HUMAN ANTI-ALPHA-GALACTOSYL IGM ELISA

Product Data Sheet

Cat. No.: RD199178110R
For Research Use Only

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CONTENTS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

INTENDED USE
STORAGE, EXPIRATION
INTRODUCTION
TEST PRINCIPLE
PRECAUTIONS
TECHNICAL HINTS
REAGENT SUPPLIED
MATERIAL REQUIRED BUT NOT SUPPLIED
PREPARATION OF REAGENTS
PREPARATION OF SAMPLES
ASSAY PROCEDURE
CALCULATIONS
PERFORMANCE CHARACTERISTICS
DEFINITION OF THE CALIBRATOR
PRELIMINARY POPULATION DATA
METHOD COMPARISON
TROUBLESHOOTING AND FAQS
REFERENCES
EXPLANATION OF SYMBOLS

3
3
4
5
5
6
6
7
7
9
10
12
13
16
17
18
19
20
22

This kit is manufactured by:

BioVendor Laboratorn medicna a.s.
Use only the current version of Product Data Sheet enclosed with the kit!

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1.

INTENDED USE

The RD199178100R Human Anti-Alpha-Galactosyl IgM ELISA is an enzyme immunoassay for

the quantitative measurement of human anti-alpha-galactosyl antibodies in the IgM class.
Features
It is intended for research use only
The total assay time is less than 3 hours
The kit measures IgM anti-alpha-galactosyl antibodies in human serum and plasma (EDTA,
citrate, heparin)
Assay format is 96 wells
Calibrator is human serum based
Components of the kit are provided ready to use, concentrated or lyophilized

2.

STORAGE, EXPIRATION

Store the complete kit at 2 - 8 C. Under these conditions, the kit is stable until the expiration
date (see label on the box).
For stability of opened reagents see Chapter 9.

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3.

INTRODUCTION

Natural antibodies circulate in serum of healthy humans naturally, without previous

immunization [1]. They can be directed against the individuals own antigens as well as against
foreign antigens [2, 3, 4]. The repertoire of natural antibodies is encoded by germ-line genes
[5, 4, 6] and is not subjected to recombination. Natural antibodies are polyreactive [2, 3, 6] and
react with low affinity but high avidity [7].
Among the natural antibodies directed against saccharide antigens, the best known are:
1) isohemagglutinins (anti-A, anti-B) which prevent the transfusion of allogeneic blood [8] and
allotransplantation [9];
2) xenoagglutinins (80% of the repertoire of human xenoreactive natural antibodies is specific
for the terminal -galactose determinant) [10] which have traditionally been detected by
agglutination test with rabbit or porcine erythrocytes [11]. The epitope against which they are
directed was identified as Gal1-3Gal1-4GlcNAc-R [12]. This epitope is present in cell
membranes of mammals with the exception of humans and Old World primates [13] in which
the gene encoding for -1,3-galactosyltransferase was inactivated during evolution [14]. This
epitope is also present in lipopolysaccharides and capsular polysaccharides of various gramnegative bacteria colonizing the gastrointestinal tract of humans [15].
In human serum, natural anti--galactosyl antibodies (anti--Gal) constitute approximately 4 8 % of total serum IgM, approx. 1 - 2 % of total serum IgG [16], and they can also be found as
the IgA isotope in serum, saliva, milk, colostrum and vaginal washings [17, 15]. About 1 % of
circulating B lymphocytes in adults is capable of producing these antibodies [18]. Their
production is thought to be induced postnatally after antigenic stimuli by intestinal microflora
[19].
The biological roles of natural antibodies are thought to be protection against infectious agents
[20], particularly at the mucosal surface [15], effector functions of the immune system, e.g. in
removing senescent or otherwise altered cells and physiologically degraded molecules [21],
and regulation of immune responses, particularly autoimmune responses [6]. Moreover, the
presence of anti--galactosyl antibodies in humans represents an important obstacle to
xenotransplantations of porcine grafts since these antibodies participate in their rejection [22].
Natural antibodies also play an important role in malignant processes, and they have potential
to be used in treatment of tumors that express these (-Gal) antigens (e.g. breast cancer) [23,
24, 25, 26].
Areas of investigation:
Immunology, Immune Response
Autoimmunity
Infection and Inflammation
Transplantation
Oncology
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4.

TEST PRINCIPLE

In the BioVendor Human Anti-Alpha-Galactosyl IgM ELISA, Calibrators and samples are
incubated in microtitration wells pre-coated with chemically synthesized molecule displaying
terminal beta-disaccharide Gal1-3Gal which is recognized by human anti-alpha-galactosyl
antibodies. After 60 minutes incubation followed by washing, polyclonal antibody against
human immunoglobulin M (IgM) conjugated with horseradish peroxidase (HRP) is added into
the wells and incubated with the captured anti-alpha-galactosyl IgM for 60 minutes. The last
washing step, the remaining conjugate is allowed to react with the substrate solution (TMB).
The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow
product is measured. The absorbance is proportional to the concentration of IgM anti-alphagalactosyl antibodies. A calibration curve is constructed by plotting absorbance values against
anti-alpha-galactosyl IgM concentrations of calibrators and concentrations of unknown samples
are determined using this calibration curve.

5.

PRECAUTIONS

For professional use only

Wear gloves and laboratory coats when handling immunodiagnostic materials
Do not drink, eat or smoke in the areas where immunodiagnostic materials are being
handled
This kit contains components of human serum origin. These materials were found nonreactive for HBsAg, HCV antibody and for HIV 1/2 antigen and antibody. However, these
materials should be handled as potentially infectious, as no test can guarantee the
complete absence of infectious agents
Avoid contact with the acidic Stop Solution and Substrate Solution, which contains
hydrogen peroxide and tetramethylbenzidine (TMB). Wear gloves and eye and clothing
protection when handling these reagents. Stop and/or Substrate Solutions may cause
skin/eyes irritation. In case of contact with the Stop Solution and the Substrate Solution
wash skin/eyes thoroughly with water and seek medical attention, when necessary
The materials must not be pipetted by mouth

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6.

TECHNICAL HINTS

Reagents with different lot numbers should not be mixed

Use thoroughly clean glassware
Use deionized (distilled) water, stored in clean containers
Avoid any contamination among samples and reagents. For this purpose, disposable tips
should be used for each sample and reagent
Substrate Solution should remain colourless until added to the plate. Keep Substrate
Solution protected from light
Stop Solution should remain colourless until added to the plate. The colour developed in
the wells will turn from blue to yellow immediately after the addition of the Stop Solution.
Wells that are green in colour indicate that the Stop Solution has not mixed thoroughly with
the Substrate Solution
Dispose of consumable materials and unused contents in accordance with applicable
national regulatory requirements

7.

REAGENT SUPPLIED

Kit Components
Antigen Coated Microtiter Strips
Conjugate Solution
Master Calibrator
Dilution Buffer
Wash Solution Conc. (10x)
Substrate Solution
Stop Solution
Product Data Sheet + Certificate of Analysis

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State
ready to use
ready to use
lyophilized
ready to use
concentrated
ready to use
ready to use
-

Quantity
96 wells
13 ml
2 vials
100 ml
100 ml
13 ml
13 ml
1 pc

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8.

MATERIAL REQUIRED BUT NOT SUPPLIED

Deionized (distilled) water

Test tubes for diluting samples
Glassware (graduated cylinder and bottle) for Wash Solution (Dilution Buffer)
Precision pipettes to deliver 5 1 000 l with disposable tips
Multichannel pipette to deliver 100 l with disposable tips
Absorbent material (e.g. paper towels) for blotting the microtitrate plate after washing
Vortex mixer
Microplate washer (optional). [Manual washing is possible but not preferable.]
Microplate reader with 450 10 nm filter, preferably with reference wavelength 630 nm
(alternatively another one from the interval 550 650 nm)
Software package facilitating data generation and analysis (optional)

9.

PREPARATION OF REAGENTS
All reagents need to be brought to room temperature prior to use
Always prepare only the appropriate quantity of reagents for your test
Do not use components after the expiration date marked on their label

Assay reagents supplied ready to use:

Antigen Coated Microtiter Strips
Stability and storage:
Return the unused strips to the provided aluminium zip-sealed bag with desicant and seal
carefully. Remaining Microtiter Strips are stable 3 months stored at 2 - 8 C and protected from
the moisture.
Conjugate Solution
Dilution Buffer
Substrate Solution
Stop Solution
Stability and storage:
Opened reagents are stable 3 months when stored at 2 - 8 C.

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 Assay reagents supplied concentrated or lyophilized:

Human IgM anti-alpha-galactosyl antibody Master Calibrator
Refer to the Certificate of Analysis for current volume of Dilution Buffer needed for
reconstitution of calibrator!!!
Reconstitute the lyophilized Master Calibrator with Dilution Buffer just prior to the assay. Let it
dissolve at least 15 minutes with occasional gentle shaking (not to foam). Vortex is
recommended. The resulting concentration of the human IgM anti-alpha-galactosyl antibodies
in the stock solution is 100 U/ml.
Prepare set of calibrators using Dilution Buffer as follows:
Volume of Calibrator
Stock
250 l of stock
250 l of 50 U/ml
250 l of 25 U/ml
250 l of 12.5 U/ml
250 l of 6.25 U/ml

Dilution Buffer
250 l
250 l
250 l
250 l
250 l

Concentration
100 U/ml
50 U/ml
25 U/ml
12.5 U/ml
6.25 U/ml
3.13 U/ml

Prepared Calibrators are ready to use, do not dilute them.

Stability and storage:
Do not store the reconstituted and/or diluted Calibrator solutions.
Wash Solution Conc. (10x)
Dilute Wash Solution Concentrate (10x) ten-fold in distilled water to prepare a 1x working
solution. Example: 100 ml of Wash Solution Concentrate (10x) + 900 ml of distilled water for
use of all 96-wells.
Stability and storage:
The diluted Wash Solution is stable 1 month when stored at 2 - 8 C. Opened Wash Solution
Concentrate (10x) is stable 3 months when stored at 2 - 8 C.

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10.

PREPARATION OF SAMPLES

The kit measures human IgM anti-alpha-galactosyl antibodies in serum and plasma (EDTA,
citrate, heparin).
Samples can be assayed immediately after collection, after storage for a week at 2 - 8 C, or
after long-term storage at -20 C (-70 C). Thoroughly mix thawed samples just prior to the
assay and avoid repeated freeze/thaw cycles, which may cause erroneous results. Avoid using
hemolyzed or lipemic samples.
An appropriate dilution should be assessed by the researcher (due to the high
variability of serum anti-alpha-galactosyl IgM level between individuals) in advance to
batch measurement. Recommended starting dilution is 100x.
Dilute samples 100x with Dilution Buffer just prior to the assay, e.g. 5 l of sample + 495 l of
Dilution Buffer for singlets and preferably for duplicates. Mix well (not to foam). Vortex is
recommended.
Stability and storage:
Samples should be stored at -20 C, or preferably at -70 C or lower for long-term storage.
Avoid repeated freeze/thaw cycles.
Do not store the diluted samples.
See Chapter 13 for stability of serum and plasma samples when stored at 2 - 8 C, effect of
freezing/thawing and effect of sample matrix (serum/plasma) on the concentration of human
IgM anti-alpha-galactosyl antibodies.
Note: It is recommended to use a precision pipette and a careful technique to perform the
dilution in order to get precise results.

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11.

ASSAY PROCEDURE

Pipet 100 l of diluted Calibrators, Dilution Buffer (= Blank) and diluted samples,
preferably in duplicates, into the appropriate wells. See Figure 1 for example of work
sheet.
2. Incubate the plate at room temperature (ca. 25 C) for 1 hour, no shaking.
3. Wash the wells 3-times with Wash Solution (0.35 ml per well). After final wash, invert and
tap the plate strongly against paper towel.
4. Add 100 l of Conjugate Solution into each well.
5. Incubate the plate at room temperature (ca. 25 C) for 1 hour, no shaking.
6. Wash the wells 3-times with Wash Solution (0.35 ml per well). After final wash, invert and
tap the plate strongly against paper towel.
7. Add 100 l of Substrate Solution into each well. Avoid exposing the microtiter plate to
direct sunlight. Covering the plate with e.g. aluminium foil is recommended.
8. Incubate the plate for 30 minutes at room temperature. The incubation time may be
extended [up to 45 minutes] if the reaction temperature is below than 20 C. Do not shake
the plate during the incubation.
9. Stop the colour development by adding 100 l of Stop Solution.
10. Determine the absorbance of each well using a microplate reader set to 450 nm,
preferably with the reference wavelength set to 630 nm (acceptable range: 550 - 650 nm).
Subtract readings at 630 nm (550 - 650 nm) from the readings at 450 nm. The
absorbance should be read within 5 minutes following step 9.
1.

Note: If some samples and calibrator/s have absorbances above the upper limit of your
microplate reader, perform a second reading at 405 nm. A new calibration curve, constructed
using the values measured at 405 nm, is used to determine anti-alpha-galactosyl IgM
concentration of off-scale calibrators and samples. The readings at 405 nm should not replace
the readings for samples that were in range at 450 nm.
Note 2: Manual washing: Aspirate wells and pipet 0.35 ml Wash Solution into each well.
Aspirate wells and repeat twice. After final wash, invert and tap the plate strongly against paper
towel. Make certain that Wash Solution has been removed entirely.

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strip 1+2

strip 3+4

strip 5+6

strip 7+8 strip 9+10 strip 11+12

Calibrator 100 Sample 2 Sample 10 Sample 18 Sample 26 Sample 34

Calibrator 50

Sample 3 Sample 11 Sample 19 Sample 27 Sample 35

Calibrator 25

Sample 4 Sample 12 Sample 20 Sample 28 Sample 36

Calibrator 12.5 Sample 5 Sample 13 Sample 21 Sample 29 Sample 37

Calibrator 6.25 Sample 6 Sample 14 Sample 22 Sample 30 Sample 38

Calibrator 3.13 Sample 7 Sample 15 Sample 23 Sample 31 Sample 39

Blank

Sample 8 Sample 16 Sample 24 Sample 32 Sample 40

Sample 1

Sample 9 Sample 17 Sample 25 Sample 33 Sample 41

Figure 1: Example of a work sheet.

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12.

CALCULATIONS

Most microtiter plate readers perform automatic calculations of analyte concentration.

The calibration curve is constructed by plotting the mean absorbance of Calibrators (Y) against
the known concentration of Calibrators (X) in logarithmic scale, using the four-parameter
algorithm. Results are reported as concentration of IgM anti-alpha-galactosyl antibodies (U/ml)
in samples.
Alternatively, the logit log function can be used to linearize the calibration curve, i.e. logit of the
mean absorbance (Y) is plotted against log of the known concentration of Calibrators (X).
Concentration obtained from calibration curve is the final concentration of IgM antialpha-galactosyl antibodies in the sample. Dilution factor (100x) is calculated. The
results are not to be multiplied by dilution factor.
Dilution factor for samples with dilution higher than the basic dilution (100x) in ELISA is shown
in the Chapter 13 in the paragraph Limit of Assay.
Human Anti-Alpha-Galactosyl IgM ELISA
Calibration Curve

Absorbance (A450 nm - A630 nm)

3,0

2,5

2,0

1,5

1,0

0,5

0,0
1

10

100

Concentration of anti-alpha-galactosyl IgM (U/ml)

Figure 2: Typical Calibration Curve for Human Anti-Alpha-Galactosyl IgM ELISA.

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13.

PERFORMANCE CHARACTERISTICS
Typical analytical data of BioVendor Human Anti-Alpha-Galactosyl IgM ELISA are
presented in this chapter

Sensitivity
Limit of detection (LOD) (defined as concentration of analyte giving absorbance higher than
mean absorbance of blank* plus three standard deviations of the absorbance of blank: Ablank +
3xSDblank) is calculated from the real anti-alpha-galactosyl IgM values in wells and is:
1.35 U/ml.
* Dilution Buffer is pipetted into blank wells.

Limit of Assay

Samples with absorbances exceeding the absorbance of the highest calibrator should be
measured again with higher dilution. The final concentration of samples calculated from
the calibration curve must be multiplied by the dilution factor divided by 100. For
example, when the samples are diluted 200 times, the results must be multiplied by 200/100 (=
2).

Specificity
The antibodies used in this ELISA are specific for human IgM antibodies.
Sera of several mammalian species were measured in the assay. See results below.
For details please contact us at info@biovendor.com
Mammalian serum
Sample
Bovine
Cat
Dog
Goat
Hamster
Horse
Monkey
Mouse
Pig
Rabbit
Rat
Sheep

Page 13 of 28

Observed
crossreactivity
no
no
no
no
no
no
yes
no
no
no
no
no

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Precision
Intra-assay (Within-Run) (n=8)
Sample

Mean
(U/ml)
33.61
5.89

1
2

SD
(U/ml)
0.91
0.18

CV
(%)
2.7
3.1

SD
(U/ml)
0.74
1.14

CV
(%)
8.4
4.4

Inter-assay (Run-to-Run) (n=5)

Sample

Mean
(U/ml)
8.76
26.02

1
2

Spiking Recovery
Serum samples were spiked with different amounts of human anti-alpha-galactosyl IgM and
assayed.
Sample

Observed
(U/ml)
6.50
11.33
16.04
26.81
11.64
15.38
19.37
27.87

Expected
(U/ml)
12.32
18.14
29.77
16.43
21.23
30.82

Recovery O/E
(%)
92.0
88.4
90.0
93.6
91.2
90.4

Linearity
Serum samples were serially diluted with Dilution Buffer and assayed.
Sample

Dilution

2x
4x
8x
2x
4x
8x

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Observed
(U/ml)
25.98
12.23
6.68
3.26
66.11
33.22
16.20
8.11

Expected Recovery O/E

(U/ml)
(%)
12.99
94.2
6.49
102.9
3.25
100.2
33.06
100.5
16.53
98.0
8.26
98.2

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Effect of sample matrix
EDTA, citrate and heparin plasma samples were compared to respective serum samples from
the same 10 individuals. Results are shown below:
Volunteer
No.
1
2
3
4
5
6
7
8
9
10
Mean (U/ml)
Mean Plasma/Serum
(%)
Coefficient of
determination R2

Serum
(U/ml)
15.12
59.21
17.92
25.51
70.97
128.80
134.08
43.83
19.17
27.85
54.25

Plasma (U/ml)
EDTA Citrate Heparin
15.51
12.30
14.50
52.60
33.33
51.23
17.85
16.46
18.65
24.38
21.81
24.53
61.65
49.72
62.61
124.16 115.20 135.93
128.80 117.52 140.03
42.91
39.27
45.78
17.97
14.78
17.09
27.73
23.55
29.67
51.36
44.39
54.00
94.7

81.8

99.5

1.00

0.97

0.99

Effect of Sample Matrix

Serum
EDTA Plasma

160

Citrate Plasma
Heparin Plasma

Concentration of
Anti-Alpha-Galactosyl IgM (U/ml)

140
120
100
80
60
40
20
0
1

10

Volunteers

Figure 3: Human anti-alpha-galactosyl IgM levels measured using Human Anti-AlphaGalactosyl IgM ELISA from 10 individuals using serum, EDTA, citrate and heparin plasma,
respectively.
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Stability of samples stored at 2 - 8 C
No significant decline in the concentration of human anti-alpha-galactosyl IgM was observed in
serum and plasma samples after 28 days when stored at 2 - 8 C. To avoid microbial
contamination, samples were treated with -aminocaproic acid and sodium azide, resulting in
the final concentration of 0.03% and 0.1%, respectively. Samples should be stored at -20 C or
preferably at -70 C or lower for long-term storage.
Sample Storage Conditions
-20 C
2 - 8 C, 14 days
2 - 8 C, 28 days
-20 C
2 - 8 C, 14 days
2 - 8 C, 28 days
-20 C
2 - 8 C, 14 days
2 - 8 C, 28 days

1
2
3

Serum
(U/ml)
9.79
9.38
9.86
22.56
21.43
21.80
33.22
33.56
36.36

Plasma (U/ml)
EDTA Citrate Heparin
9.37
7.88
9.60
9.49
7.39
8.40
9.08
7.55
8.99
21.07 17.12 20.95
21.24 17.27 20.52
20.61 16.53 20.03
34.68 29.88 36.74
34.92 32.08 37.09
36.44 31.82 38.24

Effect of Freezing/Thawing
No decline was observed in concentration of human anti-alpha-galactosyl IgM in serum and
plasma samples after repeated (5x) freeze/thaw cycles. However it is recommended to avoid
unnecessary repeated freezing/thawing of the samples.
Sample
1
2
3

14.

Number of f/t
cycles
1x
3x
5x
1x
3x
5x
1x
3x
5x

Serum
(U/ml)
17.91
19.26
17.74
12.24
13.35
12.67
28.86
27.05
27.98

Plasma (U/ml)
EDTA Citrate Heparin
17.85
15.61
19.43
17.43
14.67
18.06
16.23
14.69
18.26
11.45
9.70
12.64
12.69
9.94
12.84
12.26
9.67
13.29
28.21
23.02
26.84
26.71
21.12
23.43
25.77
20.23
25.01

DEFINITION OF THE CALIBRATOR

The Calibrator used in this kit is IgM anti-alpha-galactosyl antibody of human serum origin.

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15.

PRELIMINARY POPULATION DATA

The following results were obtained when serum samples from 168 unselected donors (91 men
+ 77 women) 20 - 65 years old were assayed with the BioVendor Human Anti-Alpha-Galactosyl
IgM ELISA in our laboratory.
Sex

Age
(years)

Mean

Median
SD
Min
Max
Anti-Alpha-Galactosyl IgM (U/ml)

Men

20-29
30-39
40-49
50-65

18
30
33
10

26.54
32.05
33.04
24.64

21.34
24.91
24.38
20.75

19.27
25.03
26.85
10.02

3.60
7.16
1.61
10.45

65.80
123.85
113.54
42.46

Women

22-29
30-39
40-49
50-61

14
29
27
7

45.14
52.02
44.19
39.11

45.15
29.35
19.82
29.95

21.67
92.51
51.02
32.55

16.00
7.70
5.53
3.86

83.75
520.32
222.03
91.16

Age and Sex Dependent Distribution

of Hu Anti-Alpha-Galactosyl IgM
Concentration of
Hu Anti-Alpha-Galactosyl IgM (U/ml)

600
500

men

400

women
300
200
100
0
0

10

20

30

40

50

60

70

Age (years)

Figure 4a: Human anti-alpha-galactosyl IgM concentration plotted against donor age and sex.

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Age and Sex Dependent Distribution

of Hu Anti-Alpha-Galactosyl IgM
Concentration of
Hu Anti-Alpha-Galactosyl IgM (U/ml)

200
180
160
140

men

120

women

100
80
60
40
20
0
0

10

20

30

40

50

60

70

Age (years)

Figure 4b: Human anti-alpha-galactosyl IgM concentration plotted against donor age and sex.
Same data as in 4a except magnified (values 200 U/ml or less).

Reference range
It is recommended that each laboratory include its own panel of control sample in the assay.
Each laboratory should establish its own normal and pathological reference ranges for antialpha-galactosyl IgM levels with the assay.

16.

METHOD COMPARISON

The BioVendor Human Anti-Alpha-Galactosyl IgM ELISA has not been compared to any
commercial immunoassay.

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17.

TROUBLESHOOTING AND FAQS

Weak signal in all wells

Possible explanations:
Omission of a reagent or a step
Improper preparation or storage of a reagent
Assay performed before reagents were allowed to come to room temperature
Improper wavelength when reading absorbance
High signal and background in all wells
Possible explanations:
Improper or inadequate washing
Overdeveloping; incubation time with Substrate Solution should be decreased before
addition of Stop Solution
Incubation temperature over 30 C
High coefficient of variation (CV)
Possible explanation:
Improper or inadequate washing
Improper mixing Calibrators or samples

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18.

REFERENCES
References to anti-alpha-galactosyl antibodies:

1. Coutinho A, Kazatchkine MD, Avrameas S. Natural autoantibodies. Curr Opin Immunol;

7(6):812-8. (1995)
2. Avrameas S, Dighiero G, Lymberi P, Guilbert B. Studies on natural antibodies and
autoantibodies. Ann Immunol (Paris); 134D(1):103-13. (1983)
3. Avrameas S, Guilbert B, Mahana W, Matsiota P, Ternynck T. Recognition of self and nonself constituents by polyspecific autoreceptors. Int Rev Immunol; 3(1-2):1-15. (1988)
4. Tomer Y, Shoenfeld Y. The significance of natural autoantibodies. Immunol Invest;
17(5):389-424. (1988)
5. Dighiero G, Lymberi P, Guilbert B, Ternynck T, Avrameas S. Natural autoantibodies
constitute a substantial part of normal circulating immunoglobulins. Ann N Y Acad Sci;
475:135-45. (1986)
6. Avrameas S. Natural autoantibodies: from 'horror autotoxicus' to 'gnothi seauton'. Immunol
Today; 12(5):154-9. (1991)
7. Ternynck T, Avrameas S. Murine natural monoclonal autoantibodies: a study of their
polyspecificities and their affinities. Immunol Rev; 94:99-112. (1986)
8. Landsteiner K. The Specificity of Serological Reactions. Harvard University Press.
Cambridge, 1945.
9. Starzl TE, Marchioro TL, Holmes JH, Hermann G, Brittain RS, Stonington OH, Talmage
DW, Waddell WR. Renal homografts in patients with major donor-recipient blood group
incompatibilities. Surgery; 55:195-200. (1964)
10. Parker W, Bruno D, Holzknecht ZE, Platt JL. Characterization and affinity isolation of
xenoreactive human natural antibodies. J Immunol; 153(8):3791-803. (1994)
11. Tnder O, Natvig JB, Matre R. Antibodies in human sera to rabbit erythrocytes.
Immunology; 12(6):629-37. (1967)
12. Galili U, Macher BA, Buehler J, Shohet SB. Human natural anti-alpha-galactosyl IgG. II.
The specific recognition of alpha (1----3)-linked galactose residues. J Exp Med;
162(2):573-82. (1985)
13. Galili U, Clark MR, Shohet SB, Buehler J, Macher BA. Evolutionary relationship between
the natural anti-Gal antibody and the Gal alpha 1----3Gal epitope in primates. Proc Natl
Acad Sci U S A; 84(5):1369-73. (1987)
14. Galili U, Swanson K. Gene sequences suggest inactivation of alpha-1,3galactosyltransferase in catarrhines after the divergence of apes from monkeys. Proc Natl
Acad Sci U S A; 88(16):7401-4. (1991)

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15. Hamadeh RM, Galili U, Zhou P, Griffiss JM. Anti-alpha-galactosyl immunoglobulin A (IgA),
IgG, and IgM in human secretions. Clin Diagn Lab Immunol; 2(2):125-31. (1995)
16. McMorrow IM, Comrack CA, Sachs DH, DerSimonian H. Heterogeneity of human anti-pig
natural antibodies cross-reactive with the Gal(alpha1,3)Galactose epitope.
Transplantation; 64(3):501-10. (1997)
17. Galili U. Evolution and pathophysiology of the human natural anti-alpha-galactosyl IgG
(anti-Gal) antibody. Springer Semin Immunopathol; 15(2-3):155-71. (1993)
18. Galili U, Anaraki F, Thall A, Hill-Black C, Radic M. One percent of human circulating B
lymphocytes are capable of producing the natural anti-Gal antibody. Blood; 82(8):2485-93.
(1993)
19. Galili U, Mandrell RE, Hamadeh RM, Shohet SB, Griffiss JM. Interaction between human
natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect
Immun; 56(7):1730-7. (1988)
20. Ochsenbein AF, Zinkernagel RM. Natural antibodies and complement link ikate and
acquired immunity. Immunol Today; 21(12):624-30. (2000)
21. Grabar P. Autoantibodies and the physiological role of immunoglobulins. Immunol Today;
4(12):337-340. (1983)
22. Good AH, Cooper DK, Malcolm AJ, Ippolito RM, Koren E, Neethling FA, Ye Y, Zuhdi N,
Lamontagne LR. Identification of carbohydrate structures that bind human antiporcine
antibodies: implications for discordant xenografting in humans. Transplant Proc; 24(2):55962. (1992)
23. Castronovo V, Colin C, Parent B, Foidart JM, Lambotte R, Mahieu P. Possible role of
human natural anti-Gal antibodies in the natural antitumor defense system. J Natl Cancer
Inst; 81(3):212-6. (1989)
24. Galili U, LaTemple DC. Natural anti-Gal antibody as a universal augmenter of autologous
tumor vaccine immunogenicity. Immunol Today; 18(6):281-5. (1997)
25. Galili U, Wigglesworth K, Abdel-Motal UM. Intratumoral injection of alpha-gal glycolipids
induces xenograft-like destruction and conversion of lesions into endogenous vaccines. J
Immunol; 178(7):4676-87. (2007)
26. Manches O, Plumas J, Lui G, Chaperot L, Molens JP, Sotto JJ, Bensa JC, Galili U. AntiGal-mediated targeting of human B lymphoma cells to antigen-presenting cells: a potential
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(2005)

For more references on this product see our WebPages at www.biovendor.com

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19.

EXPLANATION OF SYMBOLS

Catalogue number

Content

Lot number

See instructions for use

Biological hazard

Expiry date

Storage conditions

Identification of packaging materials

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Assay Procedure Summary

Antigen Coated Microtiter Plate

Reconstitute Master Calibrator

and prepare set of Calibrators

Dilute samples 100x

Add Calibrators and samples

100 l

Incubate at RT for 1 hour / no shaking

Prepare Wash Solution

Wash 3x

Add Conjugate Solution

100 l
Incubate at RT for 1 hour / no shaking
Wash 3x

Add Substrate Solution

100 l
Incubate at RT for 30 min
Add Stop Solution
100 l

Read absorbance and calculate results

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10

11

12

NOTES

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NOTES

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NOTES

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