Variations in Dominant Antigen Determinants of Glutaraldehyde Polymerized Human, Bovine and Porcine Hemoglobin

Figures & data

Figure 1 Analysis of the purity of purified porcine hemoglobin (A) and the molecular distribution of its glutaraldehyde-polymerized derivatives (B).

Figure 2 Silver-stained SDS-PAGE analysis of the purity of purified Hb and the molecular distribution of corresponding glutaraldehyde polymerized derivatives with 40 µg protein per lane (lane 1: pHb, lane 2: pPolyHb, lane 3: bHb, lane 4: bPolyHb, lane 5 :hHb and lane 6: hPolyHb).

Table 1. Comparison of immunogenicity of different hemoglobin and their glutaraldehyde polymerized derivatives tested in mice

Note: A shade box in the above table indicates that the result of an ELISA test using the corresponding serum with the indicated fold of dilution yielded a p/n ratio value larger than 2.

Figure 3 Western blotting analysis of cross-species binding reactions between a hemoglobin or its polymerized derivative and mouse polyclonal antibodies against a particular type of PolyHb.

Table 2. Cross-species binding reaction (%) between glutaraldehyde polymerized hemoglobin and a particular ant-PolyHb polyclonal antibodies from rabbits or rats, as tested by a protein array method

	hPolyHb	bPolyHb	pPolyHb
Rabbit anti-hPolyHb Ab	100	24.8	7.4
Rabbit anti-bPolyHb Ab	4.3	100	6.9
Rabbit anti-pPolyHb Ab	2.1	3.8	100
Rat anti-hPolyHb Ab	100	0	0
Rat anti-bPolyHb Ab	n/a	n/a	n/a
Rat anti-pPolyHb Ab	0	0.2	100

n/a: not analyzed.

Table 3. Amino acid sequence identity score among hemoglobin from different origins using ClustalW software for multiple alignment analysis

Proteins	Accession No^1	hHb	bHb	pHb	bHb/pHb^2	hHb/bHb/pHb^3
					α subunit
hHb	P6990	100	88	84
bHb	P01966	88	100	86
pHb	P01965	86	86	100
mouse Hb	P01942	85	86	82
rabbit Hb	P01948	90	82	83
rat Hb	P01946	78	77	75
					β subunit
hHb	P68871	100	83	85
bHb	P02070	83	100	81
pHb	P02067	85	81	100
mouse Hb	P02088	80	73	78
rabbit Hb	P02057	82	82	81
rat Hb	P02091	81	75	80

¹From the SWISS-PROT database accessed via the website of NCBI.

²Overall identity (%) among hHb, bHb and pHb.

³Overall identity (%) among hHb, bHb, pHb and mouse Hb, rabbit Hb or rat Hb.