Immunochemical studies on phospholipase A 2 from Naja nigricollis venom

Four PLA2 isoenzymes (N. nigricollis CM-PLA2I-IV) were purified from Naja nigricollis (N. nigricollis) venom using Sephadex G-50 gel filtration, and ion exchange chromatography on CM-Sephadex C-25. The characterization of the isolated PLA2 isoenzymes revealed similarities in molecular weights, and differences in the isoelectric points, the optimum temperature, optimum pH, optimum Ca concentration and metal ion requirements. A good correlation (r > 0.7) for the in vitro neutralization of enzymatic PLA2s activity and the ELISA titers was found for sera collected at one week from each boosting of the rabbits. The found correlations were particularly high (r>0.9) when the purified Seph-PLA2 and CM-PLA2II were used rather than the whole venom. The established correlations show the importance of the purified PLA2 enzymes as immunogens for raising therapeutic antisera and as diagnostic reagents for in vitro determination of the potency of the therapeutic antisera.


INTRODUCTION
Snakebite is a serious medical problem worldwide, especially in the tropics.The incidence of snakebite mortality is particularly high in Africa, Asia, Latin America and New Guinea (Gutierrez et al., 2006).In Africa, snakebites cause more than one thousand deaths each year and thousands of cases of permanent physical disability (Theakston et al., 2003).In Egypt, the Black-necked Spitting Cobra; Naja nigricollis (N.nigricollis) is one of the most venomous snakes distributed in the south part of Egypt (Saleh, 1997).
Biochemical characterization of the venom of a snake from a particular geographical location is of great importance because pathogenesis developing after a bite is dependent on the qualitative composition of the venom and on the quantitative distribution of different components in particular venom (Stocker, 1990;Warrell, 1997).
Venoms contain a variety of enzymes, non-enzymatic polypeptide toxins and non-toxic proteins.Toxicity of venom depends on the qualitative and quantitative distribution of different enzymes and toxins in the venom (Stocker, 1990).Phospholipase A 2 (PLA 2 ) is the single most toxic component of the venom and particularly the basic PLA 2 plays an important role in mortality and morbidity following snakebite (Bhat et al., 1991;Mukherjee et al., 1998;Kang et al., 2011).
The phospholipase A 2 (PLA 2 ) (EC. 3.1.1.4)superfamily consists of a broad range of enzymes defined by their ability to hydrolyze glycerophospholipids (water insoluble substrate phospholipids) at the sn-2 position of the glycerol backbone releasing equimolar amounts of lysophospholipids and free fatty acids (Kini, 2003).
The superfamily of PLA 2 enzymes currently consists of 15 groups and many subgroups (Schaloske & Dennis, 2006).
Venom sPLA 2 from Elapidae and Hydrophiidae have been classified into group IA with the exception of a few enzymes that belong to group IB.However, venom sPLA 2 from Crotalidae and Viperidae are found in group IIA, and group IIB contains some sPLA 2 from Bitis species.Finally, sPLA 2 from bee and lizard venoms belong to group III (Huang et al., 1997;Six & Dennis, 2000).
Correlation of the in vivo neutralization of lethality and the in vitro PLA 2 neutralization to efficacy of the therapeutic antivenins for a number of elapids pointed to the importance of the elapid PLA 2 enzymes as immunogens for raising therapeutic antisera and as diagnostic reagents for in vitro determination of the potency of the therapeutic antisera.Also, Good correlations between the potency of antivenoms and the enzyme-linked immunosorbent assay (ELISA) titer against crude venoms and lethal toxincontaining fractions as antigens of viperid and elapid antivenoms were found (Theakston & Reid, 1979;Rungsiwongse & Ratanabanangkoon, 1991;Barbosa et al., 1995;Heneine et al., 1998;Maria et al., 1998;Muniz et al., 2000;Sells, 2003;Beghini et al., 2004;Rial et al., 2006;Halassy et al., 2008).
The present study was designed to isolate and characterize PLA 2 enriched fractions from N. nigricollis venom.The isolated PLA 2 fractions were used as immunogens for raising therapeutic antisera and the correlation between the in vitro neutralization of the enzymatic PLA 2 activity and ELISA titer against the homologous venom was studied.

Snake venoms
The N. nigricollis venom collected from cultivated lands in Luxor, Egypt was milked from several adult specimens.The venom was lyophilized, freeze-dried and stored at -20 °C until use.The venom samples were kept at -20 °C, thawed and centrifuged before use.

Purification of PLA 2 enzyme
The N. nigricollis (200 mg) venom dissolved in 1 ml equilibration buffer (0.05 M Tris-HCl, pH 6.8), was loaded on Sephadex G-50 column (2.6 × 50 cm).The sample-dissolving buffer was used for equilibration of the Sephadex column, and elution of the loaded samples.Fractions of 4 ml were collected at a flow rate of 48 ml/hr using fraction collector (Pharmacia LKB, Sweden).Fractions with phospholipase A 2 activity recovered from the previous step were pooled and directly applied to a CM-Sephadex C-25 column (1.6 × 25 cm) pre-equilibrated with the same buffer and eluted with a linear KCl gradient from 0 to 1.3 M in the same buffer.The enzymatic PLA 2 activity was detected by the indirect hemolytic assay according to Al-Abdulla et al. (1991).The active fractions were pooled, dialyzed, freeze-dried, and stored as N. nigricollis CM-PLA 2 I, II, III and IV.

Preparation of rabbit anti-PLA 2 antisera
Eight healthy rabbits (1.2-1.5 Kg body mass) were divided into two groups: Group A. Six Rabbits; were injected intramuscularly with doses containing 15 µg of the individual N. nigricollis Seph-PLA 2 and N. nigricollis CM-PLA 2 (I, II) dissolved in 0.5 ml isotonic saline and Group B. Two rabbits pre-immunized with the individual N. nigricollis Seph-PLA 2 were boosted with the homologous venom with doses containing 8 µg of whole venom dissolved in 0.5 ml isotonic saline.The primer dose was emulsified with 0.5 ml of complete Freund's adjuvant (CFA), whereas the first and second booster doses were emulsified with equal volume of incomplete Freund's adjuvant (IFA).Blood samples of the individual rabbits were collected at one week intervals and sera were separated, pooled and stored at -20 °C.Rabbit sera before immunization were used for control purposes.

Polyacrylamide gel electrophoresis (SDS-PAGE)
The electrophoresis analysis was performed in the Mini-Protean II Dual-Slab Cell (Bio Rad).Preparation of gels, samples, and electrophoresis were performed according to the conditions described by Laemmli (1970).Proteins were stained with 0.2% Coomassie Brilliant Blue R-250 in fixative solution [methanol/acetic acid/water (45/10/45) (v/v/v)] and destained by the same solution.

Analytical isoelectric focusing (IEF)
The analytical isoelectric focusing was performed in the MultiphorII unit (80-1106-20) connected to thermostatic circulator and the temperature was set in the range of (4-6) °C.Preparation of gels, samples and electrophoresis were performed according to Garfin (1990) with minor modification.

Phospholipase A 2 activity
Three methods were used to measure the PLA 2 activity of the crude venoms and the isolated fractions according to (Marinetti, 1965;Gutierrez et al., 1988 andMoreno et al., 1988) with slight modifications.

Method of Marinetti (1965)
This method was used to study the kinetics of the isolated N. nigricollis PLA 2 fractions.Briefly, a stock of egg yolk suspension was prepared by shaking one egg yolk in 0.9% NaCl to give a final volume of 100 ml.The working suspension used for the study of phospholipase A 2 was prepared by making 5-fold dilution of this stock suspension in 0.9% NaCl.The enzymatic activity was measured as follow: To 1 ml of the working egg yolk suspension, 4.9 ml of isotonic saline was added.The suspension was mixed, and stood for 5 min during this time the suspension equilibrated to a temperature of 41°C, then to the suspension, 0.1 ml of the test solution in saline was added.The contents were rapidly mixed and the absorbancy at 925 nm was recorded each 5 min for 15 min.The control was adjusted to give an absorbency of 0.6 (at 925 nm).The reaction velocity was determined as the absorbancy change at 925 nm per 5 min and was taken as a parameter for the relative enzyme activity.

Method of Gutierrez et al. (1988)
This method was used to quantify the specific PLA 2 activity in the enzyme isolation process.Briefly, Serial dilutions of the venom or the isolated fractions in 10 μl of physiological saline were added into 3 mm wells in agarose gels containing 4% washed human erythrocytes, 4% egg yolk suspension as a source of lecithin and 10 mM CaCl 2 and incubated for 20 hr at 37 °C.The square diameter of each hemolytic halo was measured then, the standard dose response curve for the PLA 2 activity was plotted (venom concentration versus the square diameter of the hemolytic halos).One PLA 2 unit was defined as the venom concentration (g) which induce a hemolytic halo of one cm 2 square diameter (r 2 =1).The in vitro neutralization of the N. nigricollis venom PLA 2 activity was measured as follow; a constant amount of venom (one PLA 2 unit) was incubated with serial dilutions of anti-N.nigricollis venom and anti-N.nigricollis CM-PLA 2 antivenoms for 30 min at 37 °C.Then, 10 µl of each mixture was added to duplicate wells in agarose-erythrocyte-egg yolk gels; a nonimmune rabbit serum was used as control.The square diameters of the hemolytic halos were measured and the neutralization of the enzymatic PLA 2 activity was calculated from the following formula: The % neutralization of enzymatic PLA 2 activity = 100% PLA 2 -the % inhibition of PLA 2 activity.(The 100 % PLA 2 was the percentage of the haemolysis caused by the venom alone).

Method of Moreno et al. (1988)
After isoelectrofocusing (pH 3.5-10), or electrophoresis in 14% SDS-PAGE, the resolved proteins were eletrotransferred to nitrocellulose paper; the transfer was performed in the Semidry LKB -BROMMA -2117-250 NOVABLOT Electrophoresis Transfer Kit under constant current conditions of 100-150 mA for 1 hr according to Towbin et al., (1979), the nitrocellulose paper was rinsed and incubated in incubation buffer (1% casein hydrolysate dissolve in 0.07 M barbiturate buffer, pH 8.6) for 2 hr at room temperature, the nitrocellulose membrane was then incubated with a substrate-fortified gel (1% agarose dissolved in PBS, pH 7.4, containing 4% of packed human erythrocytes, 4% of egg yolk as source of lecithin and 10 mM CaCl 2 ); the plate was kept at 37 °C in a humid chamber.After 3 hr incubation, the nitrocellulose paper was removed and the substrate gel was further incubated at 4°C for additional 21 hr, then the plate was photographed and the PLA 2 activity was indicated by the hemolytic bands (clear bands) in the substrate-fortified gel.

Effect of substrate concentration
Serial concentrations of the working egg yolk suspension (0.5-3 ml complete with isotonic saline) were added to constant concentration of the individual PLA 2 fraction then the reaction velocity was determined and plotted against substrate concentrations.The substrate Concentration (ml) that gives half-maximum velocity (V max /2) was taken as the endpoint for calculation of relative K m .

Effect of temperature
The optimum temperature was examined by incubating the reaction mixture at different temperatures ranging from 25 to 70 °C under standard assay conditions.The temperature value that gives the maximum reaction velocity was defined as the optimal temperature.

Effect of pH
The optimum pH was examined using Tris-HCl buffer over a broad pH range from 3 to 10.The pH value that gives the maximum reaction velocity was defined as the optimal pH.

Effect of Ca +2 concentration
Ca +2 concentrations between 0-100 mM of CaCl 2 were added to dialyzed substrate suspension (1 ml of dialyzed stock egg yolk suspension against 0.9% NaCl for 72 hr at 4 °C and 5 ml of isotonic saline) instead of substrate suspension as mentioned above.Reaction velocity of the individual PLA 2 fraction was measured and plotted against Ca +2 concentrations values.The Ca +2 concentration value that gives the maximum reaction velocity was defined as the optimal Ca +2 concentration.

Enzyme linked immunosorbent assay (ELISA)
ELISA was performed according to the method of Maria et al. (1998) using N. nigricollis venom (0.5 µg/well) as coating antigen, the conjugate (anti-rabbit IgG peroxidase) was used at 1:5000 dilution.A standard curve between log antisera dilution and log optical density (OD) at 490 nm was plotted.The dilution that gives 0.5 OD at 490 nm was taken as ELISA titer.

Protein determination
Protein concentrations were determined according to the method of Bradford (1976), using bovine serum albumin as standard protein.

Purification of N. nigricollis PLA 2 s
The PLA 2 enzymes were isolated from N. nigricollis venom using gel filtration and ion exchange chromatography.In the First step, the N. nigricollis venom (200 mg protein) was applied on Sephadex G-50 gel filtration column (2.6 × 50 cm) and separated into three main protein peaks (Fig. 1), the PLA 2 activity fraction was detected under the first two peaks and designated as N. nigricollis Seph-PLA 2 with 75% recovery (Table 1).In the second step, the N. nigricollis Seph-PLA 2 fraction was applied on a CM-Sephadex C-25 column (1.6 × 25 cm), four major protein peaks were resolved as one unbound and three bound peaks.Four PLA 2 fractions were detected in the bound proteins and designated as CM-PLA 2 I, II, III and IV (Fig. 2).The N. nigricollis CM-PLA 2 II fraction was found to possess the highest specific PLA 2 enzymatic activity and recovery (40 U/mg and 19%), respectively (Table 1).One PLA 2 unit was defined as the venom concentration (g) which induce a hemolytic halo of one cm 2 (r 2 =1).

Characterization of the N. nigricollis CM-PLA 2 s
The electrophoretic separation of the The N. nigricollis CM-PLA 2 variants (I-IV) shows that the molecular weights of N. nigricollis CM-PLA 2 variants were between 14 and 15 kDa (Fig. 3), the activity patterns of the N. nigricollis CM-PLA 2 (I-IV) variants demonstrated that the molecular weights of the N. nigricollis CM-PLA 2 variants were of about 14 kDa (Fig. 4).The N. nigricollis CM-PLA 2 fractions (I-IV) were subjected to isoelectrofocusing technique using a wide range of ampholine (from 3-10), transferred to nitrocellulose paper, the PLA2 activity of the four fractions was then measured as mentioned above.The results indicated that these fractions have a range of isoelectric points (pIs) between 6 and 8 (Fig. 5).

Km values
The Km values of the N. nigricollis CM-PLA 2 fractions (I-IV) possessed close relative K m values as shown in (Fig. 6 and Table 2).The Km values were 1, 1.3, 1.8 and 1.6 for N. nigricollis CM-PLA 2 I, II, III and IV, respectively (Table 2).

Effect of temperature
The effect of temperature on the N. nigricollis CM-PLA 2 fractions was measured at incubation temperatures between (25-70° C), the results indicated that the N. nigricollis venom, N. nigricollis CM-PLA 2 (I, II) and N. nigricollis CM-PLA 2 (III, IV) fractions have significantly different optimal temperature values.The crude venom possessed its maximum PLA 2 activity at 65° C, the CM-PLA 2 (I, II) fractions exhibited their maximum activities at 41 and 44° C while the CM-PLA 2 (III, IV) fractions exhibited their maximum activity at 52°C (Fig. 7 A and Table 2).

pH optimum
The optimal pH values for N. nigricollis CM-PLA 2 fractions were measured at pH values between (3-10), the results revealed that the optimal pH values of N. nigricollis CM-PLA 2 (I, II) and N. nigricollis CM-PLA 2 (III, IV) fractions were different.the pH optimum of both N. nigricollis CM-PLA 2 (I, II) were 7, while that of N. nigricollis CM-PLA 2 (III, IV) were 8 (Fig. 7 B, Table 2).

Effect of metal ions and solvents
The optimal Ca +2 concentration values for N. nigricollis CM-PLA 2 (I, II) and N. nigricollis CM-PLA 2 (III, IV) fractions were different (Fig. 8, 9 and Table 2).The Ca +2 at 10 mM increased the PLA 2 activity of the fractions in the order of (N.nigricollis CM-PLA 2 I, II III, IV).K + ion activated all N. nigricollis CM-PLA 2 fractions except N. nigricollis CM-PLA 2 I, whereas Mg +2 ion activated only N. nigricollis CM-PLA 2 II fraction (Fig. 9).Ethanol and phenol (1%) had no effect on the PLA 2 activity of the four N. nigricollis CM-PLA 2 fractions, whereas phenol (5%) and formaldehyde (0.6%) had inhibitory effect on the PLA 2 activity of the four N. nigricollis CM-PLA 2 fractions (Fig. 9).Immunogenicity of the isolated PLA 2 s: Crude N. nigricollis venom and the corresponding PLA 2 isolated fractions were used as immunogens for production of the corresponding rabbit antisera.
ELISA and neutralization of enzymatic PLA 2 activity confirmed the elicited rabbit antibodies (Abs) in vitro (Tables 3,  4).Potency of the rabbit anti-N.nigricollis and its corresponding isolated PLA 2 s The ELISA titer of the rabbit antisera to the venom antigens (presumably the venom PLA 2 ) were detected after three weeks from the first injection and progressively increased over the immunization schedule.The ELISA titers of the rabbit anti-CM-PLA 2 I reached the highest peak after one week from the third injection (12775 EU), while the ELISA titers of the rabbit anti-Seph-PLA 2 and rabbit anti-CM-PLA 2 II showed the highest ELISA titers in the eighth (60191 EU) and the sixth (727307 EU) weeks, respectively (Table 3).The results revealed that the CM-PLA 2 II was the most immunogenic PLA 2 fractions, whereas CM-PLA 2 I was the least immunogenic.

In vitro neutralization of the enzymatic PLA 2 activity
Antisera to the elapid venoms and the isolated venom PLA 2 isoenzymes were raised and investigated for its efficacy to neutralize the enzymatic PLA 2 activity of crude venom.The neutralization of the venom PLA 2 activity by the various harvests of the rabbit antisera revealed a progressive increase in the PLA 2 neutralization potency over the immunization period.Also, Neutralization by antisera to the isolated PLA 2 s was found to be more efficient than neutralization by rabbit antisera to the whole venoms.Furthermore, compared to the crude venom, the isolated PLA 2 s were found to be more immunogenic (Table 4).

Correlation between ELISA antibody titers and in vitro neutralization of enzymatic PLA 2 activity
The neutralization of the venom enzymatic PLA 2 activity and the levels of antibodies measured by ELISA were correlated, a good correlation was found for the rabbit anti-N.nigricollis (r 0.7).However, attempts to correlate the same traits for all the samples from an individual immunized animal or for timed samples from different animals were not successful (r 0.6).The found correlations were particularly high (r 0.95) when the isolated Seph-PLA 2 fraction were used rather than the whole venom for establishing the correlations.Most pronouncedly the correlation for neutralization of the enzymatic PLA 2 activity of the N. nigricollis with the rabbit anti-N.nigricollis CM-PLA 2 II was the best (r > 0.98) (Table 5).

DISCUSSION
The occurrence of PLA 2 from snake venom as multiple isoenzymes is a common observation (Singh et al., 2000;Kini, 2003;Ramirez-Avila et al., 2004;Cogo et al., 2006;El Hakim et al., 2008;Romero-Vargas et al., 2010).These isoenzymes have been reported to share high identity in their amino acid sequence and possess similar threedimensional structure (Arni & Ward, 1996;Scott, 1997;Tan et al., 2003).However, the exact number of the isoenzymes in each venom is not certain.The uncertainty could be explained in terms of the fact that the specific substrate for each of the variants is not yet known.
Three different assays were used in the present study to assess the PLA 2 activity of one of the most medically important Egyptian snake venom and its isolated PLA 2 fractions.A sensitive assay described by Gutierrez et al. (1988), was used in the present study to quantities the specific PLA 2 activity of N. nigricollis venom and the coressponding isolated PLA 2 fractions, this assay based on the measurement of hemolytic haloes induced by venom on the agaroseerythrocyte-egg yolk (fortified-substrate) gel.A standard curve for PLA 2 activity of snake venom or isolated fractions was established by plotting protein concentration versus the square diameter of hemolytic halo using selected reference venom (data not shown).The enzymatic PLA 2 specific activity was calculated as U/mg proteins.Another assay of Moreno et al. (1988) was used to measure the enzymatic PLA 2 activity based on the transfer of the resolved venom to a substrate-fortified gel.This assay was highly useful in identifying the molecular weights and the pIs of the PLA 2 isoenzymes present in biological fluids in general and snake venoms in particular.
At the molecular exclusion step, almost all of the N. nigricollis venom enzymatic PLA 2 activities were recovered in the pool designated as N. nigricollis Seph-PLA 2 fraction representing 75 % of the venom.At the cationic-exchange chromatography step the N. nigricollis Seph-PLA 2 was separated into four N. nigricollis-PLA 2 (named N. nigricollis CM-PLA 2 I, II, III and IV) with recovery of (11.3, 19, 5.4 and 4.6 %), respectively.
The specific activity of N. nigricollis CM-PLA 2 I, II, III, IV fractions were 6.7, 40, 10 and 15.4 U/mg protein, respectively, demonstrating that enzymatic PLA 2 specific activity of the isoenzymes are quite different.In addition, the results showed that the highest PLA 2 specific activities were 40 U/mg for N. nigricollis CM-PLA 2 II indicating four-fold increase in the N. nigricollis venom PLA 2 specific activity.
The molecular weights of the N. nigricollis CM-PLA 2 isoenzymes resolved from N. nigricollis venom were close to 14 kDa which agree with the range generally reported for snake venom PLA 2 (Kemparaju et al., 1999;Bonfim et al., 2008;Landucci et al., 2012;Zou et al., 2012).The kinetics of the PLA 2 isoenzymes revealed that, the relative K m values for N. nigricollis CM-PLA 2 isoenzymes were close (1, 1.3, 1.6 and 1.8) for N. nigricollis CM-PLA 2 (I, II, IV and III), respectively.
The optimal temperature values of the N. nigricollis venom, N. nigricollis CM-PLA 2 (I, II) and N. nigricollis CM-PLA 2 (III, IV) fractions were different.Very weak PLA 2 activities were observed between 10 and 22° C for all N. nigricollis CM-PLA 2 isoenzymes except N. nigricollis CM-PLA 2 I, while the PLA 2 activity of these isoenzymes were completely inhibited at 80° C. The crude N. nigricollis venom possessed maximum activity at 65° C, which agrees with Nair et al. (1976) who found that the venom of several Naja species (Elapidae) showed their maximum activity at 65°C.
The optimal pH values at 41° C for N. nigricollis CM-PLA 2 (I, II) and N. nigricollis CM-PLA 2 (III, IV) were different.The catalytic activity of the N. nigricollis CM-PLA 2 I was completely inhibited at pH 8. Whereas, the catalytic activity of all the other fractions was completely inhibited at pH 9.These differences may be related to the enzyme conformational change by pH (Viljoen et al., 1975;Arni & Ward, 1996).
Effect of metal ions on enzymatic PLA 2 activity of the isolated fractions of N. nigricollis is markedly different.As most of the PLA 2 s (Murakami et al., 2006), the Ca +2 ion was found to be essential for the PLA 2 mediated hydrolysis of phospholipids (Ca +2dependent).
The optimal Ca +2 concentration values were different, 10 mM for all fractions except 5 mM for the N. nigricollis CM-PLA 2 III and IV.These differences in the concentrations of Ca +2 are probably related to the enzyme conformational change produced by this divalent ion.The catalytic activity was slightly activated by the Na + and variably inhibited by the AL +3 , Co +2 , Cu +2 , Fe +2 , Mn +2 and Ni +2 ions.The N. nigricollis CM-PLA 2 (II, III) were activated by K + , whereas, the N. nigricollis CM-PLA 2 II was activated by Mg +2 ion.However, all the other fractions were completely inhibited by the K + , Mg +2 and Zn +2 .This diversity in metal requirements might reflect different requirements for the different PLA 2 s functions.The data obtained from the present study provide biochemical information about the N. nigricollis phospholipase A 2 that help in studying their pharmacological effects.
In attempt to investigate whether or not the potency of antisera correlates with the inhibition of PLA 2 enzymatic activity of the homologues venoms.Antisera to N. nigricollis venom and the corresponding PLA 2 fractions were raised in rabbits using the same immunization schedule.
In the present study, the elicited antibodies to the PLA 2 isoforms contribute differently but significantly to the in vitro neutralization of enzymatic PLA 2 activity of the corresponding venom.A highly significant correlation between the in vitro neutralization of enzymatic PLA 2 activity and the ELISA antibody titers elicited to selected partially isolated PLA 2 was established.
The in vitro neutralization of PLA 2 activity and ELISA titers have been exploited to evaluate the neutralizing ability of antivenoms, therefore, avoiding large-scale use of mice, antigen and antiserum and to simplify and cut down the expenses for potency evaluation (Alape-Giron et al., 1997;Maria et al., 1998;Rial et al., 2006), in addition to adherence to ethical concepts.
There is a strong body of evidence indicating that the functional activity of some PLA 2 is independent of its in vivo activity (Kini & Evans, 1989;Kini, 2003).In spite of these observations, Successful correlates for testing the potency of horse antivenins were established (Alape-Giron et al., 1997;Maria et al., 1998;Rial et al., 2006).These correlates include the in vivo neutralization of lethality, neutralization of the hemolytic activity (PLA 2 ) and levels of antibodies measured by ELISA.In the present study, a good correlation for neutralization of the enzymatic PLA 2 activity and the ELISA titers was found for sera collected at one week from each boosting of the rabbits (r>0.9).The found correlations were particularly high when the isolated Seph-PLA 2 fraction were used rather than the whole venom for establishing the correlations.However, attempts to correlate the same traits for all the samples from an individual immunized animal or from timed samples from different animals were not successful.
In the present study, the indirect hemolytic assay developed by Gutierrez et al. (1988) for in vitro neutralization of PLA 2 activity was applied.The assay has several advantages, which include sensitivity and detection of very low levels of antibodies (Abs), easy, fast, and many samples can be studied simultaneously.This allows testing of the immune response of individual animals during extended immunization schedule in order to monitor antibodies production (Alape-Giron et al., 1997).
The results in the present study revealed that the antisera to the isolated PLA 2 were more potent than the antiwhole venom.This could be attributed to the complexity of the venom antigens compared to the isolated PLA 2 s that are antigenically less diverse (Freitas et al., 1990;Beghini et al. 2005) suggested that commercial antivenoms enriched in anti-PLA 2 antibodies could be useful for treating neurotoxicity in envenomed persons.In addition, the production of antivenoms against the main toxic component(s) of the venom of interest would reduce the amount of nonessential antibodies and proteins injected during antivenom therapy and could therefore result in safer treatment (less risk of side effects such as anaphylactic shock).
The data obtained from the present study give potential application in evaluation of the antivenom production.As the therapeutic antivenoms are traditionally produced in large animals, the established correlations helps in cutting down the production costs and overcome the ethical arguments concerning the extensive use of experimental animals.

Fig. 1 :
Fig. 1: Gel filtration of N. nigricollis venom on a Sephadex G-50.The venom (200 mg protein) dissolved in 0.05 M Tris-HCl, pH 6.8 buffer, was applied on the Sephadex G-50 column (2.6 × 50 cm) equilibrated with the same buffer.The column was eluted with the same buffer and 4 ml fractions were collected at a flow rate of 48 ml/hr, tubes from (25-58) show PLA 2 activity as assayed by the indirect hemolytic assay.

Fig. 2 :
Fig. 2: Ion-exchange chromatography of N. nigricollis Seph-PLA 2 fraction on CM-Sephadex C-25.The fraction (120 mg protein) dissolved in 0.05M Tris-HCl, pH 6.8 buffer, was applied on the CM-Sephadex C-25 column (1.6 × 25) equilibrated by the same buffer.The unbound proteins were washed with the dissolving buffer and the bound proteins were eluted with a linear gradient from 0 to 1.3 M KCl in 0.05M Tris-HCl pH 6.8 buffer.Fractions of 4 ml were collected at a flow rate of 48 ml/hr.Four CM-PLA 2 fractions were resolved as indicated by the indirect hemolytic assay.

Fig. 6 :
Fig. 6: Effect of substrate concentrations (egg yolk suspension) on the reaction velocity of four N. nigricollis CM-PLA 2 fractions.

Fig. 7 :
Fig. 7: Effect of temperature (A) and pH (B) on the reaction velocity of four N. nigricollis CM-PLA 2 fractions.

Table 1 :
The purification scheme of the PLA 2 fractions from N. nigricollis venom.

Table 2 :
The relative K m , the optimum temperature, pH and Ca +2 concentration values of the N. nigricollis CM-PLA 2 fractions.

Table 3 :
ELISA titer of different rabbit antisera

Table 4 :
Potency of the R. anti-N.nigricollis and R. anti-N.nigricollis PLA 2 s fractions against the homologous venom.

Table 5 :
The correlation between ELISA antibodies titers and the in vitro neutralization of N. nigricollis PLA 2 enzymatic activity.