".
Some ion channels are gated by extracellular ligands; some by
intracellular ligands.
In both cases, the ligand is not the substance that is transported
when the channel opens.

28. External ligands
External ligands (shown here in green) bind to a site on the
extracellular side of the channel.
Examples:
•Acetylcholine (ACh). The binding of the neurotransmitter
acetylcholine at certain synapses opens channels that admit Na
+
and
initiate a nerve impulse or muscle contraction.
•Gamma amino butyric acid (GABA). Binding of GABA at certain
synapses — designated GABA
A
— in the central nervous system
admits Cl
-
ions into the cell and inhibits the creation of a nerve
impulse.

29. Internal ligands
Internal ligands bind to a site on the channel protein exposed
to the cytosol.
Examples:
•"Second messengers", like cyclic AMP (cAMP) and
cyclic GMP (cGMP), regulate channels involved in the
initiation of impulses in neurons responding to odors and light
respectively.
•ATP is needed to open the channel that allows chloride (Cl
-
)
and bicarbonate (HCO
3
-
) ions out of the cell.
This channel is defective in patients with cystic fibrosis.
Although the energy liberated by the hydrolysis of ATP is
needed to open the channel, this is not an example of active
transport; the ions diffuse through the open channel
following their concentration gradient.

30. Mechanically-gated ion channels:
Examples:
•Sound waves bending the cilia-like projections on the hair
cells of the inner ear open up ion channels leading to the
creation of nerve impulses that the brain interprets as sound.
 (The hair cells are located between
the
 
basilar 
and 
tectorial membranes. Vibrations of the
endolymph cause vibrations of the basilar membrane. This
moves stereocilia at the tips of the hair cells against the
tectorial membrane and open potassium channels in them. The
influx of K
+
 
from the endolymph depolarizes the cell).
•Mechanical deformation of the cells of stretch receptors
opens ion channels leading to the creation of nerve impulses
(Na+ channels).

31. Voltage-gated ion channels:
In so-called "excitable" cells like neurons and muscle cells,
some channels open or close in response to changes in the
charge (measured in volts) across the plasma membrane.
Example: As an impulse passes down a neuron, the reduction
in the voltage opens sodium channels in the adjacent portion
of the membrane. This allows the influx of Na
+
into the neuron
and thus the continuation of the nerve impulse.
Some 7000 sodium ions pass through each channel during the
brief period (about 1 millisecond) that it remains open. This
was learned by use of the patch clamp technique.

32. Light-gated ion channels:
The light receptor, of unicellular
 
green alga
 Chlamydomonas, appears to be a 7-pass
 
transmembrane protein 
named Channelopsin-1. Like
rhodopsin, it is conjugated to
 
retinal.
Channelopsin-1 is a light-gated ion channel that opens to
allow protons (H
+
) to pass through when light falls on it.
 

33. Active transport uses energy to move solutes
against their gradients
•Facilitated diffusion is still passive because the solute
moves down its concentration gradient, and the
transport requires no energy
•Some transport proteins, however, can move solutes
against their concentration gradients
Active transport is the pumping of molecules or ions
through a membrane against their concentration
gradient.
It requires:
a transmembrane protein (usually a complex of them)
called a transporter and energy.
The source of this energy is ATP.

34. The energy of ATP may be used directly or indirectly.
•Direct Active Transport. Some transporters bind ATP
directly and use the energy of its hydrolysis to drive active
transport.
•Indirect Active Transport. Other transporters use the
energy already stored in the gradient of a directly-pumped
ion. Direct active transport of the ion establishes a
concentration gradient. When this is relieved by facilitated
diffusion, the energy released can be harnessed to the
pumping of some other ion or molecule.

35. Direct Active Transport
The Na
+
/K
+
ATPase
•The cytosol of animal cells contains a concentration of potassium ions
(K
+
) as much as 20 times higher than that in the extracellular fluid.
•Conversely, the extracellular fluid contains a concentration of sodium
ions (Na
+
) as much as 10 times greater than that within the cell.
•These concentration gradients are established by the active transport
of both ions.
•And, in fact, the same transporter, called the Na
+
/K
+
ATPase, does both
jobs. It uses the energy from the hydrolysis of ATP to
actively transport 3 Na
+
ions out of the cell
for each 2 K
+
ions pumped into the cell.

37. Figure 7.18-1
EXTRACELLULAR
FLUID
[Na

] high
[K

] low
[Na

] low
[K

] high
CYTOPLASM
Na

Na

Na

1

38. Figure 7.18-2
EXTRACELLULAR
FLUID
[Na

] high
[K

] low
[Na

] low
[K

] high
CYTOPLASM
Na

Na

Na

1 2
Na

Na

Na

P
ATP
ADP

39. Figure 7.18-3
EXTRACELLULAR
FLUID
[Na

] high
[K

] low
[Na

] low
[K

] high
CYTOPLASM
Na

Na

Na

1 2 3
Na

Na

Na

Na

Na

Na

P
P
ATP
ADP

40. Figure 7.18-4
EXTRACELLULAR
FLUID
[Na

] high
[K

] low
[Na

] low
[K

] high
CYTOPLASM
Na

Na

Na

1 2 3
4
Na

Na

Na

Na

Na

Na

K

K

P
P
P
P
i
ATP
ADP

41. Figure 7.18-5
EXTRACELLULAR
FLUID
[Na

] high
[K

] low
[Na

] low
[K

] high
CYTOPLASM
Na

Na

Na

1 2 3
45
Na

Na

Na

Na

Na

Na

K

K

K

K

P
P
P
P
i
ATP
ADP

42. Figure 7.18-6
EXTRACELLULAR
FLUID
[Na

] high
[K

] low
[Na

] low
[K

] high
CYTOPLASM
Na

Na

Na

1 2 3
456
Na

Na

Na

Na

Na

Na

K

K

K

K

K

K

P
P
P
P
i
ATP
ADP

43. This accomplishes several vital functions:
•It helps establish a net charge across the plasma membrane with
the interior of the cell being negatively charged with respect to
the exterior. This resting potential prepares nerve and muscle cells
for the propagation of action potentials leading to nerve impulses
and muscle contraction.
•The accumulation of sodium ions outside of the cell draws water
out of the cell and thus enables it to maintain osmotic balance
(otherwise it would swell and burst from the inward diffusion of
water).
•The gradient of sodium ions is harnessed to provide the energy
to run several types of indirect pumps.
The crucial roles of the Na
+
/K
+
ATPase are reflected in the fact that
almost one-third of all the energy generated by the mitochondria
in animal cells is used just to run this pump.

44. The H
+
/K
+
ATPase
The parietal cells of your stomach use this pump to secrete
gastric juice.
These cells transport protons (H
+
) from a concentration of
about 4 x 10
-8
M within the cell to a concentration of about
0.15 M in the gastric juice (giving it a pH close to 1).
Small wonder that parietal cells are stuffed with mitochondria
and uses huge amounts of ATP as they carry out this three-
million fold concentration of protons.

45. CYTOPLASM
ATP
EXTRACELLULAR
FLUID
Proton pump
H

H

H

H

H

H










46. The Ca
2+
ATPases
A Ca
2+
ATPase is located in the plasma membrane of all eukaryotic cells.
It uses the energy provided by one molecule of ATP to pump one Ca
2+

ion out of the cell. The activity of these pumps helps to maintain the
~20,000-fold concentration gradient of Ca
2+
between the cytosol (~ 100
nM) and the ECF (~ 20 mM).
In resting skeletal muscle, there is a much higher concentration of
calcium ions (Ca
2+
) in the sarcoplasmic reticulum than in the cytosol.
Activation of the muscle fiber allows some of this Ca
2+
to pass by
facilitated diffusion into the cytosol where it triggers contraction.
After contraction, this Ca
2+
is pumped back into the sarcoplasmic
reticulum. This is done by another Ca
2+
ATPase that uses the energy
from each molecule of ATP to pump 2 Ca
2+
ions.

47. ABC Transporters:
ABC ("ATP-Binding Cassette") transporters are transmembrane proteins that
•expose a ligand-binding domain at one surface and a
•ATP-binding domain at the other surface.
The ligand-binding domain is usually restricted to a single type of molecule.
The ATP bound to its domain provides the energy to pump the ligand across
the membrane.
The human genome contains 48 genes for ABC transporters. Some examples:
•CFTR — the cystic fibrosis transmembrane conductance regulator
•TAP(Transporter associated with Antigen Processing), the transporter
associated with antigen processing.
•the transporter that liver cells use to pump the salts of bile acids out into the
bile.
•ABC transporters that pump chemotherapeutic drugs out of cancer cells thus
reducing their effectiveness.
ABC transporters must have evolved early in the history of life. The ATP-
binding domains in archaea, eubacteria, and eukaryotes all share a
homologous structure, the ATP-binding "cassette".

48. Indirect Active Transport
Indirect active transport uses the downhill flow of an ion to
pump some other molecule or ion against its gradient.
The driving ion is usually sodium (Na
+
) with its gradient
established by the Na
+
/K
+
ATPase.

49. Symport Pumps (also known as co-transport)
In this type of indirect active transport, the driving ion (Na
+
) and the pumped
molecule pass through the membrane pump in the same direction.
Examples:
•The Na
+
/glucose transporter. This transmembrane protein allows sodium ions
and glucose to enter the cell together. The sodium ions flow down their
concentration gradient while the glucose molecules are pumped up theirs. Later
the sodium is pumped back out of the cell by the Na
+
/K
+
ATPase.
•The Na
+
/glucose transporter is used to actively transport glucose out of the
intestine and also out of the kidney tubules and back into the blood.
•All the amino acids can be actively transported, for example out of the kidney
tubules and into the blood, by sodium-driven symport pumps.
•Sodium-driven symport pumps also return neurotransmitters to the presynaptic
neuron.
•The Na
+
/iodide transporter. This symporter pumps iodide ions into the cells of
the thyroid gland (for the manufacture of thyroxine) and also into the cells of the
mammary gland (to supply the baby's need for iodide).
•The permease encoded by the lac operon of E. coli that transports lactose into
the cell.

50. ATP
H

H

H

H

H

H

H

H

Proton pump
Sucrose-H

cotransporter
Sucrose
Sucrose
Diffusion of H










52. Antiport Pumps (also known as exchange)
In antiport pumps, the driving ion (again, usually sodium) diffuses
through the pump in one direction providing the energy for the active
transport of some other molecule or ion in the opposite direction.
Example: Ca
2+
ions are pumped out of cells by sodium-driven antiport
pumps.
Antiport pumps in the vacuole of some plants harness the outward
facilitated diffusion of protons (themselves pumped into the vacuole by
a H
+ ATPase
)
•to the active inward transport of sodium ions. This sodium/proton
antiport pump enables the plant to sequester sodium ions in its vacuole.
Transgenic tomato plants that overexpress this sodium/proton antiport
pump are able to thrive in saline soils too salty for conventional
tomatoes.
•to the active inward transport of nitrate ions (NO
3
−
).

54. •Exocytosis---Cellular secretion
•Endocytosis—
–Phagocytosis— “Cell eating”
–Pinocytosis– “Cell drinking”
–Receptor-mediated endocytosis-
specific particles, recognition.
Exocytosis and
Endocytosis

55. Exocytosis

56. Phagocytosis

57. Pinocytosis

58. Receptor-mediated
Endocytosis

59. •Some of the integral membrane proteins that a cell displays at its
surface are receptors for particular components of the ECF.
•For example, iron is transported in the blood complexed to a
protein called transferrin. Cells have receptors for transferrin on
their surface.
•When these receptors encounter a molecule of transferrin, they
bind tightly to it.
•The complex of transferrin and its receptor is then engulfed by
endocytosis.
•Ultimately, the iron is released into the cytosol.
•The strong affinity of the transferrin receptor for transferrin (its
ligand) ensures that the cell will get all the iron it needs even if
transferrin represents only a small fraction of the protein
molecules present in the ECF.
•Receptor-mediated endocytosis is many thousand times more
efficient than simple pinocytosis in enabling the cell to acquire
the macromolecules it needs.

60. Industrial fermentation
Industrial fermentation is the intentional use of fermentation by microorganisms such
as bacteria and fungi to make products useful to humans. Fermented products have
applications as food as well as in general industry. Some commodity chemicals, such
as acetic acid, citric acid, and ethanol are made by fermentation. The rate of
fermentation depends on the concentration of microorganisms, cells, cellular
components, and enzymes as well as temperature, pH and for aerobic fermentation
oxygen. Product recovery frequently involves the concentration of the dilute solution.
Nearly all commercially produced enzymes, such as lipase, invertase and rennet, are
made by fermentation with genetically modified microbes. In some cases, production
of biomass itself is the objective, as in the case of baker's yeast and lactic acid bacteria
starter cultures for cheesemaking. In general, fermentations can be divided into four
types:
•Production of biomass (viable cellular material)
•Production of extracellular metabolites (chemical compounds)
•Production of intracellular components (enzymes and other proteins)
•Transformation of substrate (in which the transformed substrate is itself the product)
These types are not necessarily disjoint from each other, but provide a framework for
understanding the differences in approach. The organisms used may be bacteria,
yeasts, molds, algae, animal cells, or plant cells. Special considerations are required for
the specific organisms used in the fermentation, such as the dissolved oxygen level,
nutrient levels, and temperature.

61. Bacterial growth curve\Kinetic Curve

62. Production of biomass
Microbial cells or biomass is sometimes the intended product of
fermentation. Examples include single cell protein, bakers yeast,
lactobacillus, E. coli, and others. In the case of single-cell protein, algae
is grown in large open ponds which allow photosynthesis to occur. If
the biomass is to be used for inoculation of other fermentations, care
must be taken to prevent mutations from occurring.

63. Production of extracellular metabolites
Microbial metabolites can be divided into two groups: those
produced during the growth phase of the organism, called
primary metabolites and those produced during the
stationary phase, called secondary metabolites. Some
examples of primary metabolites are ethanol, citric acid,
glutamic acid, lysine, vitamins and polysaccharides. Some
examples of secondary metabolites are penicillin,
cyclosporin A, gibberellin, and lovastatin.

64. Primary metabolites
Primary metabolites are compounds made during the
ordinary metabolism of the organism during the growth
phase. A common example is ethanol or lactic acid, produced
during glycolysis. Citric acid is produced by some strains of
Aspergillus niger as part of the citric acid cycle to acidify their
environment and prevent competitors from taking over.
Glutamate is produced by some Micrococcus species, and
some Corynebacterium species produce lysine, threonine,
tryptophan and other amino acids. All of these compounds
are produced during the normal "business" of the cell and
released into the environment. There is therefore no need to
rupture the cells for product recovery.

65. Secondary metabolites
Secondary metabolites are compounds made in the
stationary phase; penicillin, for instance, prevents the growth
of bacteria which could compete with Penicillium molds for
resources. Some bacteria, such as Lactobacillus species, are
able to produce bacteriocins which prevent the growth of
bacterial competitors as well. These compounds are of
obvious value to humans wishing to prevent the growth of
bacteria, either as antibiotics or as antiseptics (such as
gramicidin S). Fungicides, such as griseofulvin are also
produced as secondary metabolites. Typically secondary
metabolites are not produced in the presence of glucose or
other carbon sources which would encourage growth, and
like primary metabolites are released into the surrounding
medium without rupture of the cell membrane.

66. Production of intracellular components
Of primary interest among the intracellular components are
microbial enzymes: catalase, amylase, protease, pectinase,
glucose isomerase, cellulase, hemicellulase, lipase, lactase,
streptokinase and many others. Recombinant proteins, such
as insulin, hepatitis B vaccine, interferon, granulocyte colony-
stimulating factor, streptokinase and others are also made
this way. The largest difference between this process and the
others is that the cells must be ruptured (lysed) at the end of
fermentation, and the environment must be manipulated to
maximize the amount of the product. Furthermore, the
product (typically a protein) must be separated from all of the
other cellular proteins in the lysate to be purified.

67. Insulin

70. Monoclonal Antibodies

71. Humans (and all jawed vertebrates) have the ability to make
antibodies able to
•recognize (by binding to) virtually any antigenic
determinant (epitope)
•to discriminate between even similar epitopes.
Not only does this provide the basis for protection against
disease organisms, but it makes antibodies attractive
candidates to target other types of molecules found in the
body, such as:
•receptors or other proteins present on the surface of
normal cells
•molecules present uniquely on the surface of cancer cells.

72. Thus the remarkable specificity of antibodies
makes them promising agents for human
therapy. Imagine, for example, being able to
•make an antibody that will bind only to the
cancer cells in a patient
•coupling a cytotoxic agent (e.g. a strong
radioactive isotope) to that antibody, and then
•giving the complex to the patient so it can
seek out and destroy the cancer cells (and no
normal cells).

73. But there are problems to be solved before antibodies
can be used in human therapy.
1. The response of the immune system to any antigen,
even the simplest, is polyclonal. That is, the system
manufactures antibodies of a great range of
structures both in their binding regions as well as in
their effector regions.
2. Even if one were to isolate a single antibody-
secreting cell, and place it in culture, it would die out
after a few generations because of the limited
growth potential of all normal somatic cells.

74. What is needed is a way to make
"monoclonal antibodies":
•antibodies of a single specificity that are
•all built alike because they are being
manufactured by a single clone of plasma
cells
•that can be grown indefinitely.

75. This problem was solved for mice in 1975 with a
technique devised by Köhler and Milstein (for which
they shared a Nobel Prize in 1984).
•An antibody-secreting B cell, like any other cell, can
become cancerous. The unchecked proliferation of
such a cell is called a myeloma.
Köhler and Milstein found a way to combine
•the unlimited growth potential of myeloma cells with
•the predetermined antibody specificity of normal
immune spleen cells.
They did this by literally fusing myeloma cells with
antibody-secreting cells from an immunized mouse.
The technique is called somatic cell hybridization.
The result is a hybridoma.

76. The procedure
Mix
•spleen cells from a mouse that has been immunized with the desired
antigen with
•myeloma cells.
Use an agent to facilitate fusion of adjacent plasma membranes. Even
so, the success rate is so low that there must be a way to select for the
rare successful fusions. So,
use myeloma cells that have:
•lost the ability to synthesize hypoxanthine-guanine-
phosphoribosyltransferase (HGPRT).
–This enzyme enables cells to synthesize purines using an extracellular
source of hypoxanthine as a precursor. Ordinarily, the absence of HGPRT
is not a problem for the cell because cells have an alternate pathway that
they can use to synthesize purines.
However, when cells are exposed to aminopterin (a folic acid analog), they are
unable to use this other pathway and are now fully dependent on HGPRT for
survival.
•lost the ability to synthesize any antibody molecules of their own (so as
not to produce a hybridoma producing two kinds of antibody molecules).

77. 1. The first property is exploited by transferring the cell
fusion mixture to a culture medium — called HAT
medium because it contains:
•hypoxanthine
•aminopterin
•the pyrimidine thymidine
The logic:
•Unfused myeloma cells cannot grow because they lack
HGPRT.
•Unfused normal spleen cells cannot grow indefinitely
because of their limited life span. However,
•Hybridoma cells (produced by successful fusions) are
able to grow indefinitely because the spleen cell partner
supplies HGPRT and the myeloma partner is immortal.

78. Test the supernatants from each culture to find those producing
the desired antibody.
3. Because the original cultures may have been started with
more than one hybridoma cell, you must now isolate single
cells from each antibody-positive culture and subculture them.
4. Again, test each supernatant for the desired antibodies. Each
positive subculture — having been started from a single cell
— represents a clone and its antibodies are monoclonal. That
is, each culture secretes a single kind of antibody molecule
directed against a single determinant on a preselected
antigen.
5. Scale up the size of the cultures of the successful clones.

79. Hybridoma cultures can be maintained indefinitely:
•in vitro; that is, in culture vessels. The yield runs
from 10-60 µg/ml.
•in vivo; i.e., growing in mice. Here the antibody
concentration in the serum and other body fluids
can reach 1-10 mg/ml. However, animal welfare
activists in Europe and in the U.S. are trying to
limit the use of mice for the production of
monoclonals.