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48
Bioanalytical chemistry
5. Antibodies: use as analytical reagents
Suggested reading: Sections 6.1 to 6.3.6 of
Mikkelsen and Cortón, Bioanalytical Chemistry
Primary Source Material
• Chapters 5 & 10 of Mikkelsen, S.R. and Corton, E., Bioanalytical Chemistry (2004).
• Chapter 6 of Goldsby Immunology 5th edition (WH Freeman)
• Appendix 1 of Immunobiology 5th edition. (NCBI bookshelf).
• http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?
cmd=Search&db=books&doptcmdl=GenBookHL&term=diagnostic+AND+imm
%5Bbook%5D+AND+125975%5Buid%5D&rid=imm.section.2405
Polyclonal antibodies or polyvalent antigens
can cause agglutination
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[antigen] >> [antibody]
[antigen] ~ [antibody]
[antigen] << [antibody]
•
•
•
•
•
•
•
•
•
When the antigen is displayed on the surface of a large particle such as a bacterium, antibodies can cause the bacteria to clump or agglutinate. The same principle applies to
the reactions used in blood typing, only here the target antigens are on the surface of red blood cells and the clumping reaction caused by antibodies against them is called
hemagglutination (from the Greek haima, blood).
Precipitation reactions are similar in principle to agglutination reaction, the difference being that the antigen is a soluble, molecular species rather than a suspended particle
such as a bacterium or erythocyte.
There are 3 types of "particles" commonly used in agglutination tests: 1) erythrocytes (RBCs), 2) bacterial cells (often stained to make the clumps visible), 3) latex particles (the
antigens are chemically attached prior to running the test).
The agglutination or precipitation reaction is affected by the number of binding sites that each antibody has for antigen, and by the maximum number of antibodies that can be
bound by an antigen molecule or particle at any one time. These quantities are defined as the valence of the antibody and the valence of the antigen: the valence of both the
antibodies and the antigen must be two or greater before any precipitation can occur. Of course, a fully intact antibody has a valence of two because of the two binding sites at the
ends of its two arms. However, a Fab or ScFv would only have a valence of one.
Antigen will be precipitated only if it has several antibody-binding sites. This condition is usually satisfied in macromolecular antigens, which have a complex surface with binding
sites for several different antibodies. The site on an antigen to which each distinct antibody molecule binds is called an antigenic determinant or an epitope. Steric considerations
limit the number of distinct antibody molecules that can bind to a single antigen molecule at any one time however, because antibody molecules binding to epitopes that partially
overlap will compete for binding. Particulate antigens (erythrocytes/bacteria/latex beads) will always have many antibody binding sites.
Q. How will a polyclonal and a monoclonal antibody differ in their ability to precipitate antigens? Is it like polyclonal antibodies can form precipitation more easily whereas monoclonal
antibody needs the antigens to have at least one equivalent epitodes on each of the antigen? Could I say polyclocal antibodies have higher ability to form precipitate than monoclocal
antibody?
A. In the case where there are many epitopes per antigen, both monoclonal and polyclonal antibodies will be equally good at participating antigens. The only situation where they
would really differ is if you had a monomeric protein that you were trying to precipitate. Since it is monomeric, there can only be one copy of each epitope per protein molecule.
Accordingly, there is no way for a monoclonal antibody to form the crosslinks necessary to result in precipitation. A polyclonal antibody mixture would contain different antibodies
that recognized different epitopes. Accordingly, it would be possible to get crosslinks (i.e., antibody-antigen-antibody) and precipitation.
Q: Does every antigen has one kind of epitope or different kinds?
A: Every protein, virus, or bacterial antigen will have many different possible epitopes on their surfaces. When we start talking about smaller species, such as peptides or small
molecules, it doesn't really make any sense to speak about distinct epitopes, since the whole molecule is (more or less) involved in the binding interaction. However, different
antibodies could bind it in different orientations and with different interactions.
Red blood cells are covered in sugars that
determine your ABO blood-group
50
GTA and GTB differ by 4 mutations
GTA and O differ by 1 mutation
A-type people have gene glycosyltransferase A (GTA)
which catalyzes the addition of N-acetylgalactosamine.
B-type people have the gene for GTB which catalyzes
the addition of galactose.
Type O have an inactive mutant of GTA
•
•
•
Red blood cells, also known as erythrocytes, are flattened, doubly concave cells about 7 µm in diameter
that carry oxygen associated in the cell's hemoglobin. Mature erythrocytes lack a nucleus.
The different carbohydrate structures on the surface of RBCs are synthesized by glycosyltransferases.
Depending on which genes you inherited from your parents, you will either have the glycosyltransferases
that make the A antigen, the B antigen, both, or neither!
You have antibodies circulating in your blood against the antigens that are not present on your own cells.
Common gut bacteria bear antigens that are similar or identical to blood-group antigens, and these
stimulate the formation of antibodies to these antigens in individuals who do not bear the corresponding
antigen on their own red blood cells; thus, type O individuals, who lack A and B, have both anti-A and
anti-B antibodies, while type AB individuals have neither.
• Q: If some bacteria are responsible for making antibodies then why for example in B blood group this bacteria
do not make anti B antibody?
• A: The bacteria doesn't make the antibody of course - the B cell makes the antibody. The point I was making
in this discussion is that early in our life we are exposed to a variety of polysaccharide antigens derived from
bacteria in our gut or our mothers gut. Some of these polysaccharide antigens are similar to the blood group
antigens on our red blood cells, but we only create antibodies against the antigens that are different than our
own. This phenomenon is known as immune tolerance. Basically, our body has mechanisms to make sure that
we don't generate antibodies against our tissues. When this process is defective, autoimmune disease can
arise.
• http://www.bioc.aecom.yu.edu/bgmut/abo.htm
• http://pubs.acs.org/isubscribe/journals/cen/81/i10/html/8110sci1.html
• http://www.rcsb.org/pdb/101/motm.do?momID=156
© 2000 Nature A
tonated ammonia may diffuse across cell
cation of Robert Louis Stevenson’s novel,
membranes, the charged ion must be
wherein the two were described. In the Ammonium transport
same way that ‘Dr Jekyll’ provided insight Ammonia (NH3), is a ubiquitous gas that transported across the membrane by a
into the workings of Mr Hyde’s mind, the is both a toxin and a nutrient. Highly solu- member of the family of ammonium
recognition of a second and unexpected ble in water, it exists predominantly as the transporter proteins2.
facet of a popular molecule provides sur- ammonium ion (NH4+) in biological fluYeast cells express ammonium transporter
prising insight into a complex biological ids. In animals, ammonia is produced dur- proteins that concentrate the toxic ammoThe Rhesus
is ofaamino
transmembrane
thatmethylammonium
is found in by about
problem (ammonium
transport inantigen
mam- ing(RhD)
catabolism
acids, and is then protein
nium analogue
3,4, and genes encoding three
malian cells). On
page
341
in
this
issue,
a
thousandfold
metabolized
through
the
urea
cycle
and
the plasma membrane of red blood cells. If you have the gene for RhD
Marini and colleagues1 report that subunits excreted. Although a waste product in ani- distinct yeast ammonium transporters
you are
don’t nitrogen
you are(Mep1–3)
RhD-.have been identified5–7. Mep2 is a
mals, RhD+.
ammonia If
is you
an important
of the human Rh blood group antigens
The Rhesus antigen
complex
polysaccharide
C
E
Rh homologues
D
plasma
membrane
Rh50 (RhAG)
Bob Crimi
palmitate
Rh30
Fig. 1 M o d el o f t h e Rh co m plex in t h e m e m bra n e o f t h e re d blo o d cell. Th e Rh
co m plex co nsists o f t w o m olecules o f t h e Rh30 su b u nit (m ost co m m o nly Rh D
or RhCE), a n d t w o m olecules o f Rh50, t h e glycosyla t e d Rh-associa t e d pro t ein.
258
51
ammonium
transporters
Fig. 2 Phylog e n e tic tre e of multiple se qu e nces from hum a n Rh blood group a ntig e ns, hum a n Rh glycoprot eins, non-hum a n se qu e nces with Rh homology, a nd
a mmonium tra nsport ers from ye ast, b act eria, pla nts a nd w orms (re fs 12,15).
nature genetics
• volume 26
• november 2000
Based on sequence homology with proteins with known
function
from
other organisms, it is likely that the Rhesus complex functions as an
ammonium transporter
Heitman and Agre, Nature Genetics (2000), 26, 258-259.
•
•
The Rhesus antigen (RhD) was first identified more than 70 years ago when a baby was
stillborn when the mother had an immune attack against an red blood cell antigen that her
baby had but that she didn’t have. That is, the mother was RhD-negative and the baby
was RhD-positive
It is only in recent years that we have started to figure out what the RhD antigen actually is
and what it’s normal role is.
There are actually 5 different versions of the Rh protein, designated D, C, E, c, e. Every
individual is positive or negative for most of these proteins, resulting in about 50 possible
combinations.
About 15% of caucasian people are D- and about 0.3% of asian people are D-.
•
•
http://en.wikipedia.org/wiki/Rh_blood_group_system#Rh_system_antigens
http://www.nature.com/ng/journal/v26/n3/pdf/ng1100_258.pdf
•
•
EldonCard: simple blood typing kit
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Each well contains dried antibodies as
indicated. A hemagglutination reaction
indicates that the corresponding
antigen is present
• Hemagglutination is used to type blood groups and match compatible donors and
recipients for blood transfusion.
• These blood-group antigens are arrayed in many copies on the surface of the red blood
cell, allowing the cells to agglutinate when cross-linked by antibodies.
• The pattern of agglutination of the red blood cells of a transfusion donor or recipient with
anti-A and anti-B antibodies reveals the individual's ABO and Rhesus blood group. Before
transfusion, the serum of the recipient is also tested for antibodies that agglutinate the red
blood cells of the donor, and vice versa, a procedure called a cross-match, which may
detect potentially harmful antibodies to other blood groups that are not part of the ABO
system.
Latex-bead agglutination assay
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http://vtpb-www.cvm.tamu.edu/vtpb/vet_micro/serology/aggl/default.html
• Antibodies or antigens can be attached to a bead either by non-specific adsorption or through chemical coupling
reactions
• Latex-bead agglutination assay is a very easy, rapid (couple of minutes) way of determining the presence of a particular
antigen or antibody to an antigen. These assays would typically come as kits (much like the cat blood typing assay) in
which the latex beads are prepared with the appropriate antibody or antigen.
• Examples of commercially available kits include:
• Detection of hepatitis B virus in blood
• Detection of antibodies against the bacteria that causes syphilis
• Detection of antibodies against the HIV virus
• Pregnancy tests
• Q. Do you always use polyclonal antibodies to do the agglutination assays?
• A. Not necessarily. It really depends on the situation. Sometimes it might be necessary to use monoclonal antibodies if
you are trying to precipitate a particular antigen in the presence of another very similar antigen. I didn't disucss this in
class, but woman have another hormone (luteinizing hormone or LH) that is almost identical to hCG. The amino acid
sequences of the two hormones are essentially identical, but hCG has an extra 24 amino acids at its C-terminus.
Polyclonal antibodies raised against either LH or hCG would definitely cross-react with the other hormone. However, a
monoclonal raised against just the C-terminal 24 amino acids of hCG would definitely be specific for hCG. The
breakthrough in monoclonal antibody development in mid 1970s is what made pregnancy tests possible.
• Q. The monoclonal antibodies will not be able to produce precipitation for monomeric protein right, if so, if we use
monoclonal antibodies in Latex bead agg. assay, will it be able to crosslink between antigens bound to beads?
• A. If a monomeric protein antigen is attached to a bead, you have essentially converted a new antigen (= bead + protein)
in which there are many epitopes per antigen. The antigen coated bead is multivalent and it could be precipitated by a
monoclonal antibody.
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Pregnancy test
1920s: identification of a hormone that is only
present in pregnant woman. Designated human
chorionic gonadotropin (hCG)
hCG
Nature 369, 455 - 461 (09 June 1994)
http://www.123rf.com
http://www.occupyforanimals.org/rabbit-test-1927.html
1920-30s: A-Z pregnancy test (named for
inventors). Involved injecting small mammals or
frogs with the urine from a potentially pregnant
woman. If the animal had an ‘estrous reaction’, it
was a positive test for pregnancy. Other versions
involved sacrificing the animal to check if ovulation
had occurred (positive test). Example of Bioassay.
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Agglutination-based
pregnancy tests
1960s: Development of
hemagglutination assays for
pregnancy tests (Wide and Gemzell).
Through 60s, 70s, early 80s the
assay used sheep red blood cells
(thus hemagglutination) with hCG
covalently crosslinked to the surface.
Later versions used latex beads
rather than RBCs.
1977: First home pregnancy test
based on hemagglutination becomes
available ($10, comes with sheep
RBCs!)
A history of pregnancy tests: http://
www.history.nih.gov/exhibits/
thinblueline/timeline.html
• Also: Lateral Flow assays (http://www.cytodiagnostics.com/lateral-flowimmunoassays.php)
• First modern ‘stick type’ pregnancy was Unipath’s Clearview home pregnancy test
introduced in 1988.
1972: Sandwich radioimmunoassay pregnancy test56
Antigen ( = hCG) present
Add sample
wash
add radiolabeled antibody
I125
I125
wash
I125
Antigen absent
Add sample
wash
add radiolabeled antibody
unbound
wash
I125
washed away
Count Ɣ-radiation and compare to calibration curve
• The example shown is a radioimmunoassay (RIA). The basic principle of a RIA is the use of radiolabeled Abs detect
Ag:Ab reactions. The Abs are labeled with the 125I (iodine-125) isotope, and the presence of Ag:Ab reactions is
detected using a gamma counter.
• Because of the requirement to use radioactive substances, RIAs are frequently being replaced by other immunologic
assays, such as ELISAs which have similar degrees of sensitivity.
Radioactive probes are among the most sensitive markers used for biological detection. Iodine isotopes, 14C, 32P,
•
35S and tritium (3H) are commonly used radiolabels. 14C and 3H are β emitters. β particles are detected by the
fluorescence they generate in a dye-containing solution referred to as a scintillation cocktail. The iodine isotopes,
which are γ emitters, have several advantages, including a relatively short half-life. The maximum specific activity
that can be achieved with an isotope is inversely related to its half-life. In addition, γ rays are directly detectable
without a scintillation cocktail. Radioimmunoassay (RIA) is a common procedure that utilizes high specific-activity
radiolabeled molecules as tracers. Radioiodination is a common procedure and provides excellent sensitivity in
many applications. Iodine-125 is the isotope primarily used in radioimmunoassays because of its high, easily
detectable specific activity and low energy γ emission. Iodine-125 has a 60-day half-life, which allows labeled
material to be prepared and stored for extended time periods. Iodine-131 is rarely used for radioimmunoassays.
There are commercially available kits for labeling the tyrosines in proteins with radioactive I125.
•
• Q: For antibodies that we want to bind to a solid surface, we would want to bind at bottom of the Fc portion, so that
would mean C terminus. So could we activate the carboxylic group with NHS and have surface be covered with good
nucleophiles?
• A: That would definitely be the best way to bind antibodies to a surface. However, in practice, researchers tend not to
worry about making such perfectly arranged surface attachments. A less well-defined attachment (e.g., one that occurs
randomly through links to Asp or Glu or the C-term) is good enough for most purposes.
Competitive radioimmunoassay pregnancy test
57
Antigen ( = hCG) present
Add sample
wash
add radiolabeled hCG
I125
washed away
unbound
I125
wash
add radiolabeled hCG
wash
I125
Antigen absent
Add sample
wash
I125
I125 I125
I125 I125
I125
Count Ɣ-radiation and compare to calibration curve
• A competitive immunoassay is one where the antigen, and a labeled version of the same
antigen compete for binding to an immobolized antibody.
• In this case, more signal (i.e., gamma-radiation) means less antigen in the original sample.
That is, the signal will be inversely proportional to antigen concentration in the sample.
ELISA has replaced radioimmunoassay
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1990s: the use of radiolabeled antibodies began to be phased out as
they were replaced with enzyme labeled-antibodies
I125
I125
vs.
colored or
fluorescent
product
I125
HRP or alkaline
phosphatase (AP)
colorless
substrate
Immunoassays that use enzyme labeled antibodies (or antigens) are called
Enzyme linked immunosorbent assay (ELISA)
I125
vs.
Sandwich and competitive ELISA are analogous to the corresponding radioimmunoassays.
Substrates for alkaline phosphatase
O2N
pNPP
O
O P O
O
phosphatase
O2N
OH
O
HO P O
O
59
Soluble
chromophore
blue precipitate
dark blue
precipitate
green fluorescent
precipitate
ELF 97
http://www.probes.com/handbook/sections/1003.html
• pNPP: p-nitrophenol phosphate (pNPP): generates a soluble product which absorbs at
405 nm
• BCIP: 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) is commonly used with a number
of different chromogens in various histological and molecular biology techniques.
Hydrolysis of this indolyl phosphate, followed by oxidation, produces a blue-colored
precipitate at the site of enzymatic activity.
• NBT: A Co-Precipitant for the BCIP Reaction Nitro blue tetrazolium (NBT) is the most
commonly used electron-transfer agent and co-precipitant for the BCIP reaction, forming
a dark blue, precisely localized precipitate in the presence of alkaline phosphatase.
• ELF 97: a phosphatase substrate (ELF 97 phosphate). Upon enzymatic cleavage this
weakly blue-fluorescent substrate yields an extremely photostable green-fluorescent
precipitate that is up to 40 times brighter than the signal achieved when using either
directly labeled fluorescent hybridization probes or fluorescent secondary detection
methods in comparable applications.
ELISA-type pregnancy test
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Late 1980s: the first stick-type pregnancy test (Unipath Clearview; 1988)
based on ELISA in a lateral flow format.
anti-hCG
conjugated to AP,
not immobilized
urine sample
hCG present = color change at
both test and control line
hCG absent = color change at only
the control line
•
•
•
anti-AP attached to membrane +
precipitating substrate for AP
anti-hCG attached to membrane +
precipitating substrate for AP
A common variation on this assay is to attach a gold nanoparticle (rather than AP) to the
conjugated antibody. In this case it is the color of the captured nanoparticle that is
detected at the test and control lines.
Q. In the pregnancy test kit, there is low amount of Anti-hcG and an excess of urine right? A. I'm not sure about the relative concentrations, but probably the limiting reagent is the
amount of capture antibody at the testing line. You need to have an excess of the soluble
antibody-enzyme conjugate so that it is not all captured at the testing line. Some has to
make it to the control line. Immunoassay (but not ELISA) example for
detection of glycated hemoglobulin (A1C)
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Another method for monitoring blood glucose is to
detect glycated hemoglobulin. The A1CNow device
from Bayer enables self-testing of A1C levels.
http://www.medicographia.com
http://www.a1cnow.com
anti-A1C
The test appears to
work using lateral
flow and a sandwich
format that localizes
blue microspheres to
a capture area. It also
determines total Hb,
and provides a
readout of %A1C.
http://withfriendship.com
•
•
•
•
A1C
Blue
microparticle
immobilized anti-A1C
A1C is home test for glycosylated hemoglobin
The device probably uses the enzyme cytochrome-b5-reductase to oxidize the Fe2+ of the
heme group to Fe3+ (with reduction of NAD+ to NADH). The oxidized heme group has a
distinct absorbance that can be used to quantify the total concentration of Hb.
http://www.a1cnow.com/asset/document/A1CNow-HCP-Product-Insert-ClinicalLaboratory-Stan
Q. How does the blue microparticle relate to the signal detection which is caused by the
enzyme cytochrome-b5-reductase?
A. The blue microparticle has nothing to do with the detection of total Hb using
cytochrome-b5-reductase. This enzyme is used to quantify the total amount of Hb. The
blue microparticle is used to quantify only the glycated Hb. The readout is the ratio of
glycated Hb to total Hb, expressed as a percentage.
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Antibodies from different
species recognize each
other as being foreign
proteins
secondary antibody
In this case the goat anti-cat secondary antibodies
recognize any cat primary antibody. This is useful since
you only need to buy one batch of labeled secondary
antibody and use it for many different applications.
•
•
•
•
•
primary antibody
As we’ve seen, the whole purpose of antibodies is to recognize foreign proteins that
happen to find their way into the blood of an animal
Although antibodies from all mammals are practically identical in terms of overall structure
and function, there are still enough minor differences for them to be recognized as foreign
when introduced into another species. For example, cat antibodies are recognized as
foreign when introduced into a goat. And cow antibodies are recognized as foreign when
introduced into a rabbit.
So, immunizing an animal with antibodies from another species leads to the generation of
‘anti-antibodies’. In the first example mentioned above, the antibodies that are made (and
could be purified from the blood of the animal) would be called ‘goat anti-cat IgG’ or ‘anticat IgG produced in goat’, or something like that.
These anti-antibodies are known as secondary antibodies and incredibly useful in
bioanalytical chemistry applications.
However, this does one interesting question: where do we get antibodies for human
therapeutic applications? Many of the most sophisticated cancer therapies rely on treating
patients with antibodies that target the cancer cells (through a variety of mechanisms).
Where could these antibodies come from?
Indirect ELISA for detection of antibodies
63
Antibody present
Add sample
wash
add enzyme labeled
secondary antibody
wash
add enzyme labeled
secondary antibody
wash
immobilized
antigen
Antibody absent
Add sample
wash
Add substrate, allow to react for a while, measure absorbance or fluorescence
(typically in multiwell plate reader), and compare to calibration curve.
More signal indicates more antibody in original sample
•
The Indirect ELISA for antibody detection is analogous to the sandwich ELISA for antigen
detection.
•
•
Q.Why would you want to detect antibodies?
A. It is typically easier to detect antibodies against a virus then it is to detect the virus
itself. That is, the concentration of the specific antibodies is higher than the concentration
of the virus.
Competitive ELISA for detection of antibodies64
Antibody present
Add sample
wash
Antibody absent
Add sample
wash
add enzyme labeled
antibody against
antigen of interest
add enzyme labeled
antibody against
antigen of interest
wash
wash
Add substrate, allow to react for a while, measure absorbance or fluorescence
(typically in multiwell plate reader), and compare to calibration curve.
More signal indicates less antibody in original sample
•
Competitive ELISA for antibody detection. The competitive ELISA for antibody detection
is analogous to the competitive ELISA for antigen detection. It is based on the principle
that Abs in the sample will bind to an Ag and then inhibit binding of an enzyme-linked Ab
that reacts with the Ag (i.e., competes for binding)
•
Q: I think the competitive ELISA is only usable in case that the Ag contains only one
epitope. Isn't it? Otherwise the E-Ab will also bind to the Ag.
•
A: The most important thing about antibodies for the competitive ELISA is that the
antibody to be detected and the enzyme labeled antibody compete for the same epitope.
It doesn't really matter if the Ag has multiple epitopes, as long as the antibodies can
compete for binding to each of them.
65
Oraquick Advance
The OraQuick Advance is an FDA approved test to detect antiHIV antibodies in oral fluid or blood. This test was approved for
home use in July 2012 and can give results in 20-40 minutes.
Since more color = more antibody, this must be an
indirect ELISA. Accordingly, there must be a secondary
labeled anti-human antibody in the bottom vial.
negative
C
anti-human antibodies
T
positive
synthetic HIV antigens
•
http://www.nytimes.com/2012/07/04/health/oraquick-at-home-hiv-test-wins-fdaapproval.html?_r=1&
•
•
http://www.medhelp.org/posts/HIV-Prevention/Oraquick-Question/show/491650
•
A. The green antibody at the control line represents a different human antibody, not
necessarily one against HIV. The antibodies immobilized at the control line are just antihuman and will bind to any human antibody.
Q. Why is there is anti-HIV antibody "green color" at the control line. As far as I
understand it should not be there. Also, to make sure about my understanding ; Anti-HIV
antibody is attached to the control line or any other human antibody to capture
the secondary labelled antibody (for example goat anti-human antibody), am I correct??